Aphrastura (rayaditos) inhabiting the Diego Ramírez Archipelago, the southernmost point of the American continent. This archipelago is geographically isolated and lacks terrestrial mammalian predators as well as woody plants, providing a contrasted habitat to the forests inhabited by the other two Aphrastura spp. Individuals of Diego Ramírez differ morphologically from Aphrastura spinicauda, the taxonomic group they were originally attributed to, by their larger beaks, longer tarsi, shorter tails, and larger body mass. These birds move at shorter distances from ground level, and instead of nesting in cavities in trees, they breed in cavities in the ground, reflecting different life-histories. Both taxa are genetically differentiated based on mitochondrial and autosomal markers, with no evidence of current gene flow. Although further research is required to define how far divergence has proceeded along the speciation continuum, we propose A. subantarctica as a new taxonomic unit, given its unique morphological, genetic, and behavioral attributes in a non-forested habitat. The discovery of this endemic passerine highlights the need to monitor and conserve this still-pristine archipelago devoid of exotic species, which is now protected by the recently created Diego Ramírez Islands-Drake Passage Marine Park.We describe a new taxon of terrestrial bird of the genus  Aphrastura (Passeriformes: Furnariidae) is endemic to southwestern South America and includes two allopatric species: the Thorn-tailed Rayadito (A. spinicauda) that inhabits the temperate forest biome of South America1, and the Masafuera Rayadito (A. masafuerae) whose distribution is limited to the misty tree fern forests of the oceanic Alejandro Selkirk Island3. This island has an area of only 85 km2 and is part of the Juan Fernández Archipelago, separated from the continent by 670 km. Unlike A. masafuerae, A. spinicauda has a broad distribution along the entire latitudinal range of the South American temperate forests’ biome4. It inhabits deciduous and evergreen forest types ranging from north-central Chile to the extreme south of Chile and Argentina7. Its northernmost population is found in the evergreen relict forest of Fray Jorge National Park (30° 30′ S), whilst its southerly population reaches the world’s southernmost forests in the Cape Horn Biosphere Reserve (CHBR) (56° S)7. However, a putative population of A. spinicauda also occurs on the Diego Ramírez Archipelago (hereafter Diego Ramírez) (56° 32′ S)10 . The geographic isolation and entirely herbaceous vegetation of Diego Ramírez raises the question whether its Aphrastura population constitutes a different taxonomic unit. This possibility is supported by the fact that two other populations of A. spinicauda that occur on islands have already been recognized as subspecies, based on their morphological distinctiveness, in terms of color and size, namely A. s. fulva from Chiloé Island (42° S) and the Chonos Archipelago (44° S), and A. s. bullocki from Mocha Island (38° S)13 (Supplementary Information Appendix 1). However, in contrast to the population inhabiting Diego Ramírez, both subspecies are geographically closer to the continental population and inhabit similar forest habitats.The Diego Ramírez is the southernmost point of the South American continent. While emerging only ca. 100 km southwest of Cape Horn on the margin of the continental shelf, it is separated from it by one of the world’s roughest seas in the northern section of the Drake Passage, with harsh climatic conditions and difficult access from the continentAphrastura (A. spinicauda and A. masafuerae) are restricted to forest ecosystems, including forest edges and surrounding woody vegetation17. The association with such habitats is striking in the northern range of A. spinicauda, where it is confined to forest patches and birds rarely venture into the arid shrubland19. Individuals usually forage for adult insects and larvae in the bark of trunks and branches, but they also glean them from the canopy and understory vegetation20. A. spinicauda typically occurs in Nothofagus-dominated forests, where they are one of the most abundant bird species, nesting in narrow cavities of old-growth trees that provide protection from rain, strong wind, and predators7. Given their strong association to forests, it is noticeable to find a population of Aphrastura on Diego Ramírez, whose vegetation is completely devoid of trees and other woody plants23.The two currently recognized species of Aphrastura is absent10, but whether this led to phenotypic divergence remains unknown. Here, we present an integrative taxonomic study combining morphological, ecological, and genetic data to clarify the taxonomic status of the Diego Ramírez population. We propose that this population is sufficiently distinct from continental populations to warrant taxonomic recognition, as the endemic Aphrastura subantarctica.Gene flow between the Diego Ramírez and the continental populations of 22. The average annual rainfall is 1500 mm, and mean annual temperature is 5.6 °C, with August being the coldest month (3.3 °C on average) and March the warmest month (7.5 °C)24. The vegetation lacks trees and shrubs, and is dominated by tall (> 1 m) tussocks (Poa flabellata), a herbaceous plant that does not offer cavities for nesting22 , and is also an important nesting site for the Black-browed  and Grey-headed albatross (T. chrysostoma) , which nest in openings of the tussock grassland.The Diego Ramírez Archipelago (56° S) is subject to an oceanic climate, characterized by an even rainfall throughout the year, low annual temperature range, and strong western winds27. These sites represent the southern limit of continental Aphrastura populations and are the closest forested habitat to Diego Ramírez. These areas are dominated by evergreen forests of Magellan's beech (Nothofagus betuloides) and Winter's bark (Drimys winteri)28. The influence of the ocean moderates thermal fluctuations, allowing the growth of a lush broadleaf evergreen forest. At the eastern end of the Beagle Channel, Navarino Island has an oceanic climate with a reduced annual rainfall of 460 mm, and snowfall mostly from fall to spring28. On this island, the high deciduous beech (N. pumilio) co-dominates with Magellan's beech, generating mixed forest of evergreen and deciduous trees. Nothofagus trees reach heights of 20 m and their straight trunks often have natural cavities that are used by the Aphrastura population for nesting7. Samples for the morphological analysis included individuals captured on the sites above mentioned .To quantify morphological differences, we surveyed several sites on the north-western arm of the Beagle Channel on Tierra del Fuego Island and on Navarino Island Fig. 26,27. Thned Fig. . For theAphrastura from Diego Ramírez and two areas of the CHBR, i.e., the northwest arm of the Beagle Channel and Navarino Island  the Omora Park  collected from December 2015 to January 2016, and on April 1, 2018 (to compare with the 2018 Diego Ramírez expedition), and at (6) Guerrico  in March 2015 and 2016. To avoid seasonal morphological variability, we restricted the morphological analysis to data obtained during the austral summer (November to April).We obtained measurements of and Fig. . On Dieg7. We measured wing length, tarsus length, tail length, beak length and beak width using a caliper (0.01 mm precision), and we determined body mass using a digital scale (0.1 g precision). The same person (OB) performed all the measurements. Blood samples were obtained by puncturing the brachial vein and stored on FTA® cards for subsequent genetic analyses.In all sites, birds were captured using 2–6 mist nets of mesh size 30 mm and 2.4 × 12 m long each. We operated the nets for 6-h periods after sunrise. Given the difficulties to operate the nets on Diego Ramírez (strong winds and dense herbaceous vegetation), we cleared a transect in the tussock to set the mist nets  recorded all individuals along two transects (1.02 km length each) starting from the shoreline to the highest ridge of Gonzalo Island . These transects included locations with different tussock density (50–100% coverage) and plant heights (0.4–2.0 m), as well as other bare sites with rocky or muddy substratum. These habitats are used as nesting sites by black-browed and grey-headed albatrosses. The transects also covered areas used by other surface-nesting seabirds, like the rockhopper (Eudyptes chrysocome), and macaroni penguin (E. chrysolophus), as well as burrowing seabird species, such as the Magellanic penguin (Spheniscus magellanicus), blue petrel  and the common diving petrel (Pelecanoides urinatrix). Nesting colonies of these sea birds modify the habitat in a way that may be relevant for the Diego Ramírez Aphrastura population. Based on the transects, we calculated the relative number of Aphrastura (birds/km) on each island. Based on all observations, we qualitatively described the habitat type used by Aphrastura  and height (m), their nesting sites, and their behavior ). In addition, one observer (OB) carried out opportunistic observations during mist-netting, observing birds when walking in different directions to cover an area of approximately 0.16 km2 .To compare the well-known habitat used by other 29.To avoid age-related morphological differences, we only used measurements from adult individuals for this analysis. We compared trait values across the sites using a Kruskal–Wallis rank sum test and Conover's non-parametric all-pairs comparison test for a posteriori analysis (α = 0.05). To examine overall morphological differences among populations, we performed a Principal Components Analysis (PCA) including z scores for all measured variables. All analyses were performed using RA. spinicauda, from Fray Jorge National Park in north-central Chile to Navarino and Horn islands, and including nine individuals from Diego Ramírez  and nucleotide diversity (π per nucleotide site). For comparison of the four geographic groups . We tested for phylogeographic structure by comparing GST and NST coefficients using permutation tests implemented in PERMUT35. To infer the spatial genetic structure of Aphrastura populations, we used an analysis of molecular variance (AMOVA)36, defining groups of populations that are geographically homogeneous and maximally differentiated from each other.We used Arlequin 3.5.1.210, using 12 autosomal polymorphic loci37. The marker panel consisted of seven species-specific markers and five cross-species amplifying markers (see38). None of the loci showed evidence for deviations from Hardy–Weinberg equilibrium  and all had null allele frequencies < 0.05. We calculated measures of genetic diversity per population using the hierfstat package39 in R. To summarize overall genetic variability among individuals and to preliminarily assess genetic structure, we used a PCA as implemented in the R package adegenet40. To estimate the optimal (most likely) number of genetic clusters that were present in our sample, we used the ‘snapclust’ method in adegenet. This method relies on maximizing the likelihood of a number of panmictic populations by combining model-based and geometric approaches41. The best-supported number of groups was determined using the Akaike Information Criterion (AIC).We performed further genetic analyses based on nuclear microsatellite markers to check the conclusions from the mitochondrial phylogeographic approach. We analyzed an additional sub-sample of 153 individuals from Central Chile, Southern Chile and Argentina, and Diego Ramírez Fig. . These i42 to calculate pairwise G-statistics for the genetic clusters identified, implementing both the Nei’s standardized index (G’ST(Nei)) and the Hedrick’s standardized index corrected for small samples (G″ST). Second, we calculated the posterior membership probabilities of all sampled individuals for the newly identified genetic clusters, using the probability of assignment obtained from the ‘snapclust’ method (see41). Finally, we performed a complementary analysis for inferring individual membership to each genetic group using GeneClass243 to identify first-generation migrants. Detection of migrants was carried out using Paetkau et al.’s44 criterion for likelihood computation (Lh) and Paetkau et al.’s45 resampling method for probability calculation, setting the default frequency for missing alleles at 0.01 and using 10 replicates with 100 simulated individuals each (α = 0.01).The resulting genetic clusters were subsequently used for population structure analyses. First, we used GenAlex 6.546.The sampling methodology was approved by the Ethics Committee of the Sciences Faculty of the University of Chile, following guidelines from the Biosecurity Manual of CONICYT (version 2008) and Chilean law N°20380 about animal protection. Full permissions for sampling and animal ethics approval were granted by the Servicio Agrícola y Ganadero , the Corporación Nacional Forestal , Chile; and the Administración de Parques Nacionales (APN-DRPN research permit N° 1405), Argentina. This manuscript complies with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelinesAphrastura in the NW Beagle Channel (n = 50), Navarino Island (n = 54), and Diego Ramírez (n = 13). Morphological differences among populations were statistically supported for weight, tail length, tarsus length, beak length, and beak width . Birds of Diego Ramírez typically performed short and low flights, rarely moving above the cover of the tussock grass. On Gonzalo Island, individuals always remained in the wind-protected habitat provided by the tall grass (< 2 m), except for brief horizontal movements to and from more open foraging areas.Aphrastura on Gonzalo Island. Two of them were found in the areas with a higher concentration of breeding seabirds, which were close to the coast (11 masl), characterized by 50% coverage of small tussocks (~ 0.4 m high) and a rocky substrate. Three nesting sites were found in the higher and wind-exposed areas of the island (> 100 masl), but sheltered by 100% tussock coverage. Yet, most nesting sites (n = 5) were observed in the lower and medium altitude zones, particularly in the presence of protected creeks and areas with tussocks at least 1 m high. These five nesting sites were composed of burrows with single entrances. Four of these burrows were generated and abandoned by underground nesting seabirds, and one was a natural soil cavity. One Aphrastura nest was located below an active nest of an incubating Grey-headed albatross (for details see Supplementary Information Appendix 2).We observed 10 nesting sites of Aphrastura typically foraged about 10–30 m away from the nesting site, capturing invertebrates. Foraging areas were covered by dense tussock or were located in the periphery of the tussock-matrix. We recorded pairs or groups of up to four individuals foraging together. Birds responded to a human intruder (the observer) by active flapping displays, vertical movements between the ground and the tussock canopy, and mobbing calls that started from 25 to 15 m away from the observer and involved pairs or groups of up to five individuals.On Gonzalo Island, cytb sequences from 120 individuals across the distributional range of A. spinicauda , compared to that of populations in the center and the north of the distribution, and also of the populations in the extreme southern part of the distribution  being higher than GST values .We obtained 471 bp A. spinicauda. The Diego Ramírez haplotype is also present on Horn Island  and in low frequency in Navarino Island   Fig. .Figure 5Based on the genotypes of 153 individuals at 12 polymorphic microsatellite loci  showed that Navarino Island and the southern continental populations (Bariloche and Tierra del Fuego) formed a homogeneous group . This estimate is congruent with the number of clusters emerging from the PCA analysis: (1) a central cluster, comprising the Manquehue population in the center of the species’ distributional range; (2) a southern cluster, composed of Bariloche, Tierra del Fuego Island, and Navarino Island populations; and (3) Diego Ramírez. The pairwise G-statistics indicated that differentiation between the central and southern clusters was low to moderate Table , whereased Table . The posed Table . These rAphrastura population, as compared to the continental Aphrastura population, we propose the following new bird species.Based on the strong genetic differentiation, as well as marked morphological and behavioral differences described above for the Diego Ramírez 47.Order: Passeriformes 48.Family: Furnariidae Aphrastura 49.Genus: Aphrastura subantarctica, sp. nov. R Rozzi, CS Quilodrán, E Botero-Delgadillo, RD Crego, C Napolitano, O Barroso, JC Torres-Mura & RA Vásquez.Scientific name: Common name (English): Subantarctic Rayadito.Common name (Spanish): Rayadito Subantártico.Aphrastura structure with rounded wings, and an idiosyncratic tail morphology. Aphrastura’s distal third of the inner web of the rectrices is abruptly and deeply excised, giving the tips of the feathers a thornlike appearance. No other genus in the family has a similar tail structure. Aphrastura differs in these morphological characters from the phylogenetically closest related genera in the subfamily Synallaxinae present in southwestern South America: Leptasthenura and Sylviorthorhynchus51. In contrast to Aphrastura, Leptasthenura’s tail is not abruptly and deeply excised at the distal portion of the inner web of the rectrices; in Sylviorthorhynchus, the rectrices are denuded of barbs1. A. subantarctica differs from A. spinicauda, in having on average a larger and heavier body (~ 25%), a larger beak (~ 15%), a larger tarsus (~ 5%), and a shorter tail (~ 16%) .Diagnosis: Morphology.—Typical Holotype.—Adult male, Museo Nacional de Historia Natural, in Santiago, Chile (MNHNCL/AVE Nº 5439), collected on 22 July 2017 by OB and JCTM on Gonzalo Island, Diego Ramírez Archipelago, Magallanes and Antarctic region, Chile, at 56.52141°S 68.71647°W.1. Crown, hind neck and face sooty black, divided by a broad and conspicuous dull orange superciliary streak that extends to the back and merges with the brown color of the back. Other upperparts are fulvous brown, with dusky dark edges becoming rufous and uniform on the rump. Underparts whitish on the throat, breast, and abdomen becoming fulvous on the flanks and undertail coverts. No brood patch; no cloacal protuberance, little fat (1), no evidence of molt on head, body, wing, or tail.Description of holotype.—Color descriptions follow VaurieMeasurements of holotype.—Total length 145 mm; head and bill length 32.4 mm; exposed culmen 12.1 mm; bill depth 3.4 mm; bill width 3.4 mm; wing chord 59 mm; tarsus length 21.8 mm; tail length along central rectrices 66 mm; mass 15.1 g.Paratypes (two).—(1) Juvenile female, MNHNCL/AVE 5440, collected 22 July 2017 at Gonzalo Island, Diego Ramírez Archipelago by OB and JCTM; same coloration as the adult, tarsus and gape yellowish; white tips in primaries and secondaries. No brood patch; no cloacal protuberance, no fat (0), no head, body, wing, or tail molt. Total length 150 mm; head and bill length 33.6 mm; exposed culmen 12.6 mm; bill depth 3.6 mm; bill width 3.2 mm; wing chord 63 mm; tarsus length 23 mm; tail length along central rectrices 69 mm; mass 15.9 g.—(2) Juvenile male, MNHNCL/AVE 5441, collected 22 July 2017 on Gonzalo Island, Diego Ramírez Archipelago by OB and JCTM; same coloration as the adult, gape and tarsus yellowish; white tips in primaries and secondaries. No brood patch; no cloacal protuberance, no fat (0), no head, body, wing, or tail molt. Total length 150 mm; head and bill length 33.6 mm; exposed culmen 12.9 mm; bill depth 3.5 mm; bill width 3.3 mm; wing chord 59 mm; tarsus length 23 mm; tail length along central rectrices 72 mm; mass 15.9 g.53, the authors encourage the adoption of this small bird as a symbol for the conservation of this unique environment.Etymology. The specific epithet refers to the distribution restricted to the remote sub-Antarctic Magellanic ecoregion of Chile. By naming the new species after this region, one of the last pristine regions in the world54. For small birds, such as Aphrastura spp., an island separated from the closest island (Horn Island) by 111 km in one of the world’s windiest oceans12, can limit exchange and hence favor population differentiation. One of the best-studied examples on islands separated by short distances is the radiation of the Darwin’s finches (Geospiza spp.) on the Galapagos Islands in the Pacific Ocean55. We studied several populations of Aphrastura spinicauda, a year-round resident of forested habitats that rarely engages in long-distance flights60. While several populations are located on southern islands, our results suggest they are not reproductively isolated, except for the population on Diego Ramírez. This population is genetically differentiated from populations inhabiting the closest islands on Tierra del Fuego and the mainland, with no evidence of recent gene flow. This may explain the morphological divergence of the population in terms of body weight, beak shape, and tarsus and tail length. In addition, the distinct environmental conditions on Diego Ramírez, in particular the lack of trees or any other woody plants, strong winds, and the presence of dense tussock used by burrow-nesting seabirds, might have triggered behavioral divergence related to living in a herbaceous environment, such as nesting in cavities below or close to ground level. This markedly contrasts with the tree creeping behavior and cavity nesting displayed by their continental relatives inhabiting forest ecosystems.Oceans constitute a strong dispersal barrier for terrestrial animals but also for birdsAphrastura on Diego Ramírez are on average heavier than their continental counterparts, and their tails are shorter. These morphological shifts might represent an adaptation to shorter and lower flights in dense tall grass habitats and windy conditions. These birds tend to fly mostly short distances among the Poa flabellata tussocks, which protect them from the strong winds. These movement patterns, shaped by the habitat conditions, conspicuously contrast with those of individuals from continental populations that move within the understory (< 1 m) to the top of the canopy (> 15 m), and often fly into open spaces while crossing gaps between tree patches7. On Diego Ramírez, restricted movement within a more sheltered habitat, close to the ground level, may have driven selection for shorter tails that may facilitate moving among dense tussock. Shorter tails could also have arisen through genetic drift after a small number of birds reached the archipelago or because other selective pressures for longer tails might have been relaxed in the environmental conditions of Diego Ramírez. A larger body size is typically found in birds that have colonized islands, and is a well-documented pattern of convergent evolution61. This has been described as the “island syndrome”, in which small birds (< 1 kg) are predicted to increase their body size in response to reduced predation pressure and food limitation under the new insular conditions62. Selection on fast escape from predators may be relaxed on the archipelago, as terrestrial predators are absent63. In addition, a larger body size may have been favored on Diego Ramírez because it increases thermoregulation efficiency64. While a larger body size has also been described in the subspecies (Aphrastura s. bullocki)13 and species (Aphrastura masafuerae)66 of Aphrastura inhabiting islands, the behavior related to grass habitat use and nesting at ground level seems to be unique to Aphrastura on Diego Ramírez. Another subspecies inhabiting Patagonian islands is Aphrastura s. fulva, but because the main differences from continental Aphrastura spinicauda are related to color patterns13, it is also expected to be morphologically and behaviorally different from Aphrastura on Diego Ramírez. The findings of our study are thus consistent with the suggestion made by François Vuilleumier thirty years ago67, who characterized Aphrastura spinicauda as a geographically variable species. He also pointed out that the individuals observed on Diego Ramírez were morphologically and behaviorally different from northern populations, and more similar to wrens (Troglodytidae) than to species ecologically equivalent to A. spinicauda, such as titmice (Paridae) or treecreepers (Certhiidae).Individuals of Aphrastura individuals use burrows of different seabird species, sometimes associating with larger seabirds , which may provide protection from weather and potential aerial predators . Across the entire range of Aphrastura spinicauda, individuals respond to intruders near their nest sites with alarm calls. In the continental populations, mobbing calls have minimum and maximum frequencies of 2.82 and 13.01 kHz, respectively, with at least six notes per second in central and southern Chile68. However, based on a previous record in 2001, it seems that minimum and maximum frequencies of mobbing calls of Aphrastura subantarctica are lower, with the same number of notes (see69). While these preliminary observations have to be confirmed by future studies, the low call frequencies of Aphrastura on Diego Ramírez could be related to the high ambient noise, as well as their larger body size70.The population on Diego Ramírez uses a distinct habitat that lacks woody plants, in ways that are unusual when compared to the continental populations. The lack of trees with nesting cavities has led to an association with breeding seabirds, which nest on Diego Ramírez during the austral spring and summer. Aphrastura population from Diego Ramírez represents an isolated entity. The autosomal differentiation is substantial even when considering values of the G′ST(Nei) index, which is a more conservative statistic than Hedrick’s index71. Despite sharing a mitochondrial haplotype with populations from Navarino and the Horn islands, the Aphrastura population of Diego Ramírez is also differentiated at the mitochondrial level, as shown by the significant genetic distance between them and individuals in Navarino and the Horn islands. Furthermore, Diego Ramírez appeared as a genetically separated cluster in our analyses, indicating a complete absence of subsequent, or at least recent, migration to or from the archipelago. When excluding the Diego Ramírez individuals, the other populations of A. spinicauda have no clear genetic structure at the autosomal and mitochondrial level across their distribution, which is consistent with moderate to high levels of gene flow, as documented by other authors30. Our results support the recent observation of the absence of current gene flow between the Diego Ramírez population and their relatives inhabiting the continent and islands closer to it10. This population could thus be considered a separate evolutionary unit.The genetic structure resulting from both mitochondrial and autosomal data suggests that the 73, which has resulted in a heterogeneous environment with high level of endemism across various biological groups74. The population on Diego Ramírez was probably established from a unique colonization event of a small group of individuals that originated from the southern Patagonian islands, which are geographically nearest to Diego Ramírez, such as Navarino or Horn islands, with one shared mitochondrial haplotype among them suggesting this historical event. In addition, the presence of a single cytb haplotype on Diego Ramírez suggests a strong founder effect, while the absence of new private haplotypes suggests a scenario of recent, post-LGM colonization of Diego Ramírez. The noticeably low levels of heterozygosity, allelic richness, and number of private alleles in the microsatellite data further supports this hypothesis. In evolutionary terms, the relatively short time that has passed since a post-LGM colonization event might have been sufficient for the Diego Ramírez population to genetically differentiate from A. spinicauda. The absence of the main mitochondrial haplotype present in all A. spinicauda populations, from the northernmost to the southernmost ones, suggests that the population on Diego Ramírez is undergoing a peripatric divergence from A. spinicauda.The sub-Antarctic Magellanic ecoregion experienced variations in sea level and volume of ice sheet during the Last Glacial Maximum Aphrastura population from Diego Ramírez compose a demographically independent population that should be considered as a new conservation and taxonomic unit. This population has remained isolated and encompasses specific morphological, genetic, and behavioral features, for which reason it is now proposed as a new species75. In his influential paper, de Queiroz76 reconciled the concept of species and stated that the only nonnegotiable condition for a species to be considered as such is to separately evolve from another lineage, which according to our data has taken place for the Aphrastura population in Diego Ramírez. Whether there is complete lineage sorting and intrinsic reproductive isolation among the Aphrastura populations or not will only show how far the diverging process has gone through the speciation continuum .Our analyses indicate that the 51, may provide more evidence of differentiation of the Diego Ramírez population. Further research may include samples from A. masafuerae and A. s. bullocki, as well as full genomes for evaluating the genomic landscape of divergence during the speciation process of the genus Aphrastura. Several other bird taxa with small genetic differences have been reclassified and subsequently considered as valid species, such as the common swift (Apus apus) and pallid swift 78, as well as the greater spotted eagle (Clanga clanga) and lesser spotted eagle (C. pomarine)79, among others (see80).We have presented multiple pieces of evidence suggesting the existence of a new species by combining morphological, ecological and genetic data, and we argue that this integrative framework is crucial for defining new taxonomic units. Acoustic analysis, a powerful tool for supporting species limits in suboscine passerines, including FurnariidaeA. subantartica as a new species. The genetic, morphological, and ecological divergence of this population, which may have resulted from isolation on an island with a distinct habitat, is probably an ongoing evolutionary process. Because of the small size of the Diego Ramírez islands and the potential arrival of exotic mammal predators, it is pressing to protect this new endemic species from extinction. The Diego Ramírez Archipelago encompasses the southernmost extreme islands of the American continent and is free of invasive alien species. Measures should be put into place to keep exotic mammals, such as rats (Rattus rattus), domestic cats (Felis catus), and American minks (Neovison vison)–which are all present on other islands of the Cape Horn Biosphere Reserve81–off the Diego Ramírez islands. In particular, the rapid expansion of the American mink has impacted bird populations on other subantarctic islands that have evolved in the absence of terrestrial mammal predators83. Collaboration between the scientific community and other institutions, in particular the Chilean Navy, which has been regularly present on this archipelago since the establishment of the lighthouse on Gonzalo island in 1951, is critical for the success of scientific long-term monitoring and conservation programs9. In 2019, the Diego Ramírez-Drake Passage Marine Park was created by the Chilean government to protect one of the few archipelagos that is still free from the arrival of exotic species worldwide12. The description of A. subantarctica is also an appeal to the community to protect not only a population of a new species, but also to protect a remote natural laboratory that represents an opportunity to preserve the diversity of nature and its ecological and evolutionary processes.We propose Supplementary Information.


Biological invasion is a complex process associated with propagule pressure, dispersal ability, environmental constraints, and human interventions, which leave genetic signatures. The population genetics of an invasive species thus provides invaluable insights into the patterns of invasion. Burcucumber, one of the most detrimental weeds for soybean production in US, has recently colonized Korea and rapidly spread posing a great threat to the natural ecosystem. We aim to infer the determinants of the rapid burcucumber invasion by examining the genetic diversity, demography, and spread pattern with advanced genomic tools. We employed 2,696 genome-wide single-nucleotide polymorphisms to assess the level of diversity and the spatial pattern associated with the landscape factors and to infer the demographic changes of 24 populations (364 genotypes) across four major river basins with the east coastal streams in South Korea. Through the approximate Bayesian computation, we inferred the likely invasion scenario of burcucumber in Korea. The landscape genetics approach adopting the circuit theory and MaxEnt model was applied to determine the landscape contributors. Our data suggested that most populations have experienced population bottlenecks, which led to lowered within-population genetic diversity and inflated population divergences. Burcucumber colonization in Korea has strongly been affected by demographic bottlenecks and multiple introductions, whereas environmental factors were not the primary determinant of the invasion. Our work highlighted the significance of preventing secondary introductions, particularly for aggressive weedy plants such as the burcucumber. Biological invasion leaves traceable genetic tracks on each stage of invasion, which offer indispensable information for making proper management plans and effective control practices . ColonizTamarix spp., and Japanese knotweed, Reynoutria japonica , is an annual vine widely distributed in the east side of North America  to assess the genetic diversity pattern that might have been associated with multiple introductions and the demographic history and 2) to determine the contributing landscape factors during the rapid range expansion. Former genetic studies of burcucumber in Japan suggested the possibility of multiple introductions, but the sample size and the number of molecular markers used were limited . DespiteDuring the summer of 2021, we collected young burcucumber leaves of 383 individuals from 24 populations across four river basins  and rivers along east coast regions in South Korea Figure 1EcoRI-HF and XbaI. NheI was added for dimer cleaving . We then ligated the index adapters  in a 15-µl reaction mixture containing 100 ng DNA, 8 µl master mix, and 1 µl of each 5-µM adapter. We made sure the total volume of the master mix to be 15 µl containing 0.5 µl of each restriction enzyme and 1.5 µl of 10× FastDigest Buffer. The digestion and ligation were conducted simultaneously in a tube. The resultant products were incubated in a thermal cycler for 15 min at 37°C. We prepared 5 µl of the ligation mixture as follows: 0.5 µl of 10× Ligase Buffer, 100 units of T4 DNA ligase, 1.5 µl of 10 mM ATP, and 2 µl ultrapure water. The ligation mixture was added to each sample and incubated at 22°C for 20 min and 37°C for 10 min. We repeated the process twice then incubated the mixture at 80°C for another 20 min. The resultant products of the ligation were then examined by PCR amplification using Bioneer Multiplex PreMix. For the PCR, we prepared a 20-µl reaction volume containing 1 µl of iTru5 and iTru7 primers and 1 µl of adaptor-ligated DNA fragments. For the amplification, we used the following thermal cycler profile: 95°C for 10 min; 35 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 30 s; and 72°C for 5 min. After the ligation examination, we pooled 5 µl of the adaptor-ligated fragments from each sample in a 1.5-ml tube and purified the pooled samples using a 1:1.8 mixture of AmPure XP magnetic beads . Seventy percent of EtOH was used to wash the prepared samples, and the samples were left at room temperature for 15 min to air-dry. The dried samples were resuspended with 60 µl of TE buffer. We then eluted the purified DNA fragments from the magnetic beads. Amplification was carried out to enrich the adapter-ligated DNA fragments. For the amplification, a 50-µl reaction volume was prepared to contain 10 µl of the pooled DNA fragments, 5 µl of 5 µM iTru5 primer, 5 µl of 5 µM iTru7 primer, 10 µl of 5× HF Buffer, 1.5 µl of 10 µM dNTP, and 1 unit of Phusion DNA Polymerase. The amplification profile was as follows: 98°C for 2 min; 7 cycles of 98°C for 20 s, 60°C for 15 s, 72°C for 30 s; and 72°C for 5 min. Additional sample cleanup was conducted by using a 1:1.8 mixture of AmPure beads. Before the size selection, we washed the samples twice with 70% EtOH and resuspended the samples in 60 µl TE buffer. Following the manufacturer’s protocol, we selected the targeted size fragments (500-bp fragments ± 10%) using Pippin Prep . We performed a final amplification with a 50-µl reaction mixture containing 5 µl of size-selected DNA, 3 µl of 5 µM P5 primer, 3 µl of 5 µM P7 primer, 1.5 µl of 10 µM dNTP, 10 µl of 5× HF Buffer, and 1 unit of Phusion DNA Polymerase. The thermal cycler profile was as follows: 98°C for 2 min, 98°C for 20 s, 61°C for 15 s, and 72°C for 45 s for 12 cycles; and 72°C for 5 min. The third cleanup  for the amplicons was administered with a 1:1.8 mixture of AmPure beads. We washed the amplicons twice with 70% EtOH and resuspended them in 35 µl ultrapure water. The complete 3RAD library was evaluated for quality and quantity through Agilent 2100 Bioanalyzer . The fully prepared library was run on an Illumina HiSeq X-10 platform using 2 × 150 paired-end sequencing at Macrogen Inc., Korea.3RAD  was emplde novo. For catalog assembly, we set the parameters as −m 3 and −M 3 in the ustacks function allowing a maximum of one mismatch between sample loci , observed heterozygosity (Ho), and the number of alleles (Na) were calculated in GenAlEx 6.5 . The number of SNPs used for the population bottleneck test was reduced to ease the computational challenges. The population at mutation-drift equilibrium approximately shows an equal probability of a heterozygosity excess or a heterozygosity deficit. Populations that experienced a recent bottleneck would show widespread heterozygote excess on a significant number of loci. Excess of heterozygosity was assessed following We tested the presence of recent bottlenecks for the 24 local populations with 261 unlinked SNPs subsampled from the total 2,696 SNPs (rK) using fastSTRUCTURE, an efficient alternative of the software STRUCTURE for large-size data such as genome-wide SNPs  and Bayesian model-based assignment test. PCoA was carried out on Nei’s genetic distance computed from all 346 genotypes in GenAlEx. We inferred the number of randomly mating subgroups (ide SNPs . For theide SNPs . We ran  package .We employed the approximate Bayesian computation (ABC) approach to further infer the demographic history of burcucumber invasion in Korea using DIYABC Random Forest (DIYABC-RF) version 1.0 . For theBased on the clustering results, the current distribution, and prior information on burcucumber invasion in Korea, we constructed eight demographic scenarios of burcucumber invasion Figure 2For checking the level of errors on the scenario choice and parameter estimation accuracy, we checked the location of an observed data set over the prior data space using a PCA plot depicting the simulated data set retained from the training and observed data following 2, an approximation for the fixed effects in a mixed effect model (ST and Slatkin’s linearized FST [FST/(1 − FST)] as proposed by ST and the linearized FST by the marginal r2. FST outperformed all distance models over the linearized FST . The best model was selected using the conditional AIC  and Naturing , which generated 879 presence data points. We used 19 climate variables  to construct the current SDM. We clipped all other data sets with DEM using the “Extract by Mask” function of ArcGIS10.6.1 (ESRI) and adjusted the geographic dimensions of the data used in the analysis to the South Korea DEM by a 5-m resolution. All parameters of MaxEnt were set to default with 50 replicates and 5,000 maximum iterations. We selected the best-fitting model using the area under the receiver operating characteristic curve (AUC), which corresponds with the perfect classification model around a value of 1.0. The produced value of SDM was inverted by the “Reclassify” function of ArcGIS for resistance raster. We finally computed the pairwise resistance distances in Circuitscape ver. 4.0  to determine the habitat suitability of burcucumber. We assumed that the low habitat suitability likely impeded the dispersal between population pairs; thus, an SDM can be applied to assess resistance distances. The maximum entropy method  in the UJ population at which the number of collected samples was the smallest, whereas the highest mean He was observed in HF . Based on the sign and Wilcoxon’s rank tests, most populations sampled in our research experienced recent population bottlenecks (P > 0.05) showing that the populations are likely in the equilibrium state of population demography  was much less than the population differentiation within each river basin . In our PCoA analysis, the first three axes only accounted for ~15% variation Figure 3Based on the LDA plot for the prior model checking, the location of the observed data was within the range of the simulated data, which suggests that ca. 3,000 simulated data sets per model were suitable for inferring the model choice Figure 2ST outperform the linearized FST whereas the three different distances computed showed a similar model fit  in the study was lower than the long-lived invasive shrubs’ [Tamarix ramosissima, mean He based on 1,996 SNPs = 0.30 , which was empirically verified by a number of studies, e.g., ST in our study. Alternatively, the mating system of burcucumber  were assigned nearly to a single cluster, suggesting that those populations might be the sources of an early establishment  can be found below: S-RL and DCS conceived ideas and designed the study. DCS prepared research grant. Field sampling was planned and performed by S-RL. S-RL designed the laboratory work and performed the genetic and statistical analyses. S-RL wrote the manuscript. DCS edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a research project of the Korea National Arboretum .We are grateful to a postdoctoral fellow, Tae-Young Choi, for the laboratory and analytical assistance. We also thank the graduate assistant, Eun-Su Kang, with the undergraduate assistants, Tea-Young Eom, Hyeon-Su Kim, and Geon-Ho Kim, for their help with field sampling and laboratory work.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Terpios hoshinota was reported from coral reefs in Guam in 1973, but was only formally described in 1993. Since then, the invasive behavior of this encrusting, coral-killing sponge has been observed in many coral reefs in the West Pacific. From 2015, its occurrence has expanded westward to the Indian Ocean. Although many studies have investigated the morphology, ecology, and symbiotic cyanobacteria of this sponge, little is known of its population genetics and demography. In this study, a mitochondrial cytochrome oxidase I (COI) fragment and nuclear ribosomal internal transcribed spacer 2 (ITS2) were sequenced to reveal the genetic variation of T. hoshinota collected from 11 marine ecoregions throughout the Indo-West Pacific. Both of the statistical parsimony networks based on the COI and nuclear ITS2 were dominated by a common haplotype. Pairwise FST and Isolation-by-distance by Mantel test of ITS2 showed moderate gene flow existed among most populations in the marine ecoregions of West Pacific, Coral Triangle, and Eastern Indian Ocean, but with a restricted gene flow between these regions and Maldives in the Central Indian Ocean. Demographic analyses of most T. hoshinota populations were consistent with the mutation-drift equilibrium, except for the Sulawesi Sea and Maldives, which showed bottlenecks following recent expansion. Our results suggest that while long-range dispersal might explain the capability of T. hoshinota to spread in the IWP, stable population demography might account for the long-term persistence of T. hoshinota outbreaks on local reefs.The first occurrence of the cyanobacteriosponge  Competition among sessile organisms acts as one of the ecological factors for shaping the biodiversity and community structure of coral reefs  and aggressiveness, resulting in the overgrowth and killing of corals . Terpiososhinota    . The occ in 1973 . Among 31,000 m2 , and in sent day .T. hoshinota- associated rapid declines in coral cover have been reported from reefs in Okinawa, Japan  and nuclear ribosomal 28S gene (28S). Two COI haplotypes that differed from the COI sequence in GenBank were discovered within the archipelago. Recent surveys on coral reef biodiversity revealed the first report of T. hoshinota in Kimberley inshore reefs, Western Australia  sequences. By revealing population genetic structure and demographic history, potential hypotheses of how T. hoshinota disperses and colonizes in the IWP are proposed.In this study, we sampled Terpios hoshinota were examined in this study  field workers not identifying T. hoshinota in the field, and (3) sampling restrictions in some cases . After collection, macro-invertebrates and commensals were carefully removed. All samples were preserved in 75% ethanol inside centrifuge tubes and stored at −20 °C until extraction.All specimens were collected with permission and sampling permits where required . A total of 234 specimens of is study , Table 1For Guam, samples were collected with permission of the Department of Agriculture Division of Aquatic and Wildlife Resources (DAWR) Guam and certificate of origin from DAWR was obtained prior to shipping. Permit is not required to collect sponges in Green Island and in China. For Indonesia, permit was issued by the Indonesian Ministry of Research, Technology & Higher Education  No. 011/SP2H/LT/DRPM/IV/2017. For Malaysia, sampling permit was issued by the Marine Park and Resource Management Division, Department of Fisheries Malaysia  with a Sampling permit: JTLM 630-7 JLD. 4(8). Samples from Australia were part of Census of Marine Life/Census of Coral Reefs, with permit No. G32313.1 issued by the Great Barrier Reef Marine Park Authority.n = 12), Lanyu (3), Wanlitung (1), Guam (2), Maldives (5), Miyako (2), Bise (6), Shiraho (1), Yakomo (2), Lizard Island (1), and Xisha (3). A fragment containing 650-bp COI of T. hoshinota was amplified by the universal primer set (http://www.missionbio.com.tw).Genomic DNA was extracted from 25 mg of clean sponge tissue using the DNAeasy-Tissue Kit (Qiagen) according to the manufacturer’s instructions. Specimens used for COI sequencing were from Green Island  LiuMaGou (2), ChaiKou (1), BiTouXi (2), ZhaoRi hot spring (2), YouZi Lake (1) and Shiraho, Japan (1). DNA samples were amplified by PCR with 1 cycle at 94 °C for 3 min, 94 °C for 30 s, 54 °C for 90 s, and 72 °C for 60 s at 35 cycles, followed by a final cycle of 72 °C for 10 min. The length of the PCR products (∼650 bp) was then confirmed by gel electrophoresis (1% agarose). PCR products were then cloned by using an Invitrogen TA cloning kit  following the manufacturers protocol. Treated plasmids containing Ampicillin (50 µL), IPTG (0.5 mM), X-Gal (50 µg/ml) were then added into Luria Bertani agar plates for cloning. Positive clones were selected during the transformation step and a minimum of 20 clones were picked up for each set of sponge samples, followed by PCR with universal primer . A total of 111 positive clones were selected and amplified for the completed ITS region. Cloned ITS DNA sequences were submitted to GenBank using the blastn option, and sequences that matched 100% to non-poriferan species were excluded from the following analyses. Of the 111 ITS clones, 72 sequences matched sponge species  with 80 to 89% similarity values .In addition, universal primers for amplifying the entire nuclear ribosomal internal transcribed spacer (ITS),  , were usy values . Primersy values  module wT. hoshinota in the IWP. DNA sequences were cleaned visually based on quality and accuracy using Geneious R10 (https://www.geneious.com). Sequences were trimmed and aligned by the MAFFT algorithm . Genetic diversity indices nucleotide diversity (π), number of haplotypes (h), and haplotype diversity (hd) were calculated using DnaSP V.6.12.03 (p- distance) were calculated with MEGA 7 . Sponges that had only a few double peaks, as expected from the superposition of two ITS2 sequence types of equal lengths differing at several positions, were phased using the programs PHASE V.2.1.1  was obtained  were calculated as described in the previous section . Best-fit models for nucleotide substitution were performed by jModelTest V.2.1.10  was assessed using 10,000 permutation replicates. Global FST and average FST per population following with UPGMA clustering of population were obtained from GraphPad Prism 9. Isolation-by-distance analysis (FST estimates (FST /1- FST), and correlated geographical distances of T. hoshinota populations based on marine ecoregions using the standard Mantel test implemented in GENODIVE V.3.05  of 0.00185, haplotypic diversity (hd) of 0.416, and p-distance of 1.09  in specimens from the major marine ecoregions, except for specimens from the Torres Strait (Northern GBR). C3 was ranked as the second-most dominant haplotype  in samples from Guam, Indonesia, and southern Kuroshio. The remaining three COI haplotypes were each identified from single individual specimens from Xisha, the GBR, and the Maldives. Due to the small sample sizes and relatively low genetic variability, COI was not utilized in the historical demography and population genetic analyses.The COI sequences obtained from 37 equences  having a of 1.09 , respectπ), p-distance, haplotype (h), and haplotype diversity (hd) of ITS2 in Terpios hoshinota based on marine ecoregions are shown in π, hd, p- distance, and following demography and FST -statistics. The mean p-distances ranged from 0.269 in the Maldives to 3.132 in the South China Sea Oceanic Islands. Similar patterns were seen in nucleotide diversity, with 0.0021 in the Maldives and 0.00245 in the South China Sea Oceanic Islands. The number of haplotypes ranged from 1 in the Torres Strait, northern GBR, to 12 in South Kuroshio, with haplotype diversity ranging from 0.117 in the Maldives to 1 in Northeast Sulawesi.The number of samples (n), nucleotide diversity  present in all sampling sites except the Maldives, GBR, Xisha, and Yakomo , and present only in sites near the equator or in the southern hemisphere, including the Sulawesi Sea, Southern Java, the Maldives, and GBR. H2 was the third-most dominant haplotype , present in samples from Guam, South China Sea Oceanic Islands, Sunda Shelf, and Southern Java. The rest of the haplotypes were present either in low frequencies or restricted to single populations; for example, H22 and H23 occurred in Xisha, China, H19 in southern Java, and H18 in Papua, Indonesia , with negative FST values in South Kuroshio - Sulawesi Sea (−0.003), South Java - Northeast Sulawesi (−0.1017), and Maldives –GBR (−0.286). Negative FST values indicate more variation within than between populations or results from uneven sample sizes  in comparison with all other populations in the marine ecoregions of the eastern Indian Ocean, Coral Triangle, and West Pacific, with the largest FST (0.6687) shown between the Maldives and Sunda Shelf. Testing for a pattern of isolation-by-distance by performing a Mantel test between genetic (FST) and spatial distances revealed a significant relationship  for T. hoshinota across the IWP (Population differentiation based on marine ecoregions was examined by pairwise atistics . Eighteele sizes . The FST heatmap  clearly  the IWP .T. hoshinota has become ubiquitous in the Indo-West-Pacific (IWP) region in the past few decades. To understand its distribution, ecology, and evolution, we examined its genetic variation, historical demography, and population differentiation to infer the possible dispersion mechanisms and outbreaks of T. hoshinota across the IWP. Our results showed that; (1) T. hoshinota is a wide-spread cyanobacteriosponge in the IWP, and both the mitochondrial COI and nuclear ITS2 markers showed a dominant haplotype associated with its dispersion and outbreaks, (2) moderate gene flow existed among most populations in the marine ecoregions of West Pacific, Coral Triangle, and east Indian Ocean, but there was restricted gene flow between the central Indian Ocean and the other three regions, and (3) demographic analyses of most T. hoshinota populations were consistent with mutation-drift equilibrium, except for the Sulawesi Sea and Maldives, which had bottlenecks following recent expansions.The cyanobacteriosponge T. hoshinota. A slow evolution pattern of mitochondrial genomes has been reported in lower metazoans, including certain lineages of poriferans and anthozoans in general (p- distance of 1.09 and low nucleotide diversity (π = 0.00185) were identified from 47 T. hoshinota specimens collected across the IWP. Slow mitochondrial gene evolution was previously also found in the coralline demosponge Astrosclera willeyana across the Indo-Pacific, which contained only three COI haplotypes with a maximum p-distance of 0.418 and low nucleotide diversity (π = 0.00049) (Aplysina spp.) from the Caribbean, there was no interspecific variation in the COI fragment and only 6 nucleotide differences among species observed in the complete mitochondrial genome  .COI sequences obtained from this study showed extremely low intraspecific variation in  general . In the 0.00049) . As well,620-bp) . Conversp-distance of 1.717 and π of 0.0164) and presented 23 haplotypes across the IWP region, suggesting that ITS2 could provide more information for intraspecific inferences in T. hoshinota. However, heterogeneous levels of ITS intra-genomic polymorphisms (IGPs) in a diverse range of marine sponge taxa have been documented, and it has been recommended that caution must be taken to survey the different levels of IGPs before applying ITS in phylogenetic studies below the species level  evolved 10 times faster than COI (π = 0.00185). Both markers resolved most of the representative haplotypes from the distributional range of Terpios hoshinota in the IWP, finding 5 COI haplotypes out of 47 samples (10%) and 23 ITS2 haplotypes out of 234 samples (9%). In the COI SPN, haplotype C2 was dominant, corresponding well with the most dominant ITS2 haplotype H3 from Southern Kuroshio, H2 from Mariana Islands, H6 from Maldives, and H22 and H23 from Xisha, South China Sea Islands. Interestingly, C2 also corresponded to the COI haplotype previously identified from the Spermonde Archipelago, Indonesia  showing a dominant haplotype comprised of several mutational steps evident among sequences from specimens. Although the number of specimens and sampling locations were not the same for the two markers, our results indicated that, by count, ITS2  to the west and the Kuroshio Current to the north to invade reefs in Japan and Taiwan   . Trans-eter 2014 . Similarand 2006 .FST and isolation-by-distance (IBD) is consistent with the “recent expansion, perhaps from Guam” hypothesis. Only 32.73% of pairwise FST values were significant, suggesting that there were recent expansion events or moderate gene flow existed among marine ecoregions in the West Pacific, Coral Triangle, and eastern Indian Ocean, but not the central Indian Ocean. The FST heatmap clearly showed that the Maldives had much higher FST values compared to counterparts in other marine ecoregions , as seen in other benthic organisms, is consistent with long-range dispersal  used to examine the demographic history of T. hoshinota based on marine ecoregions showed that most populations were consistent with the model of drift–mutation equilibrium with no evidence of selection, except for populations in the Sulawesi Sea and the Maldives , and skewed unimodal mismatch distribution may suggest sudden population expansions with bottlenecks in these populations. However, Ramos-Onsins’ R2, which is more powerful for detecting past demographic events, especially in populations with small sample sizes, remained non-significant in all populations, suggesting that T. hoshinota might have existed in the sampled reefs for a long period of time in relatively low abundances, followed by local outbreaks through asexual propagation and also consequently noted during reef surveys. Previous studies have shown that T. hoshinota can increase its coverage through asexual propagation at 1.5–2 times the speed of other sponges  and demography analyses hinting that bacteria  that rete method  and nextTerpios hoshinota have been reported from the West Pacific from the early 1970s (T. hoshinota outbreaks remain uncertain. The outbreaks are suspected to be related to large-scale disturbances caused by coastal development (T. hoshinota. Once a population outbreak is established, it can occupy reefs for decades, only disappearing possibly due to strong waves caused by storms, but still having the ability to revert to outbreak proportions at a later time (T. hoshinota in the past 20 years. It is important to continue long-term ecological studies using newly developed genomic applications to unveil the outbreak mechanisms of Terpios hoshinota, particularly given the increasing impacts of climate change.Occurrences of ly 1970s , and nowly 1970s . Neverthelopment  or volcaelopment , which cter time . As suchT. hoshinota among the IWP. Our results, based on genetic variation, historical demography, and population differentiation suggest that the potential outbreak mechanisms of T. hoshinota may result of both long-range dispersal and local outbreaks. However, we advise caution due to insufficient genetic variation and the lack of Terpios-specific gene loci on conventional molecular marker. Further investigation through genomic investigation might be able to unravel more comprehensive information about this sponge.This study illustrates the possible dispersion mechanisms and outbreak of 10.7717/peerj.13451/supp-1Supplemental Information 1Click here for additional data file.


Background:Aedes albopictus is an indigenous primary vector of dengue and Zika viruses in China. Wolbachia is a gram-negative and common intracellular bacteria, which is maternally inherited endosymbionts and could expand their propagation in host populations by means of various manipulations. Compared with research on the dispersion of Ae. albopictus at the macrospatial level (mainly at the country or continent level), little is known about its variation and Wolbachia infection at the microspatial level, which is essential for its management. Meanwhile, no local cases of dengue fever have been recorded in the history of Nanjing, which implies that few adulticides have been applied in the city. Thus, the present study examines how the Ae. albopictus population varies and the Wolbachia infection status of each population among microspatial regions of Nanjing City.Methods: The genetic structure of 17 Aedes albopictus populations collected from urban, urban fringe, and rural regions of Nanjing City was investigated based on 9 microsatellite loci and the mitochondrial coxI gene. The Wolbachia infection status of each population was also assessed with Wolbachia A- and Wolbachia B-specific primers.Results: Nine out of 58 tested pairs of microsatellite markers were highly polymorphic, with a mean PIC value of 0.560, and these markers were therefore chosen for microsatellite genotyping analysis. The Na value of each Ae. albopictus population was very high, and the urban area populations (7.353 ± 4.975) showed a lower mean value than the urban fringe region populations (7.866 ± 5.010). A total of 19 coxI haplotypes were observed among 329 Ae. albopictus individuals via haplotype genotyping, with the highest diversity observed among the urban fringe Ae. albopictus populations (Hd = 0.456) and the lowest among the urban populations (Hd = 0.277). Each Ae. albopictus population showed significant departure from HWE, and significant population expansion was observed in only three populations from the urban (ZSL), urban fringe (HAJY), and rural areas (HSZY) (p < 0.05). Combined with DAPC analysis, all the Ae. albopictus populations were adequately allocated to two clades with significant genetic differences according to population structure analysis, and the best K value was equal to two. AMOVA results showed that most (96.18%) of the genetic variation detected in Ae. albopictus occurred within individuals , while no significant positive correlation was observed via isolation by distance (IBD) analysis . The TCS network of all haplotypes showed that haplotype 1 (H1) and haplotype 4 (H4) were the most frequent haplotypes among all populations, and the haplotype frequency significantly increased from urban regions (36.84%) to rural regions (68.42%). Frequent migration was observed among Ae. albopictus populations from rural to urban regions via the urban fringe region, with four direct migration routes between rural and urban regions. Furthermore, Wolbachia genotyping results showed that most of the individuals of each population were coinfected with Wolbachia A and Wolbachia B. The independent infection rate of Wolbachia A was slightly higher than that of Wolbachia B, and no significant differences were observed among different regions.Conclusion: In the microspatial environment of Nanjing City, the urban fringe region is an important region for the dispersion of Ae. albopictus populations between rural and urban areas, and Wolbachia A and Wolbachia B coinfection is the most common Wolbachia infection status in all Ae. albopictus populations among different regions. Aedes albopictus, which is distributed in southern Chaoyang, Liaoning Province and to eastern Tianshui in Gansu Province, China, is an important vector of several arboviruses, including dengue virus (DENV), Zika virus (ZIKV), and chikungunya (CHIKV), in mainland China and many other tropical and subtropical countries , restriction fragment length polymorphism (RFLP), single-strand conformation polymorphisms (SSCPs), and single nucleotide polymorphisms (SNPs), because of their per-locus advantages and high rate of polymorphism . These m gambiae . Such efortation . The strão Paulo .Wolbachia is a rickettsia microorganism that is widely found in arthropods and can cause cytoplasmic incompatibility (CI), resulting in the decreasing densities of the host  before DNA isolation . The PCR amplification program was set as follows: predenaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 45 s and elongation at 72°C for 1 min, with a final extension at 72°C for 10 min. All the PCR products were detected and separated by 2% agarose gel electrophoresis. The coxI target fragments were then cut from the gel under UV light and purified with the GenElute™ PCR Clean-Up Kit . Each purified PCR product was subsequently cloned into the pCR™2.1 vector with a TA Cloning™ Kit  and selected by bacterial liquid PCR with T7 promoter primers. Finally, at least 20 positive clones for each PCR product were subjected to the sequencing of both strands using an ABI 3730XL automatic sequencer .We genotyped a total of 510 mosquitoes, including 30 specimens  from each of the 17 sample sites. The DNA of individual mosquitoes was extracted following the steps indicated in a TAKARA® DNA kit and stored in a -40 °C freezer until required. Individuals were genotyped based on the variation at nine microsatellite loci  Table 2Table 2. et al. , coxI seIS, FST, and FIT) were estimated from allele frequencies with FSTAT 2.9.3.2 , observed (Ho) heterozygosity), the mean number of alleles (Na), and F-statistic parameters ; the exception was AaelbB51, which had a value <0.25 and was considered to be a marker with low informativeness  was lower than that of the rural area populations (7.387 ± 4.908), while the Na values of these two areas were all lower than those of the urban fringe area, which was 7.866 ± 5.010. The observed heterozygosity (Ho) values of all areas ranged from 0.543 ± 0.030 to 0.570 ± 0.030, which were lower than the expected heterozygosity (He) values (ranging from 0.566 ± 0.094 to 0.606 ± 0.096). The FIS value of each population ranged from −0.026 to 0.218, and each Ae. albopictus population showed significant departure from HWE  was greater than the FST value between the urban and rural regions (0.009) and the FST value between urban fringe and rural regions (0.007)  revealed that only three populations from the urban (ZSL), urban fringe (HAJY), and rural areas (HSZY) displayed significant bottleneck effects , while four more populations from the urban (YHXC and NQSQ), urban fringe (TPSQ), and rural areas (CHZ) were also observed to have significant bottleneck effects under the two-phase model (T.P.M.) . In contrast to the high individual variation observed, no significant positive correlation was observed among all individuals via isolation by distance (IBD) analysis   .coxI haplotypes were observed among 329 Aedes albopictus individuals based on an analysis of coxI sequences, and the haplotype indices changed dramatically across Ae. albopictus populations from different area (π) ranging from 0.0002  to 0.0024 . The average number of nucleotide differences (k) ranged from 0.0909  to 1.1636 , and the number of polymorphic sites ranged from 1 to 9 across all the populations. Overall, the urban fringe Ae. albopictus populations showed the highest diversity, with mean Hd, π, and k values of 0.4562, 0.0018 and 0.8836, respectively, whereas the urban populations showed the lowest diversity.A total of 19 ent area . The hapcoxI sequences, and the results showed that haplotype 1 (H1) and haplotype 4 (H4) were the most frequent haplotypes and were distributed equally among all three sampling regions. Nearly all the other haplotypes were derived from H1 with one or more mutations, and the haplotype frequency increased significantly from the urban region (36.84%) to the rural area (68.42%). Relative to the urban regions (only H12 and H13), more exclusive haplotypes were detected among urban fringe regions  and rural regions . No additional shared haplotypes were detected between rural and urban fringe regions except H1 and H4.As shown in Aedes albopictus populations. As expected, Ae. albopictus was observed to migrate frequently from rural to urban regions via the urban fringe region, and four direct migration routes between rural and urban regions were observed  , and the unimodal mismatch distributions were observed mainly among the urban fringe Ae. albopictus populations  . The obtAedes albopictus populations with the Wolbachia A- and Wolbachia B-specific primers, respectively, confirming Wolbachia infection. The sequencing results showed that most of the individuals of each population were coinfected with Wolbachia A and Wolbachia B, with infection rates (IR) ranging from 23.33%  to 90.00% , and the mean infection rate of the rural region (70.56%) was significantly higher than that of the urban and urban fringe regions, which were 51.67 and 48.00%, respectively. Compared with the coinfection indices of Wolbachia A and Wolbachia B, the values of Wolbachia A and Wolbachia B for all individuals were low; the independent infection rate of Wolbachia A was slightly higher than that of Wolbachia B, and no significant differences were observed among different regions  are common indices for evaluating the genetic diversity of populations . The numl areas. . The varAe. albopictus, including ZSL, YHXC, NQSQ, HAJY, TPSQ, HSZY, and CHZ, may have experienced a bottleneck effect based on the results of the S.M.M. and T.P.M. model. FIS is an index of inbreeding within populations, where significant positive values indicate serious inbreeding within a population resulting in a loss of heterozygosity and significant negative values indicate outbreeding and an associated surplus of heterozygotes. The FIS values were all positive for the ZW and SQ populations, indicating that the degree of inbreeding in the populations was severe. The Ho values were lower than the He values except for the ZW and SQ populations, which indicated that the Ae. albopictus populations showed high gene consistency and suggested that the population might be experiencing inbreeding. The neutral test results for Tajima’s D showed that all populations except for NQSQ, HAJY, and JMJY presented negative values, suggesting that Ae. albopictus populations in Nanjing were selectively eliminated or were expanding  is aimed at reducing the density of mosquito species, which could change mosquito population genetics. An IMM program was effective in lowering the density of ty tests . The IMMeduction . Nanjingeduction . BreedinAedes albopictus generally inhabits a home range with a distance of less than 50 m, and the flight distances of this species do not exceed 500 m  is an essential measure of population genetic divergence. Values within the range of 0.05–0.15 indicate moderate deviation, and values <0.05 indicate slight deviation  was greater than the FST value between urban and rural areas (0.009) and the FST value between urban fringe and rural areas (0.007), which indicates that the mosquitoes in the city come mainly from the urban fringes. Proliferation within cities is greater than direct movement from rural areas to cities, and the exchange of mosquito populations between cities and urban-rural junctions is greater than that between rural areas and urban-rural junctions. This may be because compared to rural areas, the urban-rural fringe is the main area where urban living resources are distributed, where the flow of people and logistics is intensive, with continuous transport to the city. At the same time, the corresponding resources in the city are transferred to other areas through the urban-rural fringe. Dispersal within the city has also been verified by migration analysis. The migration analysis showed that compared with the exchange of mosquitoes between the other two regions, there were 4 main mosquito communication paths between the city and the urban-rural junction, and the gene flow value along each path was greater than 0.8. A program of IMM can affect the gene flow of populations through area effects and isolation effects and ultimately lead to a decrease in the genetic diversity of populations. However, the results showed that 17 Ae. albopictus populations experienced genetic exchange, and there was no apparent loss of genetic diversity. This may have been due to the influence of the hosts, various vehicles and potential mates that different populations of Ae. albopictus may encounter.ed 500 m . Studiesed 500 m . Howevers et al. . Consistuangzhou , our fineviation . The varAedes albopictus worldwide have been found to be infected with either Wolbachia Type A or B bacterial symbionts (Ae. albopictus to be infected with different types of Wolbachia, causing them to produce few or no offspring, resulting in abnormal free mating between different populations and inbreeding within each population (Wolbachia. Wolbachia A and B sequencing results showed differences in Wolbachia infection status within each population. The genetic differentiation between populations is not obvious because Ae. albopictus individuals that are either infected with the same type of Wolbachia or not infected with Wolbachia can mate freely.Most populations of ymbionts . CI can pulation . Except Aedes albopictus population, however, the active human-aided mosquito dispersion helps recover the Ae. albopictus population in a short period of time. Therefore, the continuous dynamic monitoring of Ae. albopictus population is important and will provide a solid foundation for the scientific prevention and control of Ae. albopictus.The present mosquito control strategy helps reduce the abundance and diversity of


The factors that determine the growth and spread advantages of an alien plant during the invasion process remain open to debate. The genetic diversity and differentiation of an invasive plant population might be closely related to its growth adaptation and spread in the introduced range. However, little is known about whether phenotypic and genetic variation in invasive plant populations covary during the invasion process along invaded geographic distances.Erigeron annuus at different geographical distances from the first recorded point of introduction (FRPI) in China. We also measured growth traits in the wild and common garden experiments, and the coefficient of variation (CV) of populations in the common garden experiments.In a wild experiment, we examined the genetic variation in populations of the aggressively invasive species E. annuus populations had better growth performance  and genetic diversity, and less trait variation, in the long-term introduced region (east) than in the short-term introduced region (west). Furthermore, population growth performance was significantly positively or negatively correlated with genetic diversity or genetic variation. Our results indicate that there was parallel genetic and phenotypic differentiation along the invaded geographic distance in response to adaptation and spread, and populations that entered introduced regions earlier had consistently high genetic diversity and high growth dominance. Growth and reproduction traits can be used as reliable predictors of the adaptation and genetic variation of invasive plants.We found that  Mimulus guttatus (in Europe) and Tamarix ramosissima and Helianthus tuberosus (North America) (Alien plant invasions are considered to be a major threat to native biodiversity and ecosystems worldwide . The facAmerica) . EvolutiAmerica) . HoweverRegional expansion of invasive plant populations along geographical distance gradients provides opportunities for genetic variance and the potential for evolutionary adaptation of populations to strong selection . The proPrevious studies showed that the genetic diversity of invasive plants was very different between populations with different geographic distances , and migInvasive plants respond to evolving adaptations not only through phenotypic changes in parents but also in offspring. The transgenerational effects allow organisms to conserve long-term environmental adaptation between generations and enhance offspring performance . By remoErigeron annuus L. (Asteraceae) is an annual or biennial plant. It is an apomictic plant, producing large numbers of minute seeds that are genetically identical to the mother plant. Thus, it can maintain the dominant performance of the mother plant for a long time, and its genetic diversity is reduced within populations; however, most populations contain several dominant genotypes, suggesting that sexual reproduction does occur occasionally  in China. We also measured the growth traits in the wild and in a common garden experiment, and analyzed the coefficient of variation (CV) of growth traits in nine geographical populations. Furthermore, we examined both the genetic and phenotypic differentiation and their relationships with the distance to the FRPI. We specifically addressed the following questions: (1) Do growth traits and their CV differ among populations at different geographical distances in wild or common garden conditions, and respond to the geographical distances from the FRPI? (2) Do genetic diversity and variation decrease and differentiation increase in populations with distance from the FRPI? (3) Do phenotypic and genetic variation of populations covary in response to adaptation and spread, and can phenotypic traits be used as significant predictors of adaptation and genetic variation in invasive plants?In a wild experiment, we examined the genetic variation of E. annuus are invasive and widely distributed in anthropogenically disturbed habitats, including roadsides, grasslands, and farmlands , as we were interested in testing for variations between different geographic distances and invasion times. Therefore, the nine selected geographic populations had different distances (from 30 km to 1425 km) from Shanghai (FRPI), which were located in different cities: Jiaxing , Hangzhou  and Wenzhou  in the eastern region, Xianning , Wuhan , and Xiangyang  in the central region, and Chengkou , Nanchuan , and the main districts of Chongqing  in the western region  were selected, and the heights were measured during the flowering period. Then, we collected two to three fully developed and undamaged leaves from each plant in 50 ml tubes placed on ice for DNA extraction. The sampled leaves were air-dried and used for leaf biomass calculation of each sampled mother plant. Then, whole mother plants were harvested and brought to the laboratory, separated into envelopes, on the basis of whether they were root, stem, leaf, branch, and flower, and dried at 80°C for 72 h in an oven. The total biomass of the plants was the sum of the biomasses of the five parts. To compare the complex genetic difference and maternal effect in offspring between different We also selected five other typical mother plants and sampled fresh leaves for DNA extraction. Then, we collected seeds from the top inflorescence of the plants at the mature seed stage. Each mature seed sample was taken directly from each mother plant and then brought back to the laboratory for preservation. All seeds were cleaned, air-dried, and stored at 4°C. These seeds were used in the common garden experiment. There were 360 samples in total  for DNA extraction and microsatellite analyses.For each collected leaf sample, total genomic DNA was extracted from 50-100 mg of leaf material using either the CTAB method  or a DNeE. annuus were screened for transferability and polymorphism in E. annuus. Three of these were found to be sufficiently polymorphic Dendranthema morifolium (Asteraceae) and Lactuca sativa (Asteraceae)). The markers were able to amplify corresponding DNA in the other two related species. Similar high cross-amplification was also observed in EST-SSR markers developed from Solidago virgaurea (Asteraceae), which were successfully transferred to the invasive species Solidago canadensis (Asteraceae) and Solidago hispida (Asteraceae) (Expressed sequence tag-microsatellite sequence (EST-SSR) markers developed for eraceae) . PCRs weeraceae) .G/N), where G is the number of genets (distinct genotypes) and N is the total number of individuals sampled; for each population, the number of alleles, the percentage of polymorphic loci (PPL), Nei’s gene diversity (Nei), observed (Ho), and expected (He) heterozygosities were calculated using GenALEx 6.5.1 (FIS) and allelic richness (Rs) were calculated using FSTAT 2.9.3 (FST) was determined using Arlequin version 3.0, with significance tests based on 1,000 permutations , and estimates of null alleles with Micro-Checker . ParametEx 6.5.1 . The inbAT 2.9.3 . All indAT 2.9.3 . An anals AMOVA, .In the common garden experiment, we used the collected seeds of mother plants from the same populations in the wild to test the real growth and phenotypic differentiation among geographic populations and the response to genetic differentiation. For each of the nine geographic populations, we used the seeds from five collected mother plants within each of four populations. The experiment was carried out in a greenhouse at Huazhong Agricultural University, Wuhan, China . Hubei has a subtropical monsoon climate, the average annual temperature is 16.4°C, and the average annual precipitation is approximately 1269 mm. From 17 December 2012 to 13 January 2013, we sowed the collected seeds in plastic trays (19.5 cm × 14.6 cm × 6.5 cm) filled with peat moss as substrate . Because the time required for germination and the germination rate varied between the different populations, we sowed them on different dates to ensure that there were enough seedlings and that all the seedlings were in similar developmental stages at the start of the experiment. We placed all the trays with seeds in a greenhouse under natural light conditions, with a temperature between 20 and 26 °C.-1, a total P of 0.58± 0.04 g kg-1, and a total K of 24.27 ± 1.15 g kg-1 . We transplanted one seedling of E. annuus in the center of each pot. After transplanting, we randomly assigned all pots in the greenhouse under natural light conditions, with a temperature between 20 and 28°C. We rerandomized the positions of all the pots every 4 weeks. There was a total of 180 pots (9 geographic populations × 4 populations × 5 mother plants = 180 pots).On 25 February 2013, we transplanted similar-sized seedlings of each population into 5 L square plastic pots (24 cm × 24 cm × 15 cm) filled with field soil. The field soil included a total N of 0.78 ± 0.03 g kgOn 12 June 2014 , we first calculated the plant height and then harvested the aboveground and underground biomass of the plants in all pots. Then, they were brought to the laboratory, separated into envelopes according to root (underground), stem, leaf, branch, and flower tissue, and dried at 80°C for 72 h in an oven. The total biomass of plants is the sum of aboveground biomass and underground biomass. The CV for each phenotypic trait was calculated.E. annuus populations using a linear mixed-effects model with the ‘lme’ function in the ‘nlme’ package  of geographic populations in the wild and their genetic diversity. We used ANCOVA to compare the difference between slopes of the linear regression between the growth of the wild experiment or of the common garden experiment and geographic distance to the FRPI.E. annuus populations in both the wild and common garden experiments exhibited great differences between the three regions in China .The plant height and biomass of the in China Table 2.In the common garden experiment, the CVs of height and total biomass were lowest in the eastern region, showing great stability in growth traits in the long-term introduced populations Figure 2PPL), Nei’s gene diversity (Nei), the proportion of distinguishable genotypes (G/N), observed heterozygosity (Ho), expected heterozygosity (He), and allelic richness (Rs) were higher in the eastern region and lowest in the western region )  and inbreeding coefficient (FIS) were significantly positively related to geographic distance to the FRPI  and genetic diversity, and less variation of traits and genetic differentiation in the long-term introduced region  than in the short-term introduced (west) region in China. Furthermore, within populations, genetic diversity showed a similar pattern to growth performance, which indicated significantly positive or negative correlations between growth performance and genetic diversity or genetic differentiation, especially for common garden experiments. These results indicate that growth traits and genetic variation of populations covary in response to regional adaptation and spread, and populations that entered earlier have parallel high genetic diversity and high growth dominance in introduced environments.We found that E. annuus populations within wild and common garden conditions, growth traits were significantly more varied, whereas their CVs were significantly lower in the eastern region than in the central and western regions, indicating a decrease in growth and an increase in phenotypic differentiation with invasion time. The E. annuus populations had a better growth advantage in the long-term introduced region. This is consistent with previous findings that populations of some invasive plant species in the earlier introduced ranges grew taller and produced larger biomass than those in the later ranges  in the eastern region, and only the scatter ecotype in the western region, in both the wild and common garden experiments. The clump ecotype had more branches and total biomass, which may be an adaptive advantage to interferences in farmland habitats  through risk sharing between multiple branches . This is supported by experimental evidence showing that dominant ecotypes of Imperata cylindrica can be maintained in the early stages of invasion and can invade a wider area with more dominant traits (common ecotype [C-type] and early flowering ecotype [E-type] are found scattered in the Japanese Islands)  introduced regions in wild and common garden conditions indicated largely genetic effects. This result is in line with the variable performance of many invasive plants  in the regions they were introduced, indicating that adaptive evolution of invasive plants occurs in the new region  . Similarve range . In our w region .E. annuus significantly decreased with geographic distance, with the same pattern as growth, i.e., highest in the long-term introduced region and lowest in the short-term introduction region. The rapid genetic decline of E. annuus with geographic distance to the FRPI is consistent with previous studies showing that alien species may decrease their genetic diversity during the invasion process and maintain population genetics depending on changes in selection, genetic drift, and gene flow  of the mother plant and more successful spread, but reducing genetic diversity. Thus, E. annuus can maintain the dominant performance of the mother plant in the offspring. Therefore, eastern geographic populations had more dominant genotypes, i.e., clump ecotype, which have been well preserved for a long time  can be found in the article/Y-JW set up the experiment. Y-JW, X-PS and ZL conducted field sampling. X-PS and Y-YL conducted molecular analysis. Q-FY and Y-YL carry out the statistical analysis. Y-YL. wrote the first draft of the manuscript. Y-JW, Z-XZ, X-PS and Y-YL contributed substantially to the revisions. All authors contributed to the article and approved the submitted version.


Pleuronectes platessa)—in which putative structural variants  have been identified—has successfully colonized the brackish water ecosystem of the Baltic Sea. Thus, the species represents an ideal opportunity to investigate how the interplay of gene flow, structural variants, natural selection, past demographic history, and gene flow impacts on population (sub)structuring in marine systems. Here, we report on the generation of an annotated draft plaice genome assembly in combination with population sequencing data—following the salinity gradient from the Baltic Sea into the North Sea together with samples from Icelandic waters—to illuminate genome‐wide patterns of divergence. Neutral markers pointed at large‐scale panmixia across the European continental shelf associated with high gene flow and a common postglacial colonization history of shelf populations. However, based on genome‐wide outlier loci, we uncovered signatures of population substructuring among the European continental shelf populations, i.e., suggesting signs of ongoing selection. Genome‐wide selection analyses (xp‐EHH) and the identification of genes within genomic regions of recent selective sweeps—overlapping with the outlier loci—suggest that these represent the signs of divergent selection. Our findings provide support for genomic divergence driven by local adaptation in the face of high gene flow and elucidate the relative importance of demographic history versus adaptive divergence in shaping the contemporary population genetic structure of a marine teleost. The role of the putative inversion(s) in the substructuring—and potentially ongoing adaptation—was seemingly not substantial.Recent studies have uncovered patterns of genomic divergence in marine teleosts where panmixia due to high gene flow has been the general paradigm. These signatures of divergent selection are often impacted by structural variants, acting as “supergenes” facilitating local adaptation. The highly dispersing European plaice ( Marine teleosts are a textbook example of high gene flow species. However, a growing body of literature has identified locally adapted populations though the underlying mechanisms in shaping a population substructure in the face of high gene flow are still not fully understood in these species. We used whole‐genome sequencing information to illuminate the population genetic structure of a marine flatfish with highly dispersive pelagic stages sampled along a steep salinity gradient and to infer the demographic history of populations. While neutral markers revealed genetic homogeneity across large parts of the European continental shelf, genome‐wide distributed outlier loci pointed at a distinct population substructure even at small geographic scales. Our findings suggest genomic divergence facilitated by local adaptation and point to the relative importance of demographic history and adaptive variation in shaping the contemporary population structure. DNA was purified using HighPrep polymerase chain reaction (PCR) magnetic beads (MAGBIO). DNA integrity was assessed on a 1%‐agarose gel and DNA content was quantified using a Qubit 3.0 Fluorometer (Invitrogen). Long‐read libraries were prepared using the Pacific Biosciences 20 kb library preparation protocol and sequenced aiming at a coverage of 30× on a Pacific Biosciences RS II instrument. All library preparation and sequencing steps mentioned in this study were performed at the Norwegian Sequencing Centre (NSC), CEES, UiO, Norway. Short‐insert (550 bp) libraries were constructed using the TruSeq DNA PCR‐Free LT Library Preparation Kit (Illumina), and libraries were paired‐end sequenced with read lengths of 250 bp (Rapid Run Mode) aiming at a coverage of 120× on an Illumina HiSeq 2500 system.To assemble a reference genome of the European plaice, we combined data obtained from long‐and short‐read sequencing from a single female specimen of ea Table  since feea Table . High mosga v0.10.15  specific to Actinopterygii using BUSCO v3.0.1 , which combines multiple ab initio gene model predictions with evidence (transcripts or proteins) aligned to the genome and outputs consensus gene models. We used the Trinity assembly as transcript evidence. We further generated a second transcript evidence by mapping the RNA reads to the plaice reference genome with Hisat v2.1.0  set as seed species and a maximum intron length of 100 kb. We assessed the completeness of our transcriptome annotation with BUSCO v3.0.1  using VCFtools. Decay plots were created per population  by binning the distance between SNPs in increments of 1 kb and averaging the r2 values within each bin. SNPs with pairwise r2 values >.1 within sliding windows of 50 kb SNPs were omitted with Plink (hereafter “pruned datasets”).To investigate the population structure of European plaice, variants within spanning deletions were removed using BCFtools v1.1 Li, . SNPs wik ranging from 2 to 10, where the most likely number of genetic ancestries was defined based on the lowest cross‐validation error. Pairwise fixation indices (FST) were calculated using the package Stampp v1.5.1  were calculated pairwisely on the pruned dataset using VCFtools and loci were screened for highly differentiated SNPs . Based on this set of high FST SNPs we explored the genetic structure by conducting a PCA analysis using Eigensoft.Locus‐specific To detect genomic regions under recent selection and potential candidate genes involved in driving the heterogeneity or local adaptation between the genetic clusters, we conducted genome scans between the Icelandic (ICE) versus all the continental shelf (SHELF) samples and between the North Sea (NS) versus the Baltic Sea (BS) samples  using the R package rehh v3.0.0  and nucleotide diversity (π) between individuals representing the noninverted (HOM_R) and the inverted (HOM_I) haplotypes and linkage decay for individuals from the two haplogroups and from heterozygote individuals (HET) along scaffold_302 and scaffold_159 were calculated as described in the previous sections.For two representative scaffolds mapping to the putative structural variant on chromosome 19, individuals were genotyped and assigned to the respective haplogroup based on the PCA clusters  separately for ICE and BS using the multiple sequentially Markovian coalescent (MSMC) method as implemented in the software msmc v2.0.0  with variant sites filtered for a mean depth of 7×. Scaffolds shorter than 500 kb in length were excluded from the analysis. Variant sites falling within repeat regions were masked to include only sites at which the mapping is of high quality.We inferred past effective population sizes  to cover the known spectrum of mutation rates in marine teleosts , trimmed, and aligned against the plaice reference genome with an average alignment rate of 98% (± SD 2%) with 92% (± SD 3%) of the mapped reads properly paired Table . Variant3.3In general, we detected a rapidly decreasing linkage decay when analyzing the 20 longest plaice scaffolds for each population  Figure  with LD The population differentiation among all samples of European plaice was investigated  error at k = 2, at which all individuals from Iceland were distinguished from the remaining individuals  and the European continental shelf (SHELF) and from the North Sea (NS) and the Baltic Sea (BS), respectively. Within a distance of 50 kb surrounding the focal SNP in both directions, 167 and 116 annotated genes were identified, respectively Table .Genomic regions of divergence were further investigated by identifying signatures of recent selective sweeps. We calculated the cross‐population extended haplotype homozygosity (xp‐EHH) statistic, which compares the integrated extended haplotype homozygosity profiles between two populations at the same locus and points to alleles that have swept to near‐fixation within one of the two populations  and demographic trends were inferred using MSMC2 analyses for three different mutation rates and a fixed generation time of 3 years . Replicate runs with three randomly chosen individuals yielded consistent Ne estimates for the Icelandic and the inner Baltic Sea past history. With an assumed mutation rate of 1.5 × 10−8 mutations/site/generation, Ne fluctuated between ~300,000 and ~100,000 for the ancestral Baltic Sea and between ~400,000 and ~50,000 for the Icelandic plaice, respectively.Estimates of historical population size  across large parts of its geographical distribution, we uncovered a population substructure that was linked to genomic signatures of local adaptation despite high gene flow within the European continental shelf. This population substructure at smaller geographical scales was only revealed with SNPs potentially under strong selection, i.e., defined as high 4.1We obtained a plaice draft genome assembly of high completeness, covering 99% of the k‐mer‐based estimated genome size. The size of plaice genome assembly (583 Mb) falls within the expected range of flatfish genomes  was detected  based on the high Throughout its distribution range, plaice experiences diverse physical environments and variable abiotic conditions, such as salinity, temperature, oxygen content, and tidal currents (in the North Sea), which can ultimately lead to adaptive genomic divergence between locations. Apart from single highly differentiated loci, several genomic regions were strongly differentiated among specimens from the North Sea  and the Baltic Sea . We detected loci that stood out above the 99.95th percentile in the global comparisons considering either AFD or xp‐EHH Figure  indicatiGasterosteus aculeatus; Shimada et al., On scaffold_107 a nonsynonymous substitution was located in the estrogen receptor ESR2 Figure , which iOur approach to identify outlier loci was rather conservative, i.e., based on the empirical distribution of allele frequencies and haplotype statistics, but a commonly used approach to detect loci that are divergent among populations  have already split more than 200,000 years ago during the mid‐Pleistocene, hinting at two glacial refugia, which has also been suggested by Le Moan et al. ; visualization (lead); writing – original draft (lead); writing – review and editing (lead). Sissel Jentoft: Conceptualization (supporting); methodology (supporting); resources (supporting); supervision (supporting); writing – original draft (supporting). Ole K. Tørresen: Formal analysis (supporting); methodology (supporting); resources (supporting); writing – original draft (supporting). Franziska M. Schade: Methodology (supporting); resources (supporting); writing – original draft (supporting). Christophe Pampoulie: Resources (supporting); writing – original draft (supporting). Uwe Krumme: Conceptualization ; funding acquisition ; methodology (supporting); project administration ; resources (supporting); writing – original draft (supporting). Reinhold Hanel: Conceptualization ; funding acquisition ; project administration ; supervision (lead); writing – original draft (supporting).Appendix S1Click here for additional data file.Table S1–S11Click here for additional data file.