Pinus pinceana Gordon & Glend. tolerates extremely arid conditions. Northern Mexico became more arid during the Quaternary, modifying ecological communities. Here, we try to identify the processes underlying the demographic history of P. pinceana and characterize its genetic diversity using 3100 SNPs from genotyping by sequencing 90 adult individuals from 10 natural populations covering the species' entire geographic distribution. We inferred its population history and contrasted possible demographic scenarios of divergence that modeled the genetic diversity present in this restricted pinyon pine; in support, the past distribution was reconstructed using climate from the Last Glacial Maximum . We inferred that P. pinceana diverged into two lineages ~2.49 Ma (95% CI 3.28–1.62), colonizing two regions: the Sierra Madre Oriental (SMO) and the Chihuahuan Desert (ChD). Our results of population genomic analyses reveal the presence of heterozygous SNPs in all populations. In addition, low migration rates across regions are probably related to glacial–interglacial cycles, followed by the gradual aridification of the Chihuahuan Desert during the Holocene.Climate changes, together with geographical barriers imposed by the Sierra Madre Oriental and the Chihuahuan Desert, have shaped the genetic diversity and spatial distribution of different species in northern Mexico.  We focused on how phylogeography, historical demography, and historical migration have influenced the current genetic variation and geographic distribution of P. pinceana, a geographically restricted pinyon pine that tolerates extremely arid conditions in northern Mexico. We analyzed the genomic data of two biogeographic zones where Pinus pinceana, exhibits high differentiation among populations. We used genomic information, along with climatic projections over the Pleistocene and Holocene, to infer the past demographic history of this species as well as its spatial and temporal distribution. The ChD is the third‐largest desert in the American continent, considered the most diverse in the Western Hemisphere and one of the most diverse arid regions in the world  is a high mountain range (1480–3720 m a.s.l.) dating to the Late Cretaceous and early Tertiary Laramide  characterize its genetic diversity using genome‐wide single nucleotide polymorphisms (SNPs) data, (2) identify phylogeographic patterns across its natural distribution range, (3) infer its past demographic history during the Pleistocene and Holocene, and (4) reconstruct its potential distribution during the LGM to infer the principal climatic limitations of the species.In this study, we aim to identify how phylogeography, historical demography, and the climatic transformation during the Quaternary influenced the current genetic variation and geographic distribution of 22.1P. pinceana  and MULTIQC  was used to correct artifacts of PCR duplication.The read adapters were removed using TRIMMOMATIC  larger than 0.6 and D (deviation from even read ratios in heterozygous) outside the range of −10 to +10. These parameters were chosen according to the proportion of heterozygous individuals within a population and allelic ratios within heterozygous individuals. A filter based on linkage disequilibrium (LD) was applied using PLINK v1.9 , within‐population gene diversity (HS), and the inbreeding coefficient (FIS) using the R package Hierfstat , and pairwise population differentiation  with the 2.3P. pinceana was inferred by testing four different scenarios via the composite likelihood approach of fastsimcoal , to infer the divergence times between and within biogeographic regions , and effective population sizes (Ne). Contraction, to estimate the divergence times between the ChD (TDIV) and SMO populations, followed by the independent contraction of both groups during the Holocene. We also modeled two scenarios M, including migrations, based on the former two scenarios with the same parameter ranges but adding independent migration rates among groups  related to Pinus pinceana  from 100 to 1000 individuals in the SMO and ChD lineages, and TDIV between 275 and 62,500 generations ago. The Contraction model was set using search ranges for the effective population sizes (Ne) of SMO and ChD from 100 to 1000 individuals. TDIV was set to date the divergence event within ChD and SMO between 275 and 62,500 generations ago, and TCON SMO was set to date a contraction event in the SMO group and TCON ChD using the range of 40 and 275 generations ago. Finally, for the two migration models, we used the same range adding between 1 × 10−9 and 1 × 10−4, per base per generation, drawn from a log uniform prior distribution.The Divergence without migration model was set using search ranges for the effective population sizes  and performed an outlier analysis using Mahalanobis distances using the package JMP .We compiled 37 records verified from Escalante , FigueroTo reduce model overfitting, we selected independent bioclimatic variables before constructing the ENMs with a Principal Component Analysis using JMP . We retained the variables that had the higher loading values for the first three principal components (>0.7) and had a correlation coefficient of <0.8: Annual Mean Temperature (Bio1), Isothermality (Bio3), Temperature Seasonality (Bio4), Annual Precipitation (Bio12), and Precipitation in the Driest Month (Bio14) from PC1; Precipitation Seasonality (Bio15), and Precipitation of Warmest Quarter (Bio18) from PC2; and Minimum Temperature of Coldest Month (Bio6) from PC3.To construct a potential niche model, we used Maxent , and a mean depth per site of 64×.HO) was 0.0663, the within‐population gene diversity (HS) was 0.0541, and the inbreeding coefficient (FIS) was −0.2247 (Table HS) were similar among populations and ranged from 0.0388  to 0.0686 . Levels of HO were highest for the ChD location, General Cepeda (0.0824), and the lowest for Maguey Verde (0.0463) in SMO. Estimates for the inbreeding coefficient FIS were negative for all populations, ranging from −0.0874  to −0.1439 , while the highest nucleotide diversity was found in Sierra de Parras (0.002), and the lowest in La Florida (0.0004).Globally, the observed heterozygosity  showed two clear population clusters divided by PC1, describing 17.14% of the total variance while PC2 describes 4.3% of the total variance. PC2 helps to divide the SMO cluster with one subcluster including Tolantongo, San Joaquín, and Maguey Verde and another including La Florida and Nuñez Figure .K = 2 supports the clustering in two biogeographic areas. For K = 2, 42 of the 45 samples from SMO were classified 100% in one cluster  showed sub fragmentation within these two biogeographic areas but with less likelihood.The ADMIXTURE analysis showed that r Figure . Forty‐t3.3We used the entire 3100 SNP dataset to model the population divergence time hypotheses from the demographic simulations. Three of the four scenarios resulted in a divergence time estimated during the Early Pleistocene while the Contraction M scenario estimated the divergence between clusters during the Miocene Tables  and S2.TDIV) between them was estimated in the Early Pleistocene . Effective population sizes (Ne) for groups were similar between groups, SMO with 4367 individuals (95% CI: 4958.59–3775.41), and ChD with 6740 individuals (95% CI: 7531.42–5948.58). Migration rates per generation (m) were estimated to be significantly lower 3.93 × 10−6 (95% CI: 1.364 × 10−5–5.78 × 10−6) from SMO to ChD, than in the opposite direction .The best‐supported model was the divergence with migration according to the ΔAIC that considers the split into two lineages according to the biogeographical regions Figure . The dat3.4P. pinceana, as the areas identified as current or past water bodies have not been occupied by the species. The areas that remained stable, that is, with suitable conditions for P. pinceana since the LGM include the current populations of General Cepeda and Mazapil, covering an area of 55,582 pixels (1 pixel is around 1 km2).The LGM model showed a reduced suitable area compared with the current model Figure , centere44.1P. pinceana between the Chihuahuan Desert and the Sierra Madre Oriental. A similar differentiation has been described in several desert scrub plant species in this area, as we discuss below. This structure is apparently related to climate dynamics during the Quaternary  patterns using SNPs were similar to the values described in conifer species with broad geographic ranges in Mexico, like Pinus patula, Abies religiosa, and Juniperus blancoi , like that typically reported for conifer taxa with wide distribution ranges , followed by the division of both biogeographic regions (~127.7–~539.2 kya) during the interglacial cycles. The climatic conditions in the habitat of P. pinceana are more suitable at present than they were during the LGM; this is in contrast to what has been described in other conifers like Picea chihuahuana, which has become more restricted due to the disappearance of a suitable habitat making it susceptible to the loss of genetic variability via genetic drift, inbreeding depression and strong selection . These lineages later colonized two biogeographic regions, the SMO and the ChD, while interglacial cycles modified the Chihuahuan Desert as the aridification increased and paleolakes shrank. This division probably occurred during the climatic changes of the Pleistocene and was related to the glacial–interglacial cycles.P. pinceana is likely explained by climate dynamics that left perceptible marks in the patterns of genetic diversity and structure observed in the species today.Thus, the phylogeographic history of Laura Figueroa‐Corona: Data curation (lead); formal analysis (lead); investigation (lead); methodology ; resources ; software ; visualization ; writing – original draft ; writing – review and editing . Alejandra Moreno‐Letelier: Formal analysis ; methodology ; visualization ; writing – review and editing . Ortega‐Del Vecchyo Diego: Data curation ; formal analysis ; methodology . Pablo Pelaez: Data curation ; formal analysis ; software . David S. Gernandt: Formal analysis ; funding acquisition ; investigation ; writing – original draft ; writing – review and editing . Luis E. Eguiarte: Formal analysis ; investigation ; supervision ; writing – review and editing . Jill L. Wegrzyn: Formal analysis ; methodology ; software ; writing – review and editing . Daniel Piñero: Conceptualization ; data curation ; formal analysis (supporting); funding acquisition (lead); investigation ; project administration (lead); resources ; supervision (lead); writing – original draft (lead); writing – review and editing (lead).All authors contributed to the manuscript writing and revisions, and declared no conflict of interest.https://www.ncbi.nlm.nih.gov/bioproject/PRJNA719106/.This article has earned Open Data and Open Materials badges. Data and materials are available at Appendix S1Click here for additional data file.


Liriomyza sativae, the vegetable leafminer, is an important agricultural pest originally from the Americas, which has now colonized all continents except Antarctica. In 2015, L. sativae arrived on the Australian mainland and established on the Cape York Peninsula in the northeast of the country near the Torres Strait, which provides a possible pathway for pests to enter Australia and evade biosecurity efforts. Here, we assessed genetic variation in L. sativae based on genome‐wide single nucleotide polymorphisms (SNPs) generated by double digest restriction‐site‐associated DNA sequencing (ddRAD‐seq), aiming to uncover the potential origin(s) of this pest in Australia and contribute to reconstructing its global invasion history. Our fineRADstructure results and principal component analysis suggest Australian mainland populations were genetically close to populations from the Torres Strait, whereas populations from Asia, Africa, and Papua New Guinea (PNG) were more distantly related. Hawaiian populations were genetically distinct from all other populations of L. sativae included in our study. Admixture analyses further revealed that L. sativae from the Torres Strait may have genetic variation originating from multiple sources including Indonesia and PNG, and which has now spread to the Australian mainland. The L. sativae lineages from Asia and Africa appear closely related. Isolation‐by‐distance (IBD) was found at a broad global scale, but not within small regions, suggesting that human‐mediated factors likely contribute to the local spread of this pest. Overall, our findings suggest that an exotic Liriomyza pest invaded Australia through the Indo‐Papuan conduit, highlighting the importance of biosecurity programs aimed at restricting the movement of pests and diseases through this corridor. For barcoding, legs of flies were taken from specimens for Chelex DNA extraction with protocols following previous work except in 70 μl of 5% Chelex 100 resin  . ddRAD‐seq libraries were prepared according to the protocol from Rašić et al.  and NlaIII , NEB CutSmart buffer, and water. Digestions were incubated at 37°C for 3 h, and then the products were cleaned with 1.5× volume of AMpure XP paramagnetic beads . After that, the digestion fragments were ligated to modified adapters containing 6 bp indexes at both ends overnight at 16°C with T4 ligase , followed by a heat deactivation step at 65°C for 10 min. Adapter ligated DNA fragments from all individuals were pooled and cleaned with 1.5× bead solution afterward. A Pippin Prep cassette of 2% agarose with marker B  was applied for size selection of fragments between 350–450 bp. Lastly, 1 μl size‐selected DNA was added in a 10 μl PCR as a template with 5 μl of the Phusion High Fidelity 2× Master mix (New England Biolabs) and 2 μl of 10 μM Illumina PCR primers. PCR programs were run at 98°C for 30 s, followed by 15 cycles of 98°C for 10 s, 60°C for 30 s, 72°C for 90 s, and a final elongation step at 72°C for 5 min. We pooled 10 such PCR products together and cleaned them with a 1.5× bead solution for making the final library. The ddRAD‐seq libraries were sequenced on an Illumina NovaSeq 6000 platform using 2 × 150 bp chemistry by Novogene HK Company Limited, Hong Kong.We built three ddRAD libraries for 200 ć et al. . In brie2.3L. sativae, we used the Stacks workflow  to build a catalog of loci de novo  due to a high level of missing data (>50%) and generated VCF files containing SNPs called in 75% of individuals (‐r 0.75) from each population (‐p 24) with a minor allele count of 3 (‐‐min‐mac 3) for downstream analyses , by using the “prcomp” function  implemented in the ade4 R package  with fineRADstructure . The co‐ancestry matrix of 193 individuals was based on a summary of nearest neighbor haplotype relationships, which were calculated from multiple SNPs per RAD locus. The individuals were assigned to corresponding populations, and a phylogenetic tree was constructed using the FineRADstructure MCMC clustering algorithm with default settings. R scripts fineradstructureplot.r and finestructurelibrary.r (available at http://cichlid.gurdon.cam.ac.uk/fineRADstructure.html) were used for visualization.We performed a co‐ancestry analysis on all 2.4.3K). This tool is based on a maximum likelihood method to estimate individual ancestries, and a cross‐validation (CV) test was applied to evaluate the optimal K .Admixture v.1.3.0  and geographic distances (km) based on 999 permutations implemented in adegenet 2.1 , we used a Mantel test to explore the correlation between genetic distances  from 2 through 10 to examine patterns of ancestry based on the 193 individuals   Figure . IndividL. sativae from Boigu Island and Masig Island contained higher levels of genetic diversity (including new genotypes) that are likely associated with multiple introduction sources .Interestingly, the admixture analysis suggests Bali and PNG contributed most to the ancestries of two (of the three) Torres Strait populations: Boigu Island and Masig Island. New genotypes were detected leading to novel ancestry for Thursday Island and Seisia. Admixture results also indicate recent co‐ancestry from the Torres Strait to mainland Australia. Compared with Seisia, K = 8 , there are additional hierarchical structure levels, primarily involving Kenya, but also within Australian L. sativae populations, and within Hawaii . The highest FST value (0.3269) was between Kawamata Farms  and Seisia . On the contrary, the lowest FST value (0.0045) was between Heyuan  and Chiayi . Pairwise FST values within Hawaiian populations ranged from 0.018 to 0.177, suggesting high genetic diversity within Hawaii. Specifically, the pairwise FST values between Paradise Park , Hilo , and USDA‐ARS‐PBARC  ranged from 0.0181 to 0.0502, while the pairwise FST values between Kawamata Farms  and all other Hawaiian populations ranged from 0.148 to 0.1775.To evaluate the possible association between genetic and geographic distances, we built matrices of genetic distances among 21 populations (excluding three populations with sample size less than four) by calculating pairwise 69 Table . Relativp = 0.002; correlation = 0.412; R2 = 0.166) based on 999 replicates . Pairwise FST between these populations ranged from 0.0414 to 0.2169 . The highest FST value (0.2169) was between Wewak and Seisia . On the contrary, the lowest FST value (0.0414) was between Thursday Island  and Seisia. A Mantel test indicated no relationship between pairwise genetic (FST) and geographic distances (p = 0.241 based on 999 replicates) , we found support for isolation‐by‐distance, with a Mantel test being significant  Figure , suggest4Liriomyza sativae represents one such threat, having invaded many countries around the world in the past 40 years  population is nearly identical to that from Seisia . However, these two populations are genetically different from Masig Island and Boigu Island (both from the Torres Strait), although all these populations still cluster together as a group when compared to other localities. Our study, therefore, provides evidence that the incursion of L. sativae into mainland Australia is directly linked to Torres Strait populations rather than arising from incursions from other countries. We also found support for multiple invasions from different countries  into the Torres Strait populations as reflected by results from the admixture analyses. Furthermore, we found that L. sativae populations in Hawaii are genetically distinct from other countries, whereas samples from Kenya, China, Vietnam, and Timor‐Leste cluster as a group that is related to Bali and PNG populations.We found expected signals of gene flow among Liriomyza sativae was first described from specimens collected from alfalfa (Medicago sativa) in Argentina in 1937  was reported to be damaging crops in mainland USA before World War 1  and eastern (remaining) groups. Unfortunately, our study did not have samples from the suspected native range of South America as a reference. Populations of L. sativae from Hawaii might be strongly differentiated from mainland USA due to genetic drift, although this might also reflect repeated ongoing introductions from the mainland.Spencer, . By 198021 Table , where iSpencer, . Given HSpencer, , importeL. sativae to Hawaii (first recorded in 1921) and then to several South Pacific islands  has largely displaced L. sativae in southern parts of the USA , which would be required to adequately assess these possibilities. Further sampling in southeast countries near Timor‐Leste may help reveal this part of the incursion history in more detail. Genetic variation in Hawaii, Asia, and Africa was relatively low based on admixture results, which suggests a possible demographic bottleneck at the time of introduction and/or a period of limited population growth after establishment. The pattern of genetic differentiation across these populations indicates a limited contribution to the L. sativae expansion in Australia.Our population genetic results are broadly congruent with the biogeographical history of L. sativae populations globally (Ben Abdelkrim et al., Despite these connections between distant locations, IBD was still detected overall, suggesting that geographical distance, wind currents, ocean currents, and obstructing landmasses at a broader scale may have affected genetic connectivity of L. sativae to other regions, but further spread seems inevitable given that many other regions on the mainland are suitable for this species to exist (Maino et al., L. sativae such as through the introduction of insecticide resistance genes. The geographical location of the Torres Strait is particularly suitable for multiple incursions as detected in species like the Asian tiger mosquito, Aedes albopictus (Beebe et al., Eumetopina flavipes (Anderson & Congdon, Culicoides biting midges (Eagles et al., Although the SNP data provide a detailed picture of relatedness that is not available from mtDNA data, our results are nevertheless consistent with some previous CO1 results. These indicated a link between the Torres Strait Islands and the mainland incursion as well as the possibility of multiple incursions into Torres Strait populations (Blacket et al., 5L. sativae provide evidence of connections among populations that are mostly consistent with historical observations. Our study is the first to apply high‐density SNP markers to determine the population structure of Liriomyza and provide a baseline and foundation to further track leafminer movements across the region and further into the Australian mainland. The data provide a basis for quarantine detections to identify local versus international sources (c.f. Schmidt et al., In summary, our SNP‐based analyses of None declared.Appendix S1Click here for additional data file.


Rattus norvegicus and Rattus tanezumi are dominant species of Chinese house rats, but the colonization and demographic history of two species in China have not been thoroughly explored. Phylogenetic analyses with mitochondrial DNA including 486 individuals from 31 localities revealed that R. norvegicus is widely distributed in China, R. tanezumi is mainly distributed in southern China with currently invading northward; northeast China was the natal region of R. norvegicus, while the spread of R. tanezumi in China most likely started from the southeast coast. A total of 123 individuals from 18 localities were subjected to 2b‐RAD analyses. In neighbor‐joining tree, individuals of R. tanezumi grouped into geographic‐specific branches, and populations from southeast coast were ancestral groups, which confirmed the colonization route from southeast coast to central and western China. However, individuals of R. norvegicus were generally grouped into two clusters instead of geographic‐specific branches. One cluster comprised inland populations, and another cluster included both southeast coast and inland populations, which indicated that spread history of R. norvegicus in China was complex; in addition to on‐land colonization, shipping transportation also have played great roles. ADMIXTURE and principal component analyses provided further supports for the colonization history. Demographic analyses revealed that climate changes at ~40,000 to 18,000 years ago and ~4000 years ago had led to population declines of both species; the R. norvegicus declined rapidly while the population of R. tanezumi continuously expanded since ~1500 years ago, indicating the importance of interspecies' competition in their population size changes. Our study provided a valuable framework for further investigation on phylogeography of two species in China. Rattus tanezumi and Rattus norvegicus in China; 486 individuals collected from 31 localities were initially analyzed with mitochondrial DNA and 10 microsatellite loci.We investigated the phylogeography of  Hybridization between species of RrC has been confirmed in both south‐east Asia and America, causing discordance between mitochondrial and nuclear phylogenetic trees .Both i Figure  are disti Figure , and R. ts Wang, . Based ona Wang, . Some sta Allen, . From ita Allen, . The genCOI, Cyt b & D‐loop), microsatellite loci, and 2b‐RAD sequences provided insights on population structure, migration route, and demographic history of R. tanezumi and R. norvegicus in China.Herein, 486 house rat samples were randomly collected from 31 localities in China. Analyses based on mitochondrial sequences  from 31 localities Figure . Then, m2.22.2.1COI: 681 bp, D‐loop: 1048 bp, and cyt b: 1143 bp), extensively used in phylogenetic analyses of Rattus, were amplified separately with primers listed in Table Genomic DNA was extracted from muscle or tail tips using a DNeasy Tissue Kit (QIAGEN). Three mtDNA fragments , 10 mM of each dNTP, 75 mM MgCl2, 10 μM of each primer, 1.5 U EXTaq polymerase (Takara Biotech) and ~20–50 ng total genomic DNA. Amplifications were done as described  were separately edited, managed, and aligned with BioEdit 7.0.9.0 .All mtDNA sequences  were used as an outgroup, with downloaded sequences for R. norvegicus (KM114605) and R. tanezumi (KF011916) used as references. A Bayesian method was used to construct phylogenetic trees. MRBAYES 3.2.7a  were discarded as burn‐in values. The entire procedure was repeated twice, starting with different random trees. It was noteworthy that the same tree topologies were obtained. Mean sequence divergences among major lineages were calculated using MEGA X  for each species were estimated based on combined mtDNA sequences of COI, D‐loop & cyt b with Arlequin V 3.5.2.2  were selected based on previous information  for all loci at each site and for each population. GENEPOP 4.7.5  were estimated by maximum‐likelihood methods using ML‐Relate . Each run consisted of 1 × 106 Markov Chain‐Monte Carlo repetitions with a 100,000 repetitions burn‐in period. The ln Prob (data) for each run was recorded, and the ln Prob (data) across runs were averaged for each value of K. The ΔK metric , long homopolymer regions (˃10 bp), poor quality , or without restriction sites were removed. A total of 1,404,872 tags with BsaXI digestion site were selected from the reference genome RGSC 5.0 and used as reference sequences. Sequencing reads with high quality were mapped to reference sequences with SOAP2 , the expected heterozygosity (He), the observed heterozygosity (Ho), polymorphism information content (PIC), effective number of alleles (Ne), and the fixation index (Fst) of each SNP.The GENEPOP V4.7.5 Rousset,  was used2.4.2p‐distances model, with bootstrapping (1000). The software MEGA X  as P1 and P2, R. norvegicus (or R. tanezumi) as P3, and R. nitidus as the outgroup. All D‐statistics were computed using Dsuite  to distinguish gene flow from incomplete lineage sorting and to estimate gene flow in a four‐taxon framework  and Dxy (inter‐species nucleotide divergence) were estimated using VCFtools V 0.1.16  were done with 500‐kb sliding windows with 100‐kb stepping.Identical scores (ISs) were calculated as described . Loci without polymorphism were removed from analyses. The correlation coefficient (r2) between any two SNPs was calculated, and the average r2 with 300 kb windows was plotted against physical distance with R script.Linkage disequilibrium (LD) decay of two species was estimated using PopLDdecay . Based on the barcoding unrooted Neighbor‐joining network tree, all COI haplotypes from our samples were clustered into two groups and were nested with reference sequences of R. tanezumi or R. norvegicus .A total of 139 haplotypes were defined from 486 combined sequences of p Tables  and S4. R. tanezumi (KF011916) from GenBank and haplotypes T1–T36 (T = tanezumi) from 15 localities  clustered into one lineage. In this lineage, haplotype T17 from GZ formed a subclade; the other subclade comprised two branches, and haplotypes from the southeast coast of China  diverged firstly in each branch. The reference sequence of R. norvegicus (KM114605) from GenBank and haplotypes N1 ‐ N103 (N = norvegicus) from 24 localities  grouped into the other lineage with two sublineages. Some haplotypes from northeast China  clustered into one subclade; in the second subclade, some haplotypes from CD, GZ, JJ, KM, LZ, MY, and ZJ grouped into one branch, and the remaining haplotypes from various localities grouped into several sub‐branches in the other branch.In a phylogenetic tree constructed with combined sequences for 139 haplotypes, all haplotypes were apparently clustered into two monophyletic lineages  were shared by 12, three and four populations, respectively. For R. norvegicus, 14 haplotypes  were shared by two to four populations  was 0.9763 ± 0.0027 (R. norvegicus) and 0.8091 ± 0.0276 (R. tanezumi), nucleotide diversity (π) was 0.0089 ± 0.0041 (R. norvegicus) and 0.0052 ± 0.0023  were slightly smaller than those of He . The Fst values ranged from 0.133 to 0.247, indicating a high level of genetic divergence. The excess test for deviations of each locus from Hardy–Weinberg equilibrium (HWE) did not deviate from Hardy–Weinberg proportions for all samples (P ≈ 1).Microsatellite genotyping was successful in 462 individuals (average genotyping success for the 10 microsatellite loci was >95%). Alleles per locus ranged from 6.000 to 10.214 Table . The valn ≥ 5) ranged from 86.10% to 100%  at K = 2  = 22.21], and the 2 Figure a, which 2 Figure . In addiThe Q‐values for each individual and each population were estimated based on microsatellite data. In total, 43 individuals from 16 populations had a Q‐value < 0.9. Furthermore, the Q‐values of 8 populations  were <0.9 Table .3.33.3.16 reads with BsaXI digestion site . An average of 720,436 unique tags for each sample were obtained . The average level of sequencing depth was 26.76 for each sample , and the average mapping rate was 64.12% . Ultimately, 187,483 SNPs were defined in 123 samples. Genetic parameters of each SNP are shown  represented to R. tanezumi. In the R. norvegicus lineage, except for four individuals from DL, CD, GZ, and JO, other 45 individuals were grouped into two clusters; one cluster comprised individuals from DL and eight inland populations , whereas the other cluster included individuals from southeast coast populations  and seven inland population . In the R. tanezumi lineage, 74 individuals from Fujian province (JO), Guangdong province (GZ), Hunan province (CS), Hubei province (HS), and Sichang Province  were grouped into geographic‐specific branches, respectively. Individuals from LS (Tibet) had a close relationship with individuals from Sichang Province (MY).The NJ tree including 123 sequenced individuals featured two lineages Figure . CombiniR. norvegicus, at K = 2, one individuals from DL and 14 individuals from eight inland populations  were classified into one cluster (purple); one individuals from DL, four individuals from southeast coast populations (JO and GZ) and 22 individuals from five inland population  were classified into another cluster (gray); seven samples from four populations  showed genetic mixture of two clusters  were scattered in one plot, some individuals from DL, southeast coast populations  and seven inland populations  were scattered in the other plot  and PC2 (5.11% of variance), samples from different localities clustered into geographic‐specific plots, but seven individuals from CS differentiated greatly from other samples from CS along PC2 , all 123 samples were clearly differentiated in two distinct plots, corresponding to i Figure a,b. In ss Figure c–f. Baset Figure . The PCAt Figure a and thet Figure . For R. 2 Figure . The PCA5 Figure b and the5 Figure .p values were >.05 , and all 3.3.3R. norvegicus and R. tanezumi, both genetic diversity of the two species and genetic differentiation between them were calculated. Among 187,483 SNPs identified in 123 individuals with RGSC 5.0 as reference genome, 142,809 SNPs were specific for R. tanezumi, 19,553 SNPs were specific for R. norvegicus, and 25,121 SNPs were shared by two species. Identity score analyses clearly illustrated that the mean genomic similarity between R. norvegicus and the reference genome was 0.96190, whereas mean genomic similarity between R. tanezumi and reference genome was only 0.47539  and X chromosome  was 0.6447 in autosomes and 0.7892 in the X chromosome  and the X chromosome  and the X chromosome (mean: 0.2244), which did not support a standard population expansion event.Based on the 2b‐RAD sequences, the average Tajimas' D values of R. norvegicus) and 167,930 SNPs (R. tanezumi), respectively. The results presented confidently the population size changes within the last 100,000 years  is significantly greater than that of R. norvegicus (~2 × 105) in China.The demographic history of each species was inferred through Stairway Plot 2, using 44,674 SNPs (44.1R. norvegicus and R. tanezumi in China (this country's two dominant species of house rat) have not been thoroughly explored. In the present study of 486 Chinese rats, there were 306 individuals of R. norvegicus from 24 localities representing most of China except Tibet. The other 180 individuals belonging to R. tanezumi were distributed in 15 localities representing most areas of southern China and a part of northern China  formed a specific subclade. Furthermore, haplotypes from southeast coast  diverged firstly in both branches of the other subclade  provided additional evidence for population expansion and colonization  formed an ancestral subclade, other haplotypes from northeast China scattered in different branches of the other subclade  instead of geographic‐specific groups. The ADMIXTURE analyses also got similar clustering results  to the southeast coast  and subsequent on‐land expansion to central China  during colonization. It was proposed that the expansion of R. tanezumi in West Pacific was facilitated by shipping transportation of Austronesian speakers during their diaspora about 4000 years ago , shipping transportation along coast of China has emerged  defined only one mitochondrial haplotype (N4), and 23 individuals of R. tanezumi from Tibet plateau (LS) defined only two mitochondrial haplotypes  during the last half‐century.Both the Ancient Tea Horse Road (connecting southwest region and Tibet of China to Southeast Asia and South Asia) and the Land Silk Road  began in the Western Han Dynasty 202 BC), but the transportation using horses or camels did not promote the spread of house rats in China. There were no house rats in Xinjiang and Tibet plateau before the 1960s . Then, both species underwent rapid population recovery during the subsequent 5000 years, although neither Ne reached the initial level.Ancestral populations of a Allen, . Before a Allen, , apparenR. tanezumi began to undergo a second bottleneck event , Shanxi, Hebei, and Tianjin during recent decades.With the population expansion of e Figure . Both cue Figure  and preve Figure  indicateR. norvegicus  was greater than that of R. tanezumi  contained microsatellite sequences originated from both species  did not support gene flow between the two species ; writing – review and editing . Jing Meidong: Resources ; software ; writing – original draft ; writing – review and editing . Yingjie Chen: Resources ; software ; writing – original draft . Keying Yao: Software (supporting). Youming Wang: Software (supporting).This work was funded by grants from the National Natural Science Foundation of China (No. 31171189) and the Second Tibetan Plateau Scientific Expedition and Research Program (STEP) (2019QZKK05010303).The authors declare that they have no competing interests.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.Table S7Click here for additional data file.Table S8Click here for additional data file.Table S9Click here for additional data file.Table S10Click here for additional data file.Table S11Click here for additional data file.Table S12Click here for additional data file.


Galaxias species are interesting biogeographic models due to their distribution and different types of life cycles, with migratory and landlocked populations. To obtain a better understanding of the genetic consequences of the Quaternary glacial cycles in Galaxias maculatus, in this work we compared landlocked and migratory populations collected in areas that were differentially affected by ice advances and retreats. We included nine populations of G. maculatus, four collected from lakes (landlocked) and five from their associated estuaries/rivers (migratory) in three estuary-lake systems across southern Chile. Genetic analyses were performed using the mitochondrial control region and nine microsatellite loci. Genetic diversity measured with both markers was significantly higher in migratory than in landlocked populations across the study area. The levels of genetic differentiation showed higher differentiation among lakes than estuaries. Genetic diversity was higher in migratory populations located in areas that were less impacted by ice during Quaternary glacial processes. These results may be the consequence of recent recolonization of small freshwater bodies following the Last Glacial Maximum (LGM). Finally, the greatest differentiation was observed in populations that were exposed to continental ice advances and retreats during the LGM. Thus, in the present work we corroborate a pattern of differentiation between lakes and estuaries, using mtDNA sequences and microsatellite nuclear markers. This pattern may be due to a combination of biological factors, i.e., resident non-migratory behaviour or landlocking and natal homing-in, as well as geological factors, i.e., Expansion-Contraction Quaternary glacial biogeographic processes. Galaxias maculatus is one of the freshwater fish species with the broadest distribution in cold and temperate regions of the Southern Hemisphere, including populations in southern Australia, Tasmania, New Zealand, South America, and the Falkland/Malvinas Islands . In contrast, ice-impacted populations along the Pacific margin, between 43°S and 53°S, were all included in a single haplogroup, with lower levels of polymorphism and genetic structure  and associated estuaries (n = 5) in different biogeographical regions across the species distribution in southern Chile, in three areas that were differentially affected by Quaternary glacial processes  for DNA preservation.DNA was extracted from muscle tissue using 1) the salting-out method described by G. maculatus were edited using GENEIOUS v.9.0.4 (http://www.geneious.com) and aligned with MUSCLE (k), the number of segregating sites (S), haplotype diversity (H), the average and number of pairwise differences (∏) for each locality, each estuary-lake system, and for the whole D-loop data set using DnaSP (G. maculatus (ST) following nn), which measures how often nearest neighbours in sequences (in sequence space) are from the same locality in geographic space   . SAMOVA  HAPVIEW . We alsoG. maculatus, together with a sequence of G. platei as the outgroup. Bayesian reconstructions of haplotype relationships were done in MrBAYES v.3.1.2  and edited using the FigTree v.1.4.3 programme (http://tree.bio.ed.ac. uk/software/figtree).Finally, we performed phylogenetic reconstructions including the complete D-loop data set in  v.3.1.2  using th v.3.1.2 . BayesiaMicrosatellite fragment analysis was performed using Peak Scanner™ Software from Applied Biosystems and allele sizes were assigned to bins using FLEXIBIN . Finallyp values by the Markov chain method using GENALEX 6.501  with p-value significance, implemented in ARLEQUIN 3.5 . This type of analysis defines a model in which genetic variation is divided within and between groups, and which at the same time maximises the synthesis of the variables within groups and minimises it between groups using the information of the precedence of each sample as a priori information. Discriminant analysis (DA) achieves the best separation of the individuals within predefined groups and makes a probabilistic assignment of individuals to each group.Finally, we performed a discriminant analysis of principal components (DAPC) using ADEGENET 3.1-1  in the RG. maculatus included 266 individuals collected from nine populations, with an average of 28 individuals per locality in a range from 19 to 45 individuals per locality (G. maculatus were A—T rich (57.6%) compared to the G—C content (42.4%). Genetic diversity in G. maculatus was high with 197 variable positions (21.22%), of which 147 (74.61%) were parsimoniously informative. Levels of genetic diversity were much higher in migratory populations (estuaries) than in most of the landlocked populations (lakes), except for Llanquihue Lake, which exhibited high levels of polymorphism , were statistically significant, showing the high degree of genetic differentiation reported in the species (nn in G. maculatus (Snn = 0.815) showed a high and significant phylogeographic signal in the whole D-loop data set (p < 0.001). SAMOVA supported the general pattern of mtDNA structure and identified eight groups with maximum differences accounting for 66.09% of the total genetic variance, while only 0.06% of the variance was due to within-group variation among localities , three of which were found in more than one individual. Considering the expanded genealogy recorded in Maullín Estuary (Π = 30.33) and Llanquihue Lake (Π = 11.65), both localities exhibited multimodal distributions of pairwise differences between haplotypes. Interestingly, two individuals collected in Llanquihue Lake are closely related to the diversity recorded in Maullín Estuary. In contrast, the parsimony network for Pichilaguna Lake included a total of only 10 haplotypes. Two dominant haplotypes were recorded in this locality, the first was found in 11 specimens (37.93%) and the second in 9 (31.39%). Another eight low-frequency haplotypes were closely associated with the dominant ones  analysed here . Maximumant ones . The paiant ones  includedant ones . ConsideG. maculatus in southern South America  of the four main haplogroups previously recorded in  America . Haplogr America . Some sp America .p < 0.01) between the groups and when estuaries and lakes were compared within each E-LS (p < 0.01). In general, estuarine migratory populations showed greater allele richness than landlocked ones, except for Llanquihue Lake (E-LS2), which showed similar diversity to those recorded in migratory estuarine localities. The highest levels of allele richness were observed in migratory populations from Moncul and Lingue Estuaries  and 110 in lakes (landlocked populations). Deviation from the Hardy-Weinberg equilibrium was observed in different loci combined with different populations . Excess stuaries , and thestuaries  showed nST = 0.149. A posteriori pairwise analysis between locations based on the FST estimator . When landlocked populations (lakes) were compared with each other, a higher genetic differentiation was observed (FST = 0.228), with significant differences (p < 0.001). When the estuaries were compared with the lakes within each system, the lowest genetic differentiation was observed between the localities of E-LS 3.Comparing only migratory populations (estuaries) with each other, we observed a low although significant genetic differentiation with FBayesian analysis of genetic structuring in STRUCTURE showed two genetic groups differentiated from one another, represented by the colours distributed among the nine locations sampled  in whichThe results of the discriminant analysis of principal components (DAPC)  showed aGalaxias maculatus , where migratory populations from estuaries were grouped in a big central cloud; however, there are two clusters of higher similarity, one including Moncul, Lingue and Pascua and another one Tortel, all geographically distant from each another. These multivariate analyses showed that individuals sampled from landlocked populations could be separated from each other, and from migratory populations. Colico, Llanquihue and Pichilaguna Lakes showed the greatest similarity. Llanquihue and Pichilaguna Lakes are 3.5 km apart and are probably connected by streams. Llanquihue is the second-largest lake in South America, covering 860 km2; accordingly, it may represent a source for the G. maculatus population of Pichilaguna lake, which has an area of approximately 1 km2, although mtDNA data do not show shared genotypes. Comparative analyses using microsatellites in G. maculatus populations into two main differentiated genetic groups. However, when evaluating this analysis with a greater number of differentiated groups (K = 5), it is possible to detect a differentiation pattern with high separation between lakes while the similarity between estuaries is maintained, which is consistent with our observations in the other analyses. The low differentiation detected among lakes by this Bayesian analysis may result from the high genetic diversity detected by microsatellite markers, due to their high mutational rate, which may generate allele similarity by homoplasies  may be due to differential effects of Quaternary climate cycles in the different locations studied, since in some cases the population adopts a resident behaviour or life history, as observed in the variation to optional diadromous behaviour in a basin in Argentinian Patagonia . This co


Blumeria graminis f. sp. avenae populations. Diversity and virulence of B. graminis f. sp. avenae was assessed based on 80 single-spore isolates collected in different European countries such as Poland (40 isolates), Germany (10), Finland (10), Czech Republic (10) and Ireland (10) using ISSR (Inter-Simple Sequence Repeats) and SCoT (Start Codon Targeted) markers. This work demonstrated differences in virulence of B. graminis f. sp. avenae isolates sampled from different countries. Molecular analysis showed that both systems were useful for assessing genetic diversity, but ISSR markers were superior and generated more polymorphic products, as well as higher PIC and RP values. UPMGA and PCoA divided the isolates into groups corresponding with their geographical origin. In conclusion, the low level of genetic differentiation of the analysed isolates has suggested that the evolution of B. graminis f. sp. Avenae population is slow, and thus the evolutionary potential of the pathogen is low. This work paves the way for future studies on B. graminis f. sp. Avenae population structure and dynamics based on genetic variability.The structure and dynamics of changes in pathogen populations can be analysed by assessing the level of virulence and genetic diversity. The aim of the present study was to determine the diversity of  Avena sativa L.) is a species susceptible to many abiotic and biotic factors, among which fungal pathogens play a particular role. One of them is oat powdery mildew caused by Blumeria graminis f. sp. Avenae, an obligate biotrophic pathogen [Oat  and ISSR (Inter-Simple Sequence Repeats) systems, and SRAP (Sequence-Related Amplified Polymorphism) and SNP (Single Nucleotide Polymorphism) markers [Molecular markers play one of the key roles in understanding the virulence frequency of athogens ,12,13,14 markers ,15,16,17B. graminis f. sp. avenae. Hence, the objective of the present work was to determine the diversity of B. graminis f. sp. avenae populations from different regions of Europe. The diversity of B. graminis f. sp. avenae was assessed using ISSR and SCoT markers and based on the virulence of the pathogen collected in different European countriesStudies on the virulence of oat powdery mildew have already been reported several times in the literature ,19,20. HB. graminis f. sp. avenae isolates collected in Poland, Germany, Czech Republic, Finland and Ireland showed varying levels of virulence compared to the control forms with described resistance genes and effective sources of resistance  and Nei’s standardized coefficient of gene differentiation (Nei’s Gst) showed that the populations from Ireland and Germany were the most distant from each other. The smallest genetic distance was observed between the populations from Poland and Germany. The gene flow (Nm) value for these populations was the highest and amounted to 7.83, indicating a high migration of alleles between these regions. The lowest gene flow was observed between the populations from Ireland and Germany (1.42), which could be related to the geographic barrier between these areas .Of the 30 examined ISSR primers, 12 detected polymorphisms between individuals. A total of 175 clearly distinguishable amplification products were found. Of these, 131 were polymorphic, with an average of 10.91 bands for each primer. The percentage of polymorphic bands for all primers was higher than 50%. The resolving power of the primers ranged from 15.46 to 4.1; the PIC scores ranged from 0.37 to 0.22 .A total of 236 amplification fragments were obtained using 14 of the 30 tested SCoT primers. Of these, 122 were polymorphic, with an average of 8.71 bands for each primer. The percentage of polymorphic bands for all primers was higher than 50%. The resolving power of the primers ranged from 13.45 to 4.67; the PIC score ranged from 0.36 to 0.13 .B. graminis f. sp. avenae isolates from five different countries was determined using ISSR and SCoT markers. The analysed isolates were considered as five distinct populations according to their geographic origin. Both marker systems indicated that the population from Poland was the most diverse. The highest number of polymorphic bands and population-specific amplification products was identified in this population. Both ISSR and SCoT analyses showed that the population from Finland was the least diverse. The lowest level of band polymorphism was observed for this population and the lowest level of diversity was confirmed by the Shannon index and expected heterozygosity  of ISSR and SCoT polymorphisms revealed that the genetic divergence was mainly derived from within the population .B. graminis f. sp. avenae populations. The analysis based on ISSR markers showed that the populations form Poland and Germany were characterised by the lowest genetic distance, as well as the highest level of gene flow. The highest genetic distance was determined for the populations form Ireland and Finland. The analysis of genetic distance based on SCoT markers also indicated that the Polish and German populations were the closest to each other, while the highest distance was calculated between populations from Ireland and Germany  and a combined dendrogram based on both marker systems . It can be speculated that the repetitive regions in the B. graminis f. sp. avenae genome are mainly composed of GA and CA motifs. SCoT markers were the second system selected to analyse the genetic variability in the B. graminis f. sp. avenae population. Start codon targeted (SCoT) polymorphism is a simple and novel DNA marker technique described by Collard and Mackill [The second approach in pathogen population analysis is the assessment of genetic diversity based on molecular markers ,28,29. T or SRAP ,30,31,32equences ,34; therequences , which aequences ,37,38. T Mackill . SCoT ma Mackill ,41.B. graminis f. sp. avenae populations. However, ISSR markers amplified more polymorphic products, and were characterised by higher PIC and RP values than SCoT markers. These results indicate that ISSR markers are a better tool for studying the genetic diversity of B. graminis populations which is consistent with Liu et al. [B. graminis f. sp. avenae isolates; therefore, SCoT markers were chosen for further analyses (data not shown).Both marker systems were useful for analysing the genetic structure of u et al. . The latu et al. ,43,44. SB. graminis f. sp. avenae isolates were divided into five distinct populations depending on their place of origin. Both marker systems indicated that the population from Poland was the most diverse. However, this could be due to the large number of isolates representing this population. We observed a high variation between the Polish isolates, which was likely due to the different geographic regions from which they originated. Among the remaining populations, isolates from Germany were the most diverse. The population from Finland, for which the lowest diversity values were identified based on both ISSR and SCoT markers, was the most homogeneous.The analysed B. graminis f. sp. avenae isolates into groups according to their geographic origin. Clustering correlated with geographic origin of B. graminis f. sp. tritici isolates was also observed by Liu et al. [B. graminis f. sp. tritici population and showed that polymorphisms detected using EST-SSR markers were correlated with the pathogen virulence in 58% of the analysed isolates. However, this correlation was not observed for the remaining isolates. The authors suggested that the level of pathogen virulence depended on many factors and that the gene sequence alone could not determine pathogenicity. The complex interaction between the plant and pathogen and the environmental impact may result in the weak correlation of polymorphisms generated by molecular markers with pathogen virulence.PCoA analysis based on both ISSR and SCoT markers divided u et al. , who utiu et al. , SNP [16u et al.  used ESTB. Graminis f. sp. Avenae isolates tested was largely based on the intra-population variation. This was also indicated by the low level of gene flow between the analysed populations. Similar results were obtained for B. graminis f. sp. Tritici [B. graminis f. sp. Hordei [Grouping associated with geographic regions indicated the genetic distinctiveness of the analysed populations. This could be observed for populations separated by geographic distance and natural barriers (Ireland and Finland). The latter probably also influenced the highest similarity found between populations in Poland and Germany and not in the Czech Republic. The genetic distinctiveness of the analysed populations was also confirmed by AMOVA. This analysis showed that the variability identified among the  Tritici  and B. g. Hordei  populatiPowdery mildew-symptomatic oat leaves were collected in 2020 from five European countries, including Poland, the Czech Republic, Germany, Ireland and Finland. The sampling sites differed from one another in terms of climate and geography. These places reflected the different conditions of Europe in which the powdery mildew of oats is present. The isolates were prepared under laboratory conditions according to the previous methodology described by Hsam et al. ,46. In tB. graminis f. sp. avenae spores per 1 cm2. Then, the dishes were incubated in a growing chamber at about 17 °C and illuminance of approximately 4 kLx.To analyse the virulence of the pathogen populations, host–pathogen tests were carried out using eleven oat genotypes, with known powdery mildew resistance genes and two genotypes with effective sources of resistance identified in our previous study ,48. The Reaction type on each differential was determined 10 days after inoculation and scored according to a 0–4 modified scale : where 0B. graminis f. sp. avenae isolates were calculated on the basis of isolate virulence patterns on the set of differential genotypes. Virulence frequency (p) as p = x/n  was calculated for each isolate.Parameters for comparing all m) regarding the simple mismatch coefficient m, and the Nei gene distance (N) based on the population virulence; and genetic diversity (KWm) and distance (KBm) measured by the Kosman indices, based on the population pathotype and virulence structure [Diversity within the populations was assessed using different types of parameters: genetic diversity was measured by Simpson (Si) and Shannon (Sh) based on the pathotype structure of the populations; gene diversity was measured by the Nei index (Hs) which is equivalent to a measurement of the average dissimilarity within a population  was performed using 30 primers . A 10 µL2, 60 ng of template DNA and 0.5 U Taq DNA Polymerase (Thermo Scientific), in a final reaction mixture of 10 μL. Amplification was carried out in a Biometra T1 thermal cycler programmed for 3 min at 94 °C of initial denaturation, 35 cycles: 94 °C—1 min; 50 °C—1 min; and 72 °C—2 min, with a final extension at 72 °C for 5 min.The analysis of genetic similarity was based on SCoT marker systems . A totalTM 100 bp DNA Ladder Plus was used to establish the molecular weights of the products.Amplification products were separated by electrophoresis on 1.5% agarose gels containing 0.1% EtBr . Fragments were visualised under a UV transilluminator and photographed using the PolyDoc System. GeneRulera) were established. An effective number of alleles at the locus (Ne) [e) [PCR-amplified ISSR and SCoT products were scored as present (1) or absent (0) from the photographs. Only clear and reproducible products were scored. The level of polymorphism of the primer  and relative frequency of polymorphic products   were calcus (Ne)  and expe(Ne) [e)  were est(Ne) [e) , the lev(Ne) [e)  and a PC(Ne) [e) . Gene fl(Ne) [e) .Dendrogram representing genetic diversity was constructed by performing unweighted pair-group analysis with arithmetic averages (UPGMA) using PAST 3.16 .B. graminis f. sp. avenae population. Our research has revealed that the level of resistance to powdery mildew in oat differs depending on the region. Therefore, combining resistance genes into pyramids in oat breeding can lead to long-term resistance under different environmental conditions. Furthermore, virulence analysis showed that the B. graminis f. sp. avenae population from Poland had a similar level of virulence as the populations studied in previous years. This suggests that the pathogen population is changing slowly.Two approaches were employed in this work to analyse the diversity of the pathogen population. To our knowledge, this is the first study that has been conducted on the B. graminis f. sp. avenae isolates according to their geographic origin. Consequently, it can be concluded that the level of genetic differentiation of the pathogen does not depend on the level of virulence, but on its origin. The low level of genetic differentiation of the analysed isolates suggests that the evolution of the B. graminis f. sp. avenae population is slow, and thus the evolutionary potential of the pathogen is low, which may result from the lack of environmental pressure on the pathogen. Oat cultivars are characterised by a low level of resistance to powdery mildew [Polymorphisms of ISSRs and SCoT markers grouped y mildew ,66; ther


Paramuricea previously identified as P. clavata. We used a diverse set of molecular markers  at 49 locations. Clear segregation of Atlantic and Mediterranean lineages was found with all markers. Species‐tree estimations based on SCO strongly supported these two clades as distinct, recently diverged sister species with incomplete lineage sorting, P. cf. grayi and P. clavata, respectively. Furthermore, a second putative (or ongoing) speciation event was detected in the Atlantic between two P. cf. grayi color morphotypes (yellow and purple) using SCO and supported by microsatellites. While segregating P. cf. grayi lineages showed considerable geographic structure, dominating circalittoral communities in southern (yellow) and western (purple) Portugal, their occurrence in sympatry at some localities suggests a degree of reproductive isolation. Overall, our results show that previous molecular and morphological studies have underestimated species diversity in Paramuricea occurring in the Iberian Peninsula, which has important implications for conservation planning. Finally, our findings validate the usefulness of phylotranscriptomics for resolving evolutionary relationships in octocorals.The accurate delimitation of species boundaries in nonbilaterian marine taxa is notoriously difficult, with consequences for many studies in ecology and evolution. Anthozoans are a diverse group of key structural organisms worldwide, but the lack of reliable morphological characters and informative genetic markers hampers our ability to understand species diversification. We investigated population differentiation and species limits in Atlantic (Iberian Peninsula) and Mediterranean lineages of the octocoral genus  Paramuricea across the Atlantic‐Mediterranean biogeographic regions. We combine species tree methods (using RNA‐seq data) with population genetics (clustering methods using microsatellites) and phylogenetics  to show that the Atlantic and Mediterranean clades studied are recently diverged sister species on separate evolutionary trajectories (Paramuricea cf. grayi and P. clavata), also revealing a second putative (or ongoing) speciation event within Atlantic P. cf. grayi.Demarcating the transition between intraspecies lineages and speciation requires accurate delimitation of species boundaries, which in many marine invertebrates such as corals is notoriously difficult, leading to misinterpretations of their ecology and evolution. This study investigates species boundaries, taxonomic affinity, and lineage diversification for key structural species of branching octocorals in the genus  Despite these findings, the existence and exact location of the Atlantic‐Mediterranean break, and the level of divergence across these regions  remains unknown in P. clavata due to incomplete geographic sampling and low marker resolution . Alternatively, the Atlantic populations of P. clavata reported from Portugal  along the Atlantic‐Mediterranean transition to better understand the evolutionary forces governing population differentiation and speciation in this genus across these important biogeographic regions. The delimitation of spatially significant and unique genetic units within a species is also necessary for emerging conservation policies aiming to preserve intraspecific genetic diversity and the associated adaptive potential. Paramuricea clavata needs such efforts given its key structuring role in ecosystem functioning across the Mediterranean  and a large part of the distribution of P. clavata in the Mediterranean Sea, including the Adriatic and Aegean seas. We combined evidence from three types of molecular markers: (1) single‐copy orthologs (obtained from RNA‐seq data) for species delimitation and screening for cryptic species diversity; (2) microsatellite loci to analyze spatial genetic diversity and structure both across and within the Atlantic and Mediterranean regions considering alternatively lineage and population levels; and (3) the mitochondrial gene mt‐mutS (assembled from RNA‐seq read data) to extend taxonomic coverage of our phylogenetic analysis for the genus. We present evidence showing that previous assignments of Atlantic populations to P. clavata are incongruent with genomic data, and the taxa present along those coasts should instead be regarded as the morphospecies P. cf. grayi.The aim of this study was to test genetic boundaries and species affinities within the genus 22.12.1.1P. clavata; and Baleeira  for P. cf. grayi  and western  Portugal. This included two specimens of the yellow color morph (1 from Tavira and 1 from P39) and two of the purple color morph from P39  or the NZYol reagent (Nzytech) for tissue homogenization with the RNeasy Mini Kit (Qiagen) for RNA purification following the manufacturer's instructions. Residual DNA was removed using TurboDNAse (Invitrogen) or DNAse I (Qiagen). The quality, purity, and yield of the RNA was assessed using gel electrophoresis and a Qubit® Fluorometer and/or Nanodrop spectrophotometer. RNA‐seq library preparation and sequencing was outsourced to BGI Tech Solutions in Hong Kong (100 bp paired‐end DNBseq). A total of 34 individuals were processed for RNA‐seq and the sequences deposited in NCBI's Sequence Read Archive .2.3https://github.com/greatfireball/filter_fq). The quality of filtered sequence data was then evaluated with two complementary quality control tools, FastQC v0.11.6 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FastqPuri . The input bloom filter was constructed from nuclear rRNA sequences downloaded from NCBI GenBank for the top octocoral blastn hit of the contaminants, which corresponded to the octocoral Dendronephthya gigantea.Raw reads were preprocessed by BGI to trim adaptor sequences and low‐quality reads (<Q20) using filter_fq (2.4P. cf. grayi) and three for samples from Balun, Altare and La Vaca (P. clavata). The assemblies were subjected to multiple steps of quality control and curation. First, the quality of each assembly was evaluated with TransRate v1.0.1 using both sequence‐based and read mapping (onto the assembled contigs) metrics  that are expected to be present across higher taxonomic groups  and from a reciprocal BLASTX search between the transcriptome assembly of P. cf. grayi and that of the octocoral Eunicella verrucosa . For the reciprocal blast, we retained contigs with up to five query hits, subsequently selecting only those with a single sequence hit in both results tables (and thus putative single‐copy orthologues). Transcript .fasta sequences for orthologues were extracted for each sample from sorted .bam files after phasing (samtools phase) and variant calling (bcftools mpileup). Allele sequences were obtained from the resulting .vcf files with vcfutils.pl and seqkit fq2fa, aligned with mafft and trimmed with Gblocks in TranslatorX using a custom python script (https://github.com/cymon). The final set of loci used contained no gaps or missing data. The input .bam files were generated by mapping reads onto the reference transcriptome using the RSEM (v1.2.31) wrapper script and Bowtie2  identified from BUSCO  for a subset of the samples .Finally, to extend the taxonomic coverage of the analysis to other 2.62.6.1P. cf. grayi (heretofore Paramuricea aff. clavata) along the southern coast of Portugal was known from deep divers' records, from shallow hard bottoms down to 90 m depth  were genotyped at six microsatellite loci: Parcla_09, Parcla_10, Parcla_12, Parcla_14, Parcla_17  were extracted using a salting out procedure  within the Atlantic (see Results). We compared the observed heterozygosity (Ho), gene diversity  and private allelic richness (Apg). The Ho, He and FIS were computed for each lineage with GENETIX 4.05  from the individual genotypes without assumptions concerning population boundaries. Because of the unbalanced sampling ). CLUMPP 1.1  was analyzed with an analysis of molecular variance  for the two measures.A discriminant analysis of principal components .The evolutionary relationships among populations were characterized using the chord distance  and the Italian population of P. clavata (Altare) were superior to those obtained for the other two P. clavata populations , with an N50 of 1452 bp , 38 and 105 Gb for ly Table . Quality3.1.2P. cf. grayi and the Mediterranean P. clavata samples with high  or full (Mediterranean) support  formed a monophyletic group to the exclusion of the purple color morph  Despite the high bootstrap support for the Atlantic and Mediterranean clades, concordance analysis of the branches supporting these clades revealed very low gCFs of only 1.15% (Atlantic) and 2.3% (Mediterranean). Concordance factors for sites (sCF) were also low to moderate (45.3% and 58.3%), which together with the short branch lengths supporting these clades, suggests recent divergence with incomplete lineage sorting (ILS). We note, however, that the branch supporting the yellow morphotype clade within the Atlantic samples was somewhat longer than those supporting the Atlantic and Mediterranean clades, had full bootstrap support, and a fairly robust sCF  and another including the samples of P. cf. grayi sequenced here together with P. grayi from the northern Iberian Peninsula . These results were congruent with the PoMo ML species tree, which supported the Mediterranean (P. clavata) and Atlantic (yellow and purple morphotypes of P. cf. grayi) populations as three distinct entities with full bootstrap support  from Madeira, Portugal  at our sampling sites in Portugal.Species delimitation based on the ASTRAL‐III species tree and performed with the SODA software supported the clades formed by samples of s Figure . The samJohnson, , but whiJohnson, . This spJohnson, , as well3.23.2.1Ho) and expected (He) heterozygosity ranged between 0.44 (TAV) and 0.83  and between 0.5 (BAL) and 0.8 , respectively. Multilocus FIS were between 0  and 0.3 . Significant heterozygote deficiencies, after FDR correction, were found in 15 of 46 populations (28%), located both in the Atlantic  and in the Mediterranean . Regarding rarefied allelic (Ar(18)) and private allelic richness (Ap(18)), we found highly contrasting values among populations. Ar(18) ranged between 3.5 (BAL) and 7.92 , while Ap(18) was null for 14 populations  with a maximum of 1.52 for TAV (mean ± SD = 0.17 ± 0.3). Seven of the highest Ap(18) values were observed for Atlantic populations , Ar(18)) with the exception of FIS. Pairwise Wilcoxon rank tests showed that significant differences were mostly driven by variation between Mediterranean and the Atlantic populations, which included the two segregating lineages identified  parameter increased until K = 4, plateaued until K = 9 and decreased for higher K values  were separated from those from the Mediterranean , and extending to southern Spain (CPS) and the Balearic Islands (CAB). The three genetic clusters (K = 3) were concordant with the areas corresponding to western  and southern  Portugal and the Mediterranean , while the second axis divided the two lineages of P. cf. grayi  supported the distinction between the Atlantic and the Mediterranean populations with a bootstrap value of 85 . Pairwise GST among lineages were 0.24, 0.12, and 0.11 for P. clavata vs. yellow lineage of P. cf. grayi, yellow vs. purple P. cf. grayi lineages and P. clavata vs. purple lineage of P. cf. grayi, respectively. Overall, pairwise GST ranged between 0.01 (PPL vs. MED) and 0.46 (AYV and BAL), with only 31 nonsignificant comparisons  and 0.99 (AYV and SEG). All except 33 pairwise comparisons (<5%) were significant. Focusing on the Atlantic populations, the values of the pairwise GST and DEST were within the range of the values observed among Mediterranean populations . For the two estimators, all but one pairwise comparison (SEG vs. BAL) were significant.The AMOVA revealed highly significant genetic structuring between the three groups (i.e. lineages), among populations, and among individuals  and in the Atlantic (P. cf. grayi), where two color morphotypes (yellow and purple) remain genetically distinct in sympatry, indicating also speciation within P. cf. grayi. These discoveries result from integrating genetic data from three independent types of markers  to assess the extent of divergence between Mediterranean populations of the gorgonian P. clavata and adjacent Atlantic populations occurring along southern and western Portugal, suggested to represent the western limit of the species' distribution range , a species previously described for this region  speciation event between purple and yellow lineages. Although these lineages display considerable geographic structure, with the yellow morphotype largely dominant in the Algarve  and the purple dominating habitats on the west coast of Portugal, they can also be found in sympatry .This study discovered novel species diversity in the gorgonian genus 4.1P. cf. grayi (heretofore P. clavata) occurring along Portugal and Mediterranean populations of P. clavata identified high levels of population differentiation between the two regions, indicating highly restricted gene flow across the Atlantic‐Mediterranean transition ), in particular, was substantially higher in P. cf. grayi, which included seven of the highest Ap(18) values observed across all populations studied. This pattern is more consistent with the long‐term persistence of distinct lineages, rather than Atlantic populations being derived as marginal extensions of a Mediterranean expansion. Indeed, all phylogenetic methods, as well as a distance‐based approach, indicate that Mediterranean P. clavata is more genetically divergent from the adjacent Atlantic populations of P. cf. grayi (yellow lineage) than from the more geographically distant populations in western Portugal (purple lineage).Previous genetic studies of Atlantic s Figure , which ss Figure  and 3, amt‐mutS barcode showed that the samples of the Atlantic lineage(s) identified here have the same haplotype as the morphospecies P. grayi from northern Iberia , our results confirm the identity of all southern and western Iberian populations studied here as P. cf. grayi. This finding agrees with previous morphological identifications documenting the presence of P. grayi in southern Portugal, particularly in circalittoral rocky habitats along the Algarve and in the Gorringe Bank , with Bayesian dispersal‐extirpation‐cladogenesis modeling suggesting that divergence likely occurred in sympatry . Our STRUCTURE analysis based on microsatellite loci identified a signal of admixture between P. cf. grayi and P. clavata at sites in the Alboran sea . Here and in two additional sites east of the AOF (CPS and CAB), the coefficients of membership to either of the Atlantic/Mediterranean clusters were generally lower  into the Mediterranean  gene pool, and involves alleles found in the purple lineage of P. cf. grayi  and genetic barriers  and sites (sCFs) supporting the Atlantic and Mediterranean clades provide further evidence for a recent divergence between i Figure . Gene‐tri Figure . Introgri Figure , includii Figure . Disentai Figure . However4.2P. cf. grayi. The microsatellite analysis revealed that populations in southern Portugal  were highly differentiated from those sampled along the western coast , with highly significant pairwise GSTs and DESTs and genetic partitioning  sampling, as well as experimental approaches  to clarify the taxonomic status of the two color morphs.Our results clearly show lineage divergence within Iberian ng Table . This song Table . ImportaP. cf. grayi, we have identified significant pairwise genetic differentiation between most populations studied, including at local spatial scales, from ~20 km  to less than 400 m , this study emphasizes the need for genome‐wide multi‐locus datasets to resolve recent lineage splitting events in the face of, for example, ILS and/or introgression. The marked spatial genetic structure and differentiation observed between the P. cf. grayi/P. clavata clades reveal historical footprints of divergence across the Atlantic‐Mediterranean transition, the corresponding evolutionary scenarios of which could be tested with further population genomic data. Like recent studies, our results emphasize the urgent need to re‐evaluate species diversity and the processes underlying diversification within the genus Paramuricea ; data curation ; formal analysis ; investigation ; methodology ; resources ; validation ; visualization ; writing – original draft (lead); writing – review and editing . Gareth A. Pearson: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; validation ; visualization ; writing – original draft ; writing – review and editing . Joana R. H. Boavida: Conceptualization ; data curation (supporting); formal analysis (supporting); funding acquisition ; investigation ; methodology (supporting); resources ; validation (supporting); writing – review and editing . Diogo Paulo: Resources (supporting); writing – review and editing (supporting). Didier Aurelle: Formal analysis (supporting); resources (supporting); writing – review and editing . Sophie Arnaud‐Haond: Writing – review and editing . Daniel Gómez‐Gras: Resources (supporting); writing – review and editing . Nathaniel Bensoussan: Resources (supporting); writing – review and editing (supporting). Paula López‐Sendino: Investigation ; resources (supporting); writing – review and editing . Carlo Cerrano: Resources (supporting); writing – review and editing . Silvija Kipson: Resources (supporting); writing – review and editing . Tatjana Bakran‐Petricioli: Resources (supporting); writing – review and editing . Eliana Ferretti: Resources (supporting); writing – review and editing (supporting). Cristina Linares: Resources (supporting); writing – review and editing . Joaquim Garrabou: Funding acquisition ; resources ; writing – review and editing . Ester A. Serrao: Conceptualization ; funding acquisition ; writing – review and editing . Jean‐Baptiste Ledoux: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; resources ; validation ; visualization ; writing – original draft ; writing – review and editing .Figure S1‐S2.Click here for additional data file.Appendix S1.Click here for additional data file.Table S1.Table S2.Table S3.Table S4.Table S5.Table S6.Click here for additional data file.