NIS). The extent to which these anthropogenic habitats are sustainable sources of propagules and influence the evolution of NIS in natural habitats was examined in the edible seaweed Undaria pinnatifida, native to Asia and introduced to Europe in the 1970s. Following its deliberate introduction 40 years ago along the French coast of the English Channel, this kelp is now found in three contrasting habitat types: farms, marinas and natural rocky reefs. In the light of the continuous spread of this NIS, it is imperative to better understand the processes behind its sustainable establishment in the wild. In addition, developing effective management plans to curtail the spread of U. pinnatifida requires determining how the three types of populations interact with one another. In addition to an analysis using microsatellite markers, we developed, for the first time in a kelp, a ddRAD‐sequencing technique to genotype 738 individuals sampled in 11 rocky reefs, 12 marinas, and two farms located along ca. 1,000 km of coastline. As expected, the RAD‐seq panel showed more power than the microsatellite panel for identifying fine‐grained patterns. However, both panels demonstrated habitat‐specific properties of the study populations. In particular, farms displayed very low genetic diversity and no inbreeding conversely to populations in marinas and natural rocky reefs. In addition, strong, but chaotic regional genetic structure, was revealed, consistent with human‐mediated dispersal . We also uncovered a tight relationship between populations in rocky reefs and those in nearby marinas, but not with nearby farms, suggesting spillover from marinas into the wild. At last, a temporal survey spanning 20 generations showed that wild populations are now self‐sustaining, albeit there was no evidence for local adaptation to any of the three habitats. These findings highlight that limiting the spread of U. pinnatifida requires efficient management policies that also target marinas.Ports and farms are well‐known primary introduction hot spots for marine non‐indigenous species ( Human‐mediated dispersal has enabled this brown alga to become an extremely successful worldwide invasive species: over the past 40–50 years, this species, native to the NW Pacific, has become well established along the coastlines of North and South America, Australia, New Zealand, and Europe  might be possible through drifting mature thalli . A recent field‐based survey carried out in the English Channel showed a relationship between the distance to marinas and presence/abundance of this kelp into the wild , still relies on the dense populations found in the nearby marinas or farms. This knowledge is needed to address concerns related to possible intensification of this kelp's cultivation in Europe, as well as to define targets for management strategies to limit its spread  human‐mediated dispersal, particularly through leisure boating, plays a prominent role in connectivity patterns at a regional scale (the lack of relationship between the genetic and geographic distances would support this hypothesis); (b) the sustainable establishment of wild populations depends on immigrants from anthropogenic habitats (marinas or farms), the alternative hypothesis being that wild populations are now self‐sustaining; and (c) considering the long time elapsed since the introduction (80 generations in the study range), signs of local adaptation, either contrasting northern and southern populations  or habitats, should be observed, except if counterbalanced by high inter‐habitat or inter‐province gene flow.Considering the short life cycle, the likely limited dispersal distance by spores and gametes, and the high population density of U. pinnatifida  sequencing technique  according to the manufacturer's instructions, but with a lysis step at room temperature instead of at 65°C to avoid extracting too many polysaccharides. DNA was eluted in two successive and separate steps with 100 μl elution buffer.From 25 to 148 mature sporophytes were sampled from each of 25 localities, comprising 11 natural rocky reef sites, 12 marinas, and two farms, between January and May 2015, to examine In addition, DNA obtained in previous studies  calling. Parameters were chosen to limit the risk of assembling non‐homologous loci, a necessary precaution in the absence of a reference genome. We, thus, used a minimal stack depth of five (‐m parameter), up to two mismatches within a locus at the sample level (‐M parameter), up to two mismatches when merging loci across the 757 sample data set (‐n parameter), and up to four mismatches when aligning secondary reads to primary stacks (‐N parameter). At last, using the ‐r filter of the Stacks populations programme, the only loci selected were those that occurred in at least 75% of the 757 samples. Quality of SNP calling was assessed by comparing genotypes across replicates, with genotype congruency averaging 99.5%. Further filtering steps were performed in R‐3.3.2. (R Development Core Team 2016) on the variant calling format (VCF) file exported from populations. These included the following: (a) keeping only those loci with a maximum of two SNPs per locus; (b) randomly selecting a single SNP per locus; (c) removing three individuals with very low number of loci (missing data exceeding 95%); and (d) discarding loci with global minor allele frequency (MAF) below 0.01 or within‐population MAF below 0.1 using the filter_maf function of the stackr R package  and by the barcode with the 2.3® v. 4.0 (Applied Biosystems). All the analyses described below for the RAD‐seq panel were also performed on the microsatellite panel unless specified otherwise.A total of 1,111 individuals originating from the same 36 temporal or spatial samples genotyped with the RAD‐seq markers were genotyped with 10 microsatellite loci: Up‐AC‐1B2, Up‐AC‐1B5, Up‐AC‐1C1, Up‐AC‐1G2, Up‐AC‐1H5, Up‐AC‐2C1, Und_2E8, Up‐AC‐4G2, Up‐AC‐4C12, Up‐AC‐4E9  for the microsatellite panel.For each of the 36 temporal or spatial samples, the expected heterozygosity, Rousset,  R packags, was derived from g2, estimated in inbreedR . Selfing rate was estimated from g2 following equation 9 in David et al.  values from 2 to 24. The analyses were then run with the logistic prior for the range of K plus one that was specified by the chooseK.py script. As U. pinnatifida is a partial selfer  was performed using three different methods. First, we used discriminant analysis of principal components (DAPC), which is implemented in lfer see , may perFrançois . Besidest et al. . Using sFrancis, .K, 10 independent runs of K = 1–25 were performed with 500,000 Markov chain Monte Carlo (MCMC) iterations following a 100,000 burn‐in period. No prior information specifying the definition of populations was entered into the model, which was run assuming correlated allele frequencies and admixture. Other parameters were left at default levels. The optimal value of K was chosen following the approach of Evanno, Regnaut, and Goudet  in Arlequin 3.5.2.2   estimate for each pair of samples  model was tested on 22 natural habitat and marina localities, excluding M11‐15. Pairwise Rousset, . Geograp2.6FST was estimated amongst the three temporal samples for each of the five localities. Significance was tested via 10,000 permutations. We then ran fastSTRUCTURE and snmf (RAD‐seq panel), as well as STRUCTURE and INSTRUCT (microsatellite panel), analyses with the 12 samples from the Bay of St. Malo (no. 12), using the same parameters as described above but testing for K between 1 and 12. In this bay, sampling was performed in 2005, 2009 and 2015 at all four study localities, which allows for fine‐scale investigation of spatial versus temporal drivers of U. pinnatifida genetic structure.To investigate the importance of time versus habitat type in determining the overall genetic structure, we performed AMOVA on the five localities collected in 2005, 2009 and in 2015, and on M6 (sampled in 2005 and 2015). The samples were grouped by year within each habitat type. These analyses were performed in Arlequin using same parameters as detailed above. In addition, 2.7With the aim of detecting outliers specific to marinas or natural habitats, we used a sample set (sample set 1) of 460 individuals originating from the 22 natural habitat and marina samples (excluding M11‐15) collected in 2015. Then, to also examine farms, we analysed a second sample set (sample set 2) of 186 individuals collected in 2015 from eight localities in the Bays of Morlaix and St. Malo with long‐standing farming activities  and a locus‐specific component , implemented in BayeScan 2.1  for sample sets 1 and 250 for sample set 2. Three of the five approaches described above, relevant for microsatellites, were used: BayeScan 2.1 with default parameters and Arlequin with the default and hierarchical island models.33.1The sequencing run yielded 1,743,297,805 reads, of which 9.91% were dropped due to ambiguous barcodes. Of the 1,570,485,667 remaining reads, there were on average 2,074,618 reads per sample . The number of reads did not differ substantially between samples from different years . The Stacks catalogue was built from 1,105,640,102 reads remaining after filtering steps, with an average depth across all samples of 22.89 reads per locus. A total of 35,309 loci were present in at least 75% of the 757 individuals, of which 17,137 were polymorphic. Keeping only one randomly selected SNP per locus resulted in 14,622 SNPs. Following MAF selection, a total of 10,615 SNPs were used in subsequent analyses.3.2He ranged from 0.037 (in Rb8‐15) to 0.151 (in M12‐15) , 151 in M1‐15 . Therefore, below, we detail results obtained with the RAD‐seq panel only, except when microsatellites showed different results.Microsatellite and RAD‐seq panels provided similar results. For example, the pairwise FST estimate computed over the four 2015 Bay of St. Malo localities was 0.166 , whereas the global FST for the 2015 samples was 0.313 . Pairwise FST comparisons amongst the 2015 samples revealed only three nonsignificant estimates  than were the marina samples . The two farms displayed slight, but significant (RAD‐seq panel only) pairwise genetic differentiation with each other and were highly genetically differentiated from all the other samples except for Ra8‐15 . An AMOVA carried out on samples collected in 2015 with localities grouped according to habitat showed a low but significant difference amongst the three habitat types , natural reefs only  or marinas only .No IBD pattern was detected when analysing either the samples collected in natural reefs and marinas  and marinas   clearly confirmed this conclusion Figure b. A patt3.5q‐value of 0.05. A single locus was detected as an outlier by all four remaining approaches  revealed that these loci were specific to a bay rather than to a particular habitat. For OutFLANK, a conservative approach, we examined the 64 loci detected at a q‐value of 0.056: 40 loci behaved as outliers within a single locality—Men Guen  were identified as outliers across all methods . In an interesting manner, the remaining locus was fixed for an alternative allele only in the two study farms. Nine other loci, identified as outliers with two methods, also discriminated the two farms. For the microsatellite panel, one locus was identified as an outlier by two of the three methods but without any particular pattern.With sample set 2, which was used to compare the three habitats , 294 (3.89% of the investigated loci) outliers were found across all methods. None of the BayeScan‐identified outliers were detected by any other method  and the nearby natural reefs to which the species had spread, and determine whether some location adaptation had occurred. Our results revealed: (a) contrasting genetic properties linked to habitat; (b) absence of isolation by distance; (c) stability of the genetic composition of populations in natural habitat over ca. 20 generations; and (d) no specific signatures of adaptation to habitat type. These main findings are discussed below in the light of our initial hypotheses, which were only partially confirmed.Despite the acute need for improved understanding of the evolutionary dynamics of marine invasions, notably for setting up more effective management and for curtailing the invasion rates, few studies have examined population genomics of NIS, and most have focused on issues related to hybridization between lineages  to a level similar to that found in 2015 in many marina or natural reefs , and microscopic gametophytes are likely to be transported by various means  via boating activities  scale, t Figure a and thet Figure A, there t Figure , but clet Figure . Driftint Figure , and, tht Figure . However4.3U. pinnatifida came from a combined field and microsatellite study carried out at a local scale . The marina and rocky reef populations sampled in the same bay were frequently genetically very similar: they were assigned to the same genetic cluster(s) by fastSTRUCTURE  or life cycle  in bay no. 8 . However, the other natural population studied in this bay (Rb8) did not show such high similarity with the farm. Overall, there was no evidence that cultivated populations are contributing  to the immigrant pool reaching rocky reefs or marinas. It is assumed that at the very beginning of 4.4FST and fastSTRUCTURE analyses carried out on samples collected in 2005, 2009 and 2015 . This self‐sustaining scenario has been confirmed in a microsatellite‐based genetic survey of the colonization dynamics of a newly built marina . With limited dispersal ability, local recruitment is essential for long‐term maintenance of the population as commonly reported in native kelps  our coarse‐grained classification of the populations. These hypotheses are not mutually exclusive. The traits that may confer an advantage in the different study environments are likely determined by many genes . In addition, NIS are known to display substantial levels of phenotypic plasticity  and the Men Guen population (Rb8) sampled in bay no. 8 (Morlaix). For bay no. 4, this pattern could be due to a singular history of introduction, because a new large farm was recently established in this area. The singularity of bay no. 4 in the neutral RAD‐seq panel supports this hypothesis. Regarding sample Rb8, the hypothesis of a specific environmental feature is interesting to consider: this locality, where the species was reported for the first time 10 years ago, is exposed to strong waves, which is a rare habitat for U. pinnatifida. Further dedicated studies are needed, to characterize the fine‐grained environmental conditions  using local autonomous data loggers in the areas colonized by U. pinnatifida and then examine genomic‐environmental associations, as recently done in the invasive crab Carcinus maenas  the methodology itself, as genome scans have limited efficiency, except if few genes with strong effects are involved; (b) insufficient lapse of time since interhabitat gene flow ceased; (c) the persistence of a et al. , and sho4.5U. pinnatifida at a regional scale. Whereas farms were the primary source of the introduction of U. pinnatifida in the study area, our results clearly support the hypothesis that present‐day spillover effects from farms are negligible, compared with from marinas. We also showed that after colonization, populations eventually become self‐sustaining. Although we did not capture signatures of local adaptation after only 20–40 generations following the foundation of wild populations, we anticipate that adaptation can nevertheless occur if the populations remain self‐sustaining and protected from gene flow counterbalancing local adaptation to their local natural environment. Under the current situation in which farming activities are maintained at low level, our results demonstrate the need for management targeting marinas to reduce the risk of spread of U. pinnatifida into the natural environment. This is especially important for newly built marinas, or marinas not yet colonized by U. pinnatifida, particularly those that occur in areas where the surrounding rocky reefs are still free of this kelp. Tight controls on the (bio)fouling of the floating pontoons and of hulls of boats docking there should be established. Such management measures would be helpful to prevent spread beyond the study species as spillover from marinas might occur for other NIS of the fouling community.The results of the present study were based on over ca. 10,000 SNPs derived from ddRAD‐sequencing. As expected (Gagnaire et al., None declaredhttps://doi.org/10.5061/dryad.cd4q0ds.ddRAD‐seq and microsatellite data set are available as a VCF file and a multilocus genotype file uploaded on Dryad Digital Repository at  Click here for additional data file.


Bullacta exarata , a species endemic to the Northwestern Pacific. We sampled 14 natural populations spanning across 3800 km of the Chinese coastline, essentially covering most of the species distribution range. COI analysis identified a total of 149 haplotypes separated into two distinct groups with nine mutation steps, revealing a prominent phylogeographic structure. Nuclear microsatellite data also demonstrated a similar but weaker genetic structure. The estimated time to the most recent common ancestor between the two COI haplogroups is at ∼0.89 Ma, indicating that B. exarata populations survived the Pleistocene glaciation in the Sea of Japan and the Okinawa Trough, two marginal seas around the species range. The consistent significant patterns of isolation by distance of both COI and microsatellites suggests that limited mobility of adults and short planktonic stage of larvae may have played an important role in promoting or maintaining the genetic differentiation of B. exarata. Results from population demographic analyses support population expansion late in the Pleistocene era.Many phylogeographic studies on marine organisms in the Northwestern Pacific have supported for the biogeographic hypotheses that isolation in the marginal seas of this region during the Pleistocene glaciation lower sea level led to population genetic divergence, and thus population expansion was a common phenomenon when the sea level rebounded. However, most of these studies were based on maternally inherited mitochondrial DNA markers with limited sample sites and therefore, were unable to reveal detailed pictures encompassing paternal line information covering of the entire range. In this study, we used the mitochondrial cytochrome c oxidase subunit I (COI) and nine nuclear microsatellite loci to investigate the phylogeography of the mud snail,  Sargassum provide evidence of allopatric divergence and expansion closely associated with the sea level changes in the Northwestern Pacific during the Pleistocene era  belongs to the superfamily Haminoeoidea  and is an endemic species of the Northwestern Pacific, encompassing China from the East China Sea northwards, the Korean Peninsula, and the coast of Japan  whether the Pleistocene marginal sea refugia have left any genetic imprint on B. exarata, (2) whether the short planktonic larval stage have promoted the genetic structure of this species. By addressing these issues, we hope to gain a better understanding of the genetic diversity distribution and the evolutionary mechanisms of marine organisms along the Northwestern Pacific Ocean.The mud snail of Japan . B. exarng stage . The pelB. exarata . Bi-directional sequencing was conducted using the PCR primers and the BigDye® Terminator Cycle Sequencing Kit (v3.1) on an ABI PRISM® 3730 Genetic Analyzer .The COI gene was amplified using polymerase chain reaction (PCR) with primers HCO-2198 and LCO-1490 from B. exarata (TM-500LIZTM (Applied Biosystems) was used to determine the allele size. Fragment sizes were obtained on an Applied Biosystems 3730 and scored using GeneMapper version 3.7 (Applied Biosystems). About 30% samples were reamplified, and all samples were scored twice independently to check for genotyping consistency.Genotypes of DNA samples were scored using nine microsatellite loci developed for  exarata . PCR coc exarata . Loci ofNh), haplotype diversity (Hd), and nucleotide diversity (π) were calculated with DnaSP v.5.0  , and werSP v.5.0 .MRCAt) was estimated using BEAST v1.8.2 (−1) as estimated for the COI gene of two marine teguline snails separated by the Isthmus of Panama  with Arlequin 3.0 . The geographical distances between populations were represented by the coastline distance calculated using ArcGIS .Population genetic structure was detected with pairwise quin 3.0 . To testservice1 . The cort = τ/2 μk, where τ is calculated in the mismatch distribution analysis, μ is the mutation rate, and k is the length of sequence. Both mismatch analysis and neutrality test were performed in Arlequin 3.0. BSP . In the neutrality test, the values of 3.0. BSP  was geneHWE) at each locus and linkage disequilibrium between all pairs of loci were tested using MCMC approach implemented in GENEPOP version 4.0 (Na), the observed (Ho), and expected (He) heterozygosities and the value of pairwise STF between populations were calculated using Arlequin 3.0.Possible genotyping errors due to null allele, stuttering or large allele drop out within each locus were assessed with Micro-Checker version 2.2.3 . The depsion 4.0 . All mulsion 4.0 . GeneticK cluster of 2 ≤K ≤ 14 with 20 independent simulations running 200000 MCMC steps, with the initial 50000 steps as burn-in. The average permutated individual Q-matrices throughout the 20 simulations for the estimated number of clusters, i.e., K value, were obtained using greedy algorithm in CLUMPP v.1.1.2 ], resulting in the highest percentage of membership coefficient (q) to each cluster (K = mean[| L”(K)| ]/SD[L(K)], i.e., rate of change in the log probability of data between successive K values, was plotted against K and the optimal K identified by the largest change in log-likelihood [L(K)] values between K values , among which haplotype 49 (Hap49) was the most common and was mainly present in the southern populations from WH southward, while Hap5 was the second most common haplotype and was mainly existed in the northern populations from QD northward . None of the loci showed significant deviation from HWE, and no linkage equilibrium was found between any pairs of loci after Bonferroni corrections. Total Na, He, and Ho across 14 populations were 13.06, 0.8278, and 0.6551, respectively , a time falling into the Pleistocene era (2.58–0.01 Ma)  . The resK using microsatellite data set in the structure analysis, the best log-likelihood score approach “LnP(D)” was first applied, but no distinct plateau of the estimates of LnP(D) was observed , supporting a pattern of IBD in B. exarata (Figures Most of the pairwise differences between populations revealed by pairwise  Figures .Fu’s Fs and Tajima’s D (Supplementary Table The mismatch distribution analysis on the two COI haplogroup populations showed unimodal patterns, suggesting a history of expansion Figures . These rB. exarata. Consistent with many previous studies, our data also suggested that the species remained the imprint of divergence in the two marginal seas around their range. Population expansion and a significant pattern of IBD were also detected.In the present study, evidence from the mitochondrial COI gene and nine nuclear microsatellite loci was combined to reconstruct the phylogeographical history of B. exarata populations. Nuclear microsatellite data also demonstrated a similar but weaker genetic structure, indicating a stronger gene flow that might be mediated by paternal line. Nonetheless, the phylogeographic structure pattern implies a historical divergence, and two origins in the north and south, respectively. The estimated MRCAt for the two COI haplogroups falls into Pleistocene era, during which the global climate changed drastically with the alternation of glaciation and deglaciation periods. The dramatic climate change had profound impacts on species distribution and abundance and played an important role in reconstructing their genetic structures (B. exarata. In this study, the observed distribution pattern of the two haplogroup populations and estimated MRCAt for the two COI haplogroups imply that the ancestors of B. exarata were probably isolated and survived separately in the Sea of Japan and the Okinawa Trough, two marginal seas left during glaciation lower sea levels (The COI gene analysis unveiled a significant north-south differentiation in ructures , 2004. Iructures , Atlantiructures , as wellructures . During a levels . A simila levels , marine a levels , and mita levels  in this B. exarata populations is most likely attributable to the limited dispersal ability of 4–10 days of planktonic larval stage (Coelomactra antiquate showed highly significant genetic differentiation between populations from the Bohai Sea and East China Sea with a STF value of 0.958 based on the COI gene. This species has a pelagic larval phase of around 9 days, a time duration that might be too short for the ocean currents to transfer the larvae a long distance and mix populations of with different genetic backgrounds (Mytilus coruscus along the coast of the East China Sea based on the COI gene, with STF values from −0.036 to 0.097. This homogeneous phylogeographic structure could be explained by the same genetic background of the populations studied. The planktonic larvae stage of this species lasts up to 35 days, meaning that the ocean currents could promote the population mixture by facilitating the diffusion of planktonic larvae and generating the homogeneous phylogeographic structure of this species (On the other hand, the strong pattern of IBD in al stage . Limitedal stage . For exakgrounds . In cont species .B. exarata in the two refugia might have rapidly colonized the new habitat available and experienced expansion history. Postglacial range expansions might have brought formerly isolated haplogroups into secondary contact at suture zones (B. exarata after expansion. When the sea level rose, the survived ancestors of northern populations may have expanded from the Sea of Japan through the Tsushima Strait and occupied the coastline of the Korea Peninsula westward. It the same time period, the ancestors of southern populations may have expanded out of the Okinawa Trough and colonized the coastline of the East China Sea.All results obtained while investigating population demography support historical expansion of the two COI haplotype populations since the late Pleistocene era. The wide distributions of the two microsatellite groups could also explain the expansion from nuclear side. Interglaciation and post-glaciation expansion for marine species have been proven in many previous studies, and the expansion hypothesis is highly plausible in the Northwestern Pacific, which has many marginal seas . When thre zones . Our sture zones , as wellre zones , indicatB. exarata. The estimated MRCAt for the two COI haplogroup populations falls into the Pleistocene era, providing evidence that the ancestors of B. exarata might have been isolated and diverged in the Sea of Japan and the Okinawa Trough as refugia during Pleistocene glaciation lower sea levels. Expansion of the two genetic groups since the late Pleistocene era may have occurred via recolonization of the recovered habitat when the sea level rose in deglaciation periods. The present genetic structure could have been promoted and maintained by limited mobility of adults and a short planktonic larval stage of B. exarata.In our study, both the nuclear microsatellites and the mitochondrial COI gene revealed two genetic groups with clear geographic cline, as well as significant patterns of IBD in L-YS was responsible for the overall experiment and writing of this paper. JL and S-QW provided some help in sampling and experiment. JH provided financial support and worked on the experimental plan.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.


Siphonaria lessonii across its distribution, from northern Chile in the Pacific to Uruguay in the Atlantic. Contrary to our expectations, populations from the southwestern Atlantic, an area that was less impacted by ice during glacial maxima, showed low genetic diversity and evidence of recent expansion, similar to the patterns recorded in this study across heavily ice-impacted areas in the Pacific Magellan margin. We propose that Atlantic and Pacific shallow marine hard-substrate benthic species were both affected during the Quaternary in South America, but by different processes. At higher latitudes of the southeast Pacific, ice-scouring drastically affected S. lessonii populations compared to non-glaciated areas along the Chile-Peru province where the species was resilient. In the southwest Atlantic, S. lessonii populations would have been dramatically impacted by the reduction of near-shore rocky habitat availability as a consequence of glacio-eustatic movements. The increase of gravelly and rocky shore substrates in the southwest Atlantic supports a hypothesis of glacial refugia from where the species recolonized lower latitudes across the Atlantic and Pacific margins. Our results suggest that current patterns of genetic diversity and structure in near-shore marine benthic species do not solely depend on the impact of Quaternary glacial ice expansions but also on the availability of suitable habitats and life-history traits, including developmental mode, bathymetry and the likelihood of dispersal by rafting.The southern coastline of South America is a remarkable area to evaluate how Quaternary glacial processes impacted the demography of the near-shore marine biota. Here we present new phylogeographic analyses in the pulmonate  Under the Expansion-Contraction (E-C) model of Pleistocene biogeography, cold-temperate species contracted their distribution ranges towards lower latitude glacial refugia located in less ice-impacted or non-glaciated areas3. During interglacial periods they recolonized higher latitudes through range expansions following deglaciation processes6. This simple E-C model provides a straightforward paradigm to test population demographic hypotheses through the Quaternary, and phylogeographic studies have helped to understand better the response of species to major climate changes during this period by recognizing distribution range shifts, potential refugial areas and recolonization routes9.Quaternary ice sheet expansion and contraction during the Last Glacial Maximum (LGM), between 23,000 and 18,000 years before present, triggered major climate and environmental changes that strongly affected the distribution of the biota worldwide10. During the LGM, the Pacific margin of the Magellan province, from Chiloé Island (42°S) to Cape Horn (56°S), was almost completely covered by the Patagonian Ice Sheet12. Consequently, the availability of near-shore sheltered and hard-substrate habitats in the Pacific Magellan margin became very limited or even absent, and accordingly impeded the persistence of numerous near-shore marine species. Heavily ice-impacted areas were recolonized as the ice receded by dispersal of individuals from refugial populations. Repetitive rounds of local extinctions followed by recolonization may have enhanced the diversification of invertebrates across the region by the availability/diversity of vacant habitats and/or by survival in multiple refugia that may have led to allopatric divergence14.In southern South America, glacial periods produced major shifts in sea level, climate and landscapes, particularly in marine near-shore environments15. Accordingly, near-shore marine species in the Atlantic Magellan margin could have persisted in situ during the last glacial period, in contrast to their conspecific populations located in the Pacific.In contrast to the glacial history recorded along the Pacific Magellan margin, ice sheet expansion and contraction would have affected the marine ecosystems across the southwestern Atlantic to a much lesser extent17 and near-shore marine24 organisms. Phylogeographic studies in freshwater and terrestrial Magellan ecosystems have inferred the presence of Quaternary glacial refugia on the east side of the Andes25, as well as on the Pacific Magellan margin within26 and outside the glacier limits27. Phylogeographic studies in Magellan marine organisms have established that most of the taxa analyzed exhibited low levels of genetic diversity, absence of genetic structure and strong signals of recent demographic growth, supporting the hypothesis of postglacial expansions28. Nevertheless, although several near-shore marine species are broadly distributed across the whole Magellan province, very few phylogeographic studies have been conducted across the whole region29. In fact, most of the studies have been restricted either to the Pacific30 or to the Atlantic31 margin of the Magellan province. Therefore, a phylogeographic study conducted on a broadly distributed species would represent an important contribution to propose an integrated scenario of how coastal marine biota have responded to historical climate changes along temperate and cold areas of southern South America.During the last two decades phylogeographic and population-based studies have been carried out for different groups of Magellan freshwaterSiphonaria are distributed worldwide, except in the Arctic. In the last revision of the genus, Dayrat et al.32 recognized three distinct species in southern South America: Siphonaria lessonii Blainville, 1827, S. lateralis Gould, 1846, and the recently described S. fuegiensis33. Siphonaria lessonii is commonly found on intertidal rocky ecosystems and is widely distributed from southern Peru to Cape Horn in the Pacific and north to Uruguay in the Atlantic, as well as in the Malvinas/Falkland Islands34. Accordingly, the distribution of this pulmonate limpet extends across different ecoregions35 in temperate areas of the Chile-Peru and the Argentina provinces, as well as cold areas across the Magellan Province36. The species S. lessonii is a hermaphroditic broadcast-spawner with free-swimming veliger larvae that disperse for approximately 8 to 11 days in the water column37. Considering the geographic range and the reproductive biology of the species, S. lessonii is a suitable model to infer the impact of Quaternary glacial cycles over its distribution range, demographic inferences, recolonization pathways and its local persistence across a latitudinal gradient in areas that were differentially affected by the Quaternary glacial processes.Members of the pulmonate genus 24. However, the low number of samples from the Pacific margin did not allow them to locate the actual limit between the recorded genetic clades. More recently, new phylogeographic analyses in the species were combined with environmental niche modelling analyses along the southeast Pacific coast28. The results of this study are congruent with a historical scenario involving Quaternary demographic and distribution contractions of S. lessonii surviving in glacial refugia in the southern portion of the southeastern Pacific, followed by recolonization of the deglaciated areas. Nevertheless, the absence of information concerning the genetic diversity of the species along its Atlantic range may result in an incomplete or misinterpreted Quaternary demographic scenario. Therefore, a broader phylogeographic study of S. lessonii is required to better understand the historical biogeography of a species widely distributed across the southern Atlantic and Pacific coasts of South America.A first phylogeographic genetic approach based on a fragment of the mitochondrial DNA (mtDNA) COI gene recognized the presence of two genetic clades in the species, one on the Pacific margin of South America and one on the Atlantic side, which would have diverged between 100,000 and 1,000,000 years agoSiphonaria lessonii across different South American provinces. For this purpose we performed phylogeographic and population-based analyses using fragments of the mitochondrial cytochrome oxidase c subunit 1 (COI) and the nuclear internal transcribed spacers (ITS1 and ITS2). We hypothesize that Quaternary glacial processes have differentially affected S. lessonii, since habitat eradication would have strongly impacted populations along Pacific coasts. In contrast, southwestern Atlantic populations may have persisted through glacial cycles, as previously demonstrated in the Chile-Peru Province. Accordingly, populations from the Magallanes Province, and particularly those from the Pacific margin are expected to show lower levels of genetic diversity than temperate populations from the Chile-Peru and Argentina provinces, and should harbor a genetic signal of a recent demographic expansion process. Conversely, lower latitude populations located in non-glaciated areas of the southwestern Atlantic and the Chile-Peru province are expected to exhibit higher levels of genetic diversity and structure as a consequence of more stable demographic histories. Hence, we expect to find different genetic signatures between higher latitude Magellan populations and lower latitude temperate populations located across the Atlantic and Pacific South American margins.This study aims to evaluate the impacts of past glaciations on marine taxa using as a model species the pulmonate Siphonaria lessonii individuals were collected from 25 localities across the species’ distribution in three main South American biogeographic areas as defined by Trovant et al.29  were included in the study of Nuñez et al.25 No insertion, deletion, or stop codons were detected in the whole COI data set. The complete ITS data set included 45 new sequences, consisted of 1259 nucleotides and a total of six insertion/deletions were recorded. Mitochondrial and nuclear sequences were not saturated at any position and within the COI data set we recorded four amino acid substitutions . High levels of mtDNA genetic variability characterized S. lessonii with 115 polymorphic positions (20.35%), of which 66 (57.39%) were parsimoniously informative. Mitochondrial DNA sequences were A-T rich (63.5%) compared to the mean G-C content. Conversely, nuclear ITS sequences were G-C rich (59.3%) and more conserved than mtDNA sequences, with only 24 polymorphic positions (1.90%) of which 13 (54.1%) were parsimoniously informative.The complete COI data set of S. lessonii, with the presence of two main genetic groups. SAMOVA recovered two significantly differentiated groups (FCT = 0.64) explaining 64.48% of the total variance. In contrast, differences between localities within groups were significant (FSC = 0.03) but only explained 1.35% of the total variation (Table S. lessonii COI data set (Snn = 0.11) detected a low but significant phylogeographic signal (P < 0.001). However, when this analysis was performed considering the SAMOVA and GENELAND groupings (C1 vs. C2), Snn became very high (Snn = 0.87) and remained highly significant (P < 0.0001), demonstrating the high phylogeographic signal detected in the species across its distribution.SAMOVA and GENELAND analyses recovered similar patterns of phylogeographic structure in on Table . Values on Table . Again, on Table . The neaH) varied between 1.000  and 0.418 . The number of polymorphic sites (S) varied between 27  and 7 . Similarly, the number of haplotypes (k) varied between 30  and 6  (Table Π), and the mean nucleotide diversity (π) varied between 6.53/0.0115  and 0.6/0.001  (Table Levels of mtDNA genetic diversity were significantly higher in R1 populations across the Chile-Peru province (CP) compared to those recorded south of 42°S, across MP and AP within R2 (Table ST). Mean general values of differentiation were high, especially considering the mean NST = 0.314  showed low levels of genetic differentiation from Iquique (20°S) to Pucatrihue (40°S)  in C1 were broadly distributed across CP from Iquique (20°S) to Pucatrihue (40°S). Associated with these medium-frequency haplotypes we recorded many low- to single-frequency ones (n = 114). Conversely, the C2 parsimony network showed a typical star-like topology with a very short genealogy Fig. . A centreps Fig. . SimilarThe nuclear ITS maximum parsimony network included a total of 19 different haplotypes Fig. , with a Maximum likelihood gene flow analysis using MIGRATE with different migration models detected evidence of low levels of gene flow with signals of being asymmetric from the CP (C1) towards the MP + AP (C2). Among the tested models, the one from C1 to C2 received the highest probability Table . SimilarS. lessonii across its distribution in southern South America. Populations in the Chile-Peru province showed a more stable and older demographic history than those of the Magellan and Argentinian provinces, which showed evidence of a very recent expansion . A concordance between historic biogeographic and contemporary phylogeographic patterns supports that evolutionary forces determining species distributions are also associated with spatial patterns of populations genetic structure45. Here we corroborate previous molecular results in the species28, and enhance our understanding of the Quaternary biogeography of the species by recognizing two clearly discriminated genetic clusters currently found in: 1) temperate areas across the Chile-Peru province, north of the 42°S in the Pacific, and 2) cold and temperate areas across the Magellan and Argentina provinces, respectively. The boundary of these clusters (C1 and C2) agrees with the 42°S biogeographic break demonstrated in several marine organisms50. This break represents the southern limit for many Chile-Peru Province species and the northern border for numerous Magellan ones36. The shift in species composition recorded in marine organisms at 42°S in the southeast Pacific has been explained by changes in salinity and wave exposure51, topographical shifts of the coastline as a consequence of the increased number of fjords and channels47, differences in main oceanographic patterns50, and Quaternary glaciological histories43. In fact, this biogeographic limit coincides with the separation of the West Wind Drift into two main Pacific currents, the Humboldt Current System flowing north across the Central Chilean coast and the Cape Horn Current heading south along the Pacific Magellan margin46. Our results support that 42°S represents a main boundary in the biogeography of S. lessonii in southern South America, as recorded in other marine invertebrates including patellogastropods14, polychaetes49 and macroalgae48.How species and ecosystems responded to Quaternary glacial cycles can tell us about the evolution and biogeography of the biota during the last million years and provide useful information for understanding their responses to future climate change38. The hypothesis predicting that non-glaciated areas should exhibit higher levels of genetic diversity and complexity than those sites that were heavily impacted during Quaternary glacial cycles is well supported here for the southeast Pacific, where populations from the Chile-Peru Province (R1) showed higher levels of genetic diversity than those located south of the 42°S break (R2), across areas that were heavily impacted by ice during glacial maxima. Thus the main phylogeographic signatures in S. lessonii, together with patterns of genetic diversity, structure, connectivity, and demographic reconstructions provide clear evidence in favor or the E-C model of Quaternary biogeography3. Nevertheless, contrary to our expectations, populations along the Atlantic margin of the species distribution, an area considered less impacted by ice during glacial periods, did not exhibit higher genetic diversity than those located within heavily glaciated areas in the Pacific margin of the Magellan province. Populations along temperate areas of the Argentinean province also showed low levels of genetic diversity and represent a continuity of the diversity found along the Magellan province. Absence of genetic differentiation between areas that were differentially affected by ice expansion and contraction along the Pacific and Atlantic margins of Magellan was also recorded in the pulmonate Siphonaria lateralis21 and in the keystone patellogastropod Nacella magellanica23.Colonization toward former glacial areas involves a series of genetic bottlenecks, and accordingly recolonized areas should exhibit low genetic diversity dominated by few haplotypes and high frequency of sequences identical to or descended from the founding population52; a large portion of the Atlantic Continental Shelf (ACS) was exposed as a consequence of these glacio-eustatic movements, with the development of enormous plains10. A plausible explanation for the genetic pattern recorded in S. lessonii and in other hard-bottom invertebrates across the Atlantic portion of their distributions relies on the fact that during the LGM the coastline lay 200 to 400 km away and was mainly dominated by depositional environments. Accordingly, it is likely that suitable hard substrate habitats for these species would have been strongly reduced and dominated by abrasion platforms of friable sedimentary rocks53. During the LGM, sandy environments dominated over the entire ACS while gravels and rocks were more abundant in southern areas54. Gravel environments covered around 12.5% of the total ACS and were very abundant south of 46°S off the coasts of Santa Cruz and Tierra del Fuego in Argentina and in the Malvinas/Falkland Islands. Bedrock outcrops during the LGM covered 2% of the total ACS and also were more abundant in southern Atlantic areas of Magellan53. Accordingly, suitable refuges for S. lessonii would have been more available in the southern tip of the southwest Atlantic during the LGM, from where the species could have recolonized lower latitudes following the deglaciation process and the sea level increment, as proposed by Trovant et al.44 for the scorched mussel Perumytilus purpuratus. Thus current phylogeographic patterns in higher latitude near-shore marine invertebrates are not solely due to the impact of Quaternary glacial ice expansion and contraction but also to the availability of suitable habitats for the species.During the LGM the sea level was between 120 and 140 m below its present levelS. lessonii is the absence of genetic structure along the Chile-Peru province (R1), as well as along the Magellan-Argentina provinces (R2). Such results may be a consequence of gene flow mediated by larval dispersal through the main flows of the Humboldt Current System, heading north along the Chilean coast, as well as the Cape Horn Current, heading south across the Magellan province, as recorded in several cold and temperate near-shore marine organisms56. Strong demographic and geographic expansion following post-glacial sea level rise may also be a suitable explanation for the low levels of genetic diversity and the high degree of genetic homogeneity in S. lessonii across these provinces. This pattern of genetic structure has been found in several Magellan marine benthic near-shore organisms with dispersive potential including invertebrates55, vertebrates22 and macroalgae27. Finally, in contrast to recent phylogeographic analyses in sub-Antarctic invertebrates including Siphonaria species21, isopods57, and patellogastropods55, we found in S. lessonii a complete absence of genetic structure between continental Magellan and the Malvinas/Falkland Islands populations. The presence of a free-living dispersive stage in S. lessonii, absent in other sub-Antarctic Siphonaria species, seems to play a key role establishing genetic homogeneity across thousands of kilometers from Chiloé Island to Uruguay in the southwest Atlantic. Nevertheless, it is important to mention that the degree of resolution of the analyzed mitochondrial and nuclear markers does not allow us to distinguish whether the low divergence across these provinces are due to contemporary connectivity or is only a consequence of post-glacial demographic expansion.Another interesting result recorded in 16. Of these three, we recognize evolutionary variability around the coalescence as the main important source of uncertainty. Incomplete sampling of haplotypes is inevitable in any study of natural populations but the extensive geographical coverage we obtained for S. lessonii should ensure that most of the common haplotypes were included. Unsampled haplotypes are probably rare and terminal ones and should not likely affect the overall shape of the skyline plot or the isolation-with migration analyses. However, they may have some effect on the time of coalescence. Finally, the substitutional rate used in demographic inference analyses will also greatly determine the time estimation of population growth detected here. Nonetheless, even considering important confidence interval around the substitution rate used here, our estimations allow us to infer that divergence onset between clades and demographic events in S. lessonii were associated to Quaternary glacial processes and probably to the last glacial cycle. The paleogeographic evolution of the Magellan continental shelves (Pacific and Atlantic), together with genealogical and demographic reconstructions, as well as main patterns of genetic diversity/structure and gene flow analyses in S. lessonii allow us to propose a Quaternary biogeographic hypothesis for the species across its distribution in southern South America. At the end of the last interglacial period, the geographic distribution of S. lessonii was similar to that currently observed and extended from northern Chile to Uruguay. During glacial maxima, ice expansion over the Pacific Magellan coastline would have eradicated the species from this area, while populations in non-glaciated Pacific areas north of the 42°S break would have persisted along the Chile-Peru province. Along the Atlantic margin, a reduction of suitable habitats during glacial maxima would have led to local extinction of species associated with these hard bottom habitats, as recorded in other invertebrates during the Holocene such as the keystone Magellan gastropod Tegula atra59. As proposed by Aguirre et al.59 the absence of this important Magellan hard-bottom species along the Argentine Atlantic represents a climate-change driven shift associated with changes in Sea Surface Temperature (ca. 2 °C higher), wind velocities (less), light (less), nutrient availability (less), and intensity of cold (less) and warm (increased) shallow water currents that altered water masses and biogeographical boundaries in the region. Paleo-eustatic reconstructions in the southwest Atlantic Patagonia suggest that during glacial maxima a higher proportion of suitable habitats for S. lessonii at higher latitudes in the Atlantic Magellanic margin54 may have allowed the persistence of small populations that acted as glacial refugia located close the southern tip of South America  should be tempered by the consideration of potential effects of three different sources of uncertainty including (a) evolutionary variability around the coalescence; (b) inadequate samplings, which may have missed some haplotypes and led to underestimates of population ages and (c) uncertainty of the molecular clock of the mtDNA COIica Fig. . At the ica Fig.  until S.ion Fig. . Separation Fig. . Similarct Figs.  and S2.FS. lessonii along the Pacific margin of its distribution provide obvious evidence towards the E-C model of Quaternary biogeography proposed by Provan & Bennett3. In contrast, temperate area populations across the Argentina and Atlantic Patagonia showed very low levels of genetic diversity and structure and are clearly connected to the diversity recorded across the Pacific Magellan margin. Accordingly, the impact of Quaternary ice expansions over the demography of near-shore species does not solely explain current patterns of genetic diversity across the Atlantic Magellan margin and the Argentinean Province. Populations of S. lessonii across these areas that were less impacted by ice during glacial maxima share the diversity and structure that probably survived the last glacial maxima at higher latitudes refugia. Accordingly, ecological factors such as the availability of suitable habitats, bathymetry, developmental mode and the likelihood of dispersal through rafting are also key factors explaining main phylogeographical patterns in the species across its distribution in different South American ecoregions.Phylogeographical studies in South American near-shore marine benthic organisms are helping us to understand the recent evolution of higher latitude marine biota. Main phylogeographic signatures in S. lessoni in the Chile-Peru Province (CP), the Magellan Province (MP), and the Argentina Province (AP)  Fig. ; Table 160. Universal primers LCO1490 and HCO219861 and its-1d and its-4r62 were used to amplify partial fragments of the mitochondrial gene cytochrome c oxidase subunit I (COI) and the nuclear internal transcribed spacer region (ITS1 and ITS2), respectively. PCR procedures were performed using 2.5 μl 10X Buffer (Invitrogen), 1.0 μl of 50 mM MgCl2 (Invitrogen), 200 mM dNTPs (Invitrogen), 10 pg/μl each of dATP, dGTP, dCTP and dTTP (Invitrogen), 1 U Taq polymerase (Invitrogen), 50 ng of genomic DNA and 16.7 μl of double-distilled water. Thermal cycling parameters for the analyzed markers followed González-Wevar et al.21. PCR products were purified using QIAquick Gel Extraction Kit (QIAGEN) and sequenced in both directions with an ABI3730 x 1 Automatic Sequencer at Macrogen Inc. .Specimens were preserved in ethanol (99%) and DNA was prepared from the mantle using standard salting-out methodology following Aljanabi & MartinezS. lessonii were edited independently using GENEIOUS63 and aligned with MUSCLE64. New COI and ITS sequences have been deposited in GenBank under the following accession numbers: MN436071–MN436695 (COI), and MN436696–MN436714 (ITS). In order to evaluate how saturation of transitions is accumulated in relation to divergence we performed a saturation analysis in DAMBE 565. Following Wright66, we evaluated the codon usage of mtDNA sequences using the effective number of codon value (ENC) in DNASP v.567.Mitochondrial (COI) and nuclear (ITS) data sets of k), segregating sites (S), haplotype diversity (H), average number of pairwise differences (П), and nucleotide diversity (π) per site, genetic cluster, and for the whole COI data set.Polymorphism of mtDNA sequences was estimated in DNASP using standard diversity indices: number of haplotypes (S. lessonii were estimated following Pons and Petit68 using main pairwise differences (NST) and haplotype frequencies (GST) in ARLEQUIN v. 3.569. The statistical significance of pairwise population comparisons was determined through permutation tests  of haplotype identities following Excoffier et al.69. We also determined levels of phylogeographic differentiation using the nearest-neighbor statistic (Snn) in DNASP and the statistical significance of the analyses was determined using a permutation test  following Hudson70.Levels of genetic structure between populations of S. lessonii across its distribution was inferred using two clustering methods. First, through a Bayesian model we determined the number and composition of panmictic groups and the spatial boundaries among them using GENELAND v. 2.0.071 in the R environment 72. The best association of localities was estimated using a Markov Chain Monte Carlo (MCMC) procedure including genetic and geographic information following Guillot et al.71. Analyses were run using 50 × 106 MCMC iterations sampled each 1,000 steps. Stationarity of the analyses was identified in TRACER v.1.5 (http://beast.bio.ed.ac.uk/Tracer) and 10% of the iterations were removed as burn-in. A maximum number of clusters (K = 24) was included in the analyses to determine model parameters and posterior probabilities of group membership. Second, using SAMOVA we determined the composition and number of geographically homogeneous and maximally differentiated groups of localities following Dupanloup et al.73. This analysis allows characterization of spatial genetic structure by separating the variance into within populations, among populations within groups and among groups. This methodology seeks to maximize the proportion of total genetic variance due to differences among groups.The spatial mtDNA genetic structure of S. lessonii were constructed based on mtDNA (COI) and nucDNA (ITS) sequences using maximum parsimony networks in HAPVIEW74. Different models of gene flow between the mtDNA groups were tested for different scenarios using the software MIGRATE v.3.5 following Beerli & Felsenstein75. Four candidate models were compared constraining the presence of directionality of gene flow between the genetic groups found in S. lessonii. A first model permitted bidirectional gene flow (full-island model). Models 2 and 3 were defined based on the asymmetry of gene flow between groups. Finally, the fourth model assumed the groups were a single panmictic population. The analyses were performed using a TrN + I + G substitution model and transition-transversion ratio of 16.9927, estimated using JMODELTEST v276. The specific substitution rate for the selected marker in siphonariids78, was set to constant, following Darriba et al.76. The analysis included a long chain with 500,000 recorded parameter steps with a sampling interval of 100 and a burn-in of 10%. Following Darriba et al.76 we used a heated scheme of 1.0, 1.5, 3.0. and 1000000.0 to estimate the marginal likelihoods of the models to compared them. A thermodynamic integration approximation (T.I.) was used for the log-equivalent Bayes Factor (LBF) analyses following Beerli & Palczewski79. The associated probability of each model in relation to another was estimated through Bayes Factors following Kass and Rafery80. We also determined different demographic indices to evaluate asymmetric isolation-with-migration models between the recognized genetic groups using IMA2 software81. For this we performed several preliminary runs in the M-mode  to estimate the best set of priors. Uniform priors were used to determine the effective population size  and splitting time (t = 30); an exponential prior (mean = 20) for gene flow (m) was assumed. We performed 80 × 106 MCMC steps sampling every 100 generations, with a burn-in period of 10%. Mitochondrial sequences in S. lessonii were assumed to mutate under the HKY mutation model following Hey and Nielsen81. Simulated genealogies were performed under the L-Mode (Load Tree mode) to determine the log maximum likelihood and credibility interval (95% under HPD) estimates for migration parameters using a likelihood ratio test. Finally, under the L-Mode mtDNA data set of S. lessonii was compared against a null model of no migration81.Genealogical relationships in 82. For this we performed three independent Bayesian Monte Carlo analyses for 100 × 106 generations. Trees and parameters were sampled every 1,000 generations in each of the genetic groups of S. lessonii. Previously, JMODELTEST was used to estimate the proper substitution model using the Akaike information criterion (AIC). Following Ho et al.83 we used a rough tenfold evolutionary rate correction for population-based analyses. A time-dependency rate of molecular evolution has been described where the short-term mutation rate (1 to 2 Ma) could be tenfold higher than the long-term substitution rate used for phylogenetic reconstructions. Accordingly, BSP analyses used a 10% population-based mutation rate considering the 1.0% phylogenetic rate used for siphonariids78. Convergence between runs was confirmed with TRACER and the results for multiple runs were combined using LOGCOMBINER v1.4.784.Demographic mtDNA dynamics were determined using a Bayesian skyline plot (BSP) methodology implemented in the program BEAST v.1.8S) using DNASP for the whole COI data set and for each recognized genetic group to estimate whether sequences deviate from mutation-drift equilibrium. Demographic reconstructions were also estimated using the comparison of pairwise differences between haplotypes (mismatch distribution) for each recognized group to the expected distribution under the sudden expansion growth model85. For this we estimated three main parameters: i) τ = date of population expansion/contraction measured in units of mutational time (τ = 2 μt) where t = time in years and μ = mutational rate per sequence per year, ii) initial theta θi = 2 Niμ before the population expansion/contraction), and iii) final theta θt = 2 Ntμ after population expansion/contraction. Finally, demographic parameters were calculated in DNASP using a nonlinear least square approach following Schneider & Excoffier86.We performed statistical neutrality tests (Tajima’s D and Fu’s FSupplementary Information.


Rhyzopertha dominica. We found a substantial degree of admixture between weakly structured populations in the US. Ecological regions were more important in explaining R. dominica population structure than crop type, suggesting insect movement between wheat and rice grain distribution channels. We have also found a significant correlation between the genetic and geographical distance . However, our modeling approach combining the ecological and management variables has highlighted the importance of the volume of grain received by a location in the dispersal dynamics of the pest. The first-generation migrant analysis offered additional supported to movement over great distances that are likely associated with grain movement. Our data suggest that a multitude of factors play small but significant parts in the movement dynamics of the pest. The beetles can take advantage of the source-sink dynamic of grain movement in the US, but also engage in a high rate of movement at the local scale. Understanding population structure for R. dominica will provide insights into the potential for local processes of adaptation and broader patterns of movement that will impact management programs and the potential for spread of resistance genes.Population genetic structure of agricultural pests can be impacted not only by geographic distance and the broader ecological and physical barriers but also by patterns related to where crops are produced and how they are moved after harvest. Stored-product pests, for instance, specialize in exploiting grains such as wheat and rice from on-farm storage through transportation to final processing at often geographically distant locations; therefore human-aided movement may impact their dispersal. Although stored product insects are associated with stored grain, they can also exploit resources in the surrounding environments so different ecological regions where the grain is grown and stored may also influence population structure. Here we used 1,156 SNP markers to investigate how geographic distance, ecological and agricultural variables can impact the genetic structure and gene flow of the stored food pest beetle  Population structure of agricultural pests is likely impacted not only by geographic distance and the broader ecological and physical barriers but also by patterns related to where crops are produced and how they are moved after harvest. For example, grain commodities such as wheat and rice are typically grown in specific geographic regions where proper agronomic and environmental conditions can be found for the plant to develop. After harvest, the grain is often transported and stored multiple times, often over considerable geographic distances. Unwantedly, there is a community of organisms, including a number of significant stored product insect pests that can exploit the sizeable artificial resource patches created by storage of grain after harvest and are effective at finding and infesting these patches. The movement and storage of grain as it moves from producers to consumers generates a complex network of sources and receivers of grain that has the potential to facilitate the movement of associated pests . The human designed grain production system and its network of storage and movement can facilitate the long-range dispersal of some insects  within the United States, including diverse habitats such as prairies, plains, mountains, valley, highlands, hills, and lowlands (One can find olerance . Due to lowlands . While tvs. rice), geographical distance, and human-aided movement (rail transportation network) on R. dominica population structure within the United States. To accomplish that, we collected beetles from different wheat and rice areas within the same season and conducted a hierarchical approach to test for possible structure-causing factors , elements of the crop production system  following the manufacturer recommended protocol. We eluted the DNA using distilled water and use a vacuum concentrator process  to concentrate samples to 50 μl DNA. RAD-sequencing (restriction site-associated DNA sequencing) libraries were prepared following PstI (CTGCAG) and MseI (AATT), to create the genomic DNA fragments. After size selection targeting ∼300bp DNA fragment length, the size distribution of DNA fragments in the genomic libraries and the presence of contaminating adaptor peaks were tested on a Bioanalyzer (Agilent 2100), and real-time PCR was used to quantify libraries. Three libraries  were diluted to 10 nM concentrations and sequenced on three lanes of Illumina HiSeq2000 (100 bp single-end read run) at the University of Kansas Genome Sequencing Core facility.Genomic DNA was extracted from individual Rhyzopertha dominica libraries were demultiplexed, separating individual beetles using the process_rad-tags script in STACKS v.1.44 (S v.1.44 . FilteriS v.1.44  (Table SS v.1.44 .o) and expected heterozygosity (He), inbreeding coefficient (FIS), and nucleotide diversity (π) across the genome were estimated for each sampled location. We have calculated allelic richness (AR) and private allelic richness (PAR) for each genetic cluster using rarefaction with standardized sample size of 8, equivalent to 4 diploid samples, with a tolerance of 15% missing data using ADZE 1.0  steps. The analysis considered admixture model  and correlated allele frequencies . No prior information on the sampling location was included in the analysis. We have tested clusters from K = 1 to K = 12 with 10 replications. STRUCTURE output was analyzed using the program CLUMPP 1.1.2  using 99,999 permutations with ARLEQUIN software in two hierarchical levels . To visuPP 1.1.2  and visuPP 1.1.2 . Most liarvester . We addiST analysis in Arlequin 3.5.2.2 was used to measure levels of genetic differentiation between two populations. We also assessed the population average pairwise matrix of Slatkin linearized FST, population average pairwise differences, and population average pairwise matrix of M values (M = 2Nm). We used RColorBrewer and igraph packages to visualize the FST relations. We performed Mantel test with 10,000 permutations to correlate the genetic distances (FST) matrix with the geographic distance matrix using the R libraries ecodist  prior to analysis so the beta could be interpreted as the standardized partial regression coefficient or beta weight (iw) (glmulti (We standardized response and predictor variables (a weight . Beta wea weight . In addight (wi) . To sepaght (wi) . We used and the average percentage of quality-filtered reads (≥Q30) per library was 91.13% , giving an average depth of coverage per individual over all SNPs of 35x . Average yield per library was 20.13 GB. Eighteen beetles  had insufficient mean coverage (<5x) and were removed from further analysis. After filtering steps, 45,7018 SNPs were retained before population module in STACKS when it was filtered down to 1,156 SNP markers.0) was 0.234 ± 0.137, the expected heterozygosity (HE) was 0.259 ± 0.136, and inbreeding coefficient (FIS) was 0.133 indicating fewer heterozygotes than expected .We had not found significant differences in nucleotide diversity among sampled locations when diversity parameters were calculated using only variant positions  or usingXY - (πX + πY)/2) followed by WA and LA and WA and SC (Table S6). The population pair with the smallest overall number of differences  was KS and OK. The population pair with the largest number of pairwise differences within the population (πX)  was KS and WA whereas the population with the least amount of within population differences  were CA and TX (Table S6). The estimated number of effective migrants Nm varied from 35.6 beetles between KS and ND to 5.1 beetles between CA and WA. The places that in average exchanged most migrants per generation were KS (24.5 beetles) and OK (23.2 beetles) whereas the places that exchanged the least were WA (5.9 beetles) and TX (9.1 beetles) (Table S9). The assignment analysis successfully assigned the majority of beetles to their correct collection location (78%), but there was considerable variation among locations (range: 0% LA to 98% CA). Beetles from SC, ND, TX, and CA were more often assigned to their correct locations, while LA and one of the KS populations (FKS) did not have any beetles assigned correctly. The high heterogeneity  of central locations such as KS and OK can explain the low assignment rate found in those populations whereas TX, CA, SC were more homogenous. Two beetles were flagged as putative first-generation migrants: one beetle collected in Beaumont, TX, migrating from Jonesboro, AR, (Lh/Lmax (Threshold)= 173 (28.7)) and one collected in Orangeburg, SC, migrating from Parlier, CA (Lh/Lmax (Threshold)= 222.3 (65.5)). This result gives us strong evidence of a long-distance movement that is likely to be human-aided.The pair WA and TX showed the greatest pairwise nucleotide difference between populations  revealed a small but significant degree of genetic differentiation among ecoregion  within tK = 2 as the most likely number of genetic clusters and the second most probably partition is K = 7 indicating a weaker but a possible level of substructure . Genetic differentiation between wheat and rice regions was not apparent in the STRUCTURE analysis  considering all pairwise comparisons   tended to exchange more migrants with other locations. On the other hand, locations farther apart with a high rate of grain receipts  tended to exchange fewer migrants with other locations. The volume of grain received at each location alone has a significant effect on the average number of effective migrants (M) and explains a significant portion of the variance (A single model was selected to explain the average number of effective migrants (M) at each location . The top model elected volume of grain received and geographical distance as best predictors according to AIC= 30.48) . The twovariance .Here we used hundreds of markers to examine and extract genetic diversity information from local populations and found correlations with geographic distances, agriculture practices, and the movement of commodities across vast distances. For instance, agriculture-related variables such as the volume of grain received by a particular location helped to explain the degree of genetic isolation, and the number of effective migrants exchanged between populations. Here, we have not found statistically significant differences in genetic diversity among populations including nucleotide diversity, allele richness, and expected heterozygosity, which could be attributed to the high gene flow between areas. An interesting finding is that the most heterogeneous and better-connected populations are also located in places with the largest grain production in the United States. KS was the most heterogeneous location showing the highest value for pairwise nucleotide differences within population and lowest values of pairwise nucleotide differences between populations. More heterogenous areas tended to be large hubs of the grain transport network and highly connected to other areas. Our results support the notion that important production areas are support larger and heterogeneous populations  and a higher relatedness for individuals flying away from a storage structure(dispersing).All populations showed lower values of observed heterozygosity than expected. Similar low heterozygosity has been described in the literature for other stored product insects . This efR. dominica colonizers. For instance, it would be useful to determine if they tend to have higher levels of genetic similarity among themselves , or if they will exhibit a higher level of genetic differentiation from one another (arise from different sources) as observed in Tribolium populations  to engage in cross-continental travels.solation . Physicasolation . We haveR. dominica populations experience a considerable amount of gene flow that was reflected in the high degree of population admixture illustrated in the structure plots. Rhyzopertha dominica populations seem to exhibit a combination of patterns, both continuous genetic differentiation and discrete population structure. The subtle levels of structure can give us clues of earlier demographic processes and ongoing evolutionary dynamics and therefore should be evaluated carefully. Keeping that in mind, we explored the population genetic structure using a wide range of techniques that indicated the influence of multiple and concurrent structuring factors. It appears that structuring factors might be to some extent correlated.Data presented here demonstrate that K = 2 was the most likely number of groups, but there was also a small peak at K = 7. The smaller peak is much less important here, but it is nonetheless interesting to see that sampled location can still be identified despite the extremely high levels of gene flow. The PCA analysis, a non-model-based method, supported those two levels of structure even though it is not clear if crop type, source-sink dynamic, or another non-tested factor was responsible for the observed pattern. Scrutinizing K = 7, we confirm that KS and OK are indeed the most heterogeneous locations and CA, WA, and TX are the most homogenous. We can also spot the putative first-generation migrant in SC coming from CA. The FST values allowed the separation of the locations into two groups, one composed of relatively well-connected locations such as KS, ND, and OK and one composed by more structured locations  such as WA, CA, and TX. Interestingly, TX is very close geographically to LA but seemed to be very distant genetically suggesting the importance of other processes and dynamics more complicated than just geographic distance.We presented two estimations for the number of clusters suggesting potential substructure. According to the Evanno method, i.e., 0.63% of the total variance was explained by crop type). Given that rough rice tends not to be transported over very long distances prior to processing and is less likely to be cross-contaminated with wheat in transportation, we had predicted populations from rice-growing regions would be more isolated from wheat growing regions. However, our results are not conclusive regarding the impact of the crop because only the TX location could be considered an exclusive rice production region according to our classification (crop type within a 50 km radius). Our other rice-growing regions in AR and LA, while they can be considered rice regions predominantly, did also have some wheat grown within the established perimeter, making them mixed crop regions. That pattern of mixing areas was also apparent in the PCA plot. Texas presented a substantial degree of differentiation compared to a close location such as LA. These few cases of crop-related variation suggest that there may be a crop effect that was not detectable using our sample locations. The hypothesis that crop type could be a factor causing population structure has been investigated in the past in wheat and rice mills for Tribolium (R. dominica.We found very little structure caused by crop region (ST/1-FST) using the Mantel test, and a high rate of admixture .Because distance has not been found as a major factor affecting population structure in stored-product insect populations in the past, grain transportation is often evoked as the best explanation for the lack of IBD . In the dmixture . Howeverdmixture . We usedvs. natural habitat locations, or if it truly represents a pattern of substructure or kin-structure. Most heterogeneous and well-connected populations were in regions with the greatest wheat production. Given that after harvest wheat is typically stored locally and then transported through a series of successively larger and more distant storage locations, large production areas offer more opportunities for R. dominica to breed and these regions can potentially be a source of migrants to other less productive locations. In addition, dispersal by flight behavior and displacement from one location to another by human-aided means using trucks and railroads connecting populations can largely explain population differentiation patterns in R. dominica in the United States. This information also has a potential application to pest management and the management of insecticide resistance that needs further exploration.Here we have shown how the geographic distance, features of the environment in which local populations are present, the agriculture management, as well as storage, and transportation of commodity are likely to affect different aspects of a stored grain insect pest population. This is the first time that population structure and IBD have been shown for a stored product insect. Inbreeding coefficients gave us insights on how populations infest and develop within grain storage sites; however, the observed heterozygosity deficiency needs further evaluation to determine if it is due to sample bias, such as timing before or after fumigation or being nearer to stored grain


Oreochromis niloticus) is one of the most produced farmed fish in the world and represents an important source of protein for human consumption. Farmed Nile tilapia populations are increasingly based on genetically improved stocks, which have been established from admixed populations. To date, there is scarce information about the population genomics of farmed Nile tilapia, assessed by dense single nucleotide polymorphism (SNP) panels. The patterns of linkage disequilibrium (LD) may affect the success of genome-wide association studies (GWAS) and genomic selection (GS), and also provide key information about demographic history of farmed Nile tilapia populations. The objectives of this study were to provide further knowledge about the population structure and LD patterns, as well as, estimate the effective population size (Ne) for three farmed Nile tilapia populations, one from Brazil (POP A) and two from Costa Rica (POP B and POP C). A total of 55 individuals from each population, were genotyped using a 50K SNP panel selected from a whole-genome sequencing (WGS) experiment. The first two principal components explained about 20% of the total variation and clearly differentiated between the three populations. Population genetic structure analysis showed evidence of admixture, especially for POP C. The contemporary Ne estimated, based on LD values, ranged from 78 to 159. No differences were observed in the LD decay among populations, with a rapid decrease of r2 with increasing inter-marker distance. Average r2 between adjacent SNP pairs ranged from 0.19 to 0.03 for both POP A and C, and 0.20 to 0.03 f or POP B. Based on the number of independent chromosome segments in the Nile tilapia genome, at least 9.4, 7.6, and 4.6K SNPs for POP A, POP B, and POP C respectively, are required for the implementation of GS in the present farmed Nile tilapia populations.Nile tilapia ( Oreochromis niloticus) is one of most important farmed fish species worldwide  between SNPs and quantitative trait locus (QTL) or causative mutation. Thus, knowing the extent and decay of LD within a population is important to determine the number of markers that are required for successful association mapping and genomic prediction . Therefo2) and Lewontin’s D’ (commonly named D’). Values lower than 1 for D’ indicate loci separation due to recombination, while D’ = 1 indicates complete LD between loci, i.e. no recombination. However, this parameter is highly influenced by allele frequency and sample size. Thus, high D’ estimations are possible even when loci are in linkage equilibrium , POP C and the wild strains from Egypt and Kenya used to generate the GIFT strain. The POP C breeding population represents a combination of genetic material from Israel, Singapore, Taiwan and Thailand. Therefore, the three breeding populations are considered recently admixed populations; which are directly or indirectly related to the GIFT strain. Based on the overall identical by descent (IBD) alleles, average relatedness between individuals, within each population, was estimated using Plink v1.90  belong to the AquaAmerica (Brazil) breeding population, where the animals are evaluated in cage-based production systems and have been artificially selected during three generations for improved growth rate using daily weight gain as selection criteria. This population was imported from GIFT Malaysia in 2005 for breeding and farming purposes. Individuals from population B (POP B) and C (POP C) were obtained from Aquacorporación Internacional (Costa Rica) and correspond to fish from the seventh and eighth generation, respectively, of selection for improved growth-related traits (body weight at 400 g as selection criteria) under pond system production. The POP B breeding population is a mixture of the GIFT strain . Whole-genome sequencing was performed using multiplexing of four bar-coded samples per lane of 100bp paired-end in the Illumina HiSeq 2500 machine. The sequences were trimmed and aligned against the genome assembly O_niloticus_UMD_NMBU  to estimate the LD between each pair of markers. We used Plink v1.09 using the parameters --ld-window-kb 10000, --ld-window 99999, and --ld-window-r2 set to zero to calculate the LD between all pairs of SNPs on each chromosome. Based on the physical distance of each SNP pair, we created bins of 100 kb among all pairwise combinations. The extent and decay of the LD, for each population, were visualized by plotting the average r2 within each bin, spanning a physical distance from 0 to 10 Mb. We used the software SNeP v1.1 (Ne). Considering the LD within each population, Ne was estimated using the following equation proposed by We used the Pearson’s squared correlation coefficient  of t and mapping function f(ct), and α is the adjustment for mutation rate . Values for number and size of each bin were used as default . Based on the relatively small number of SNP per chromosome, Ne per chromosome was calculated using harmonic mean  was assessed based on the following formula proposed by Estimation of the effective number of chromosome segments  Table 1.For downstream analysis, we selected 55 animals for each population based on identity by descent analysis (IBD). We discarded a total of 4, 71 and 86 animals from POP A, POP B and POP C, respectively. Thus, the average relatedness within populations was 0.00 ± 0.01.o/He). We found values of 0.23/0.34, 0.26/0.35 and 0.26/0.36 for POP A, POP B and POP C, respectively. Similar levels of genetic differentiation were found between POP A and POP BC and POP A and POP C (Fst = 0.072 ± 0.11 and Fst = 0.070 ± 0.10), whereas a lower Fst value was observed between POP B and POP C (Fst = 0.056 ± 0.09).Upon plotting the first two eigenvectors on the PCA plot, the three populations were stratified based on the single dimensional variation between them. The first two principal components together accounted for 20.0% of the genetic variation, revealing different populations Figure 2In the admixture analysis, the posterior probability (Pr) of the fitted admixture model to the data was computed using K-values from 1 to 12 , the bes2 was similar among populations, with values of 0.06 ± 0.10 for the three populations studied  and also the lowest effective population size (<161) was reported for LG7, LG13 and LG19   . Some aupulation . In contow trout  and Atlaow trout .r2 = 0.15 for markers spaced at 20–80 Kb). Similar values were found in humans .The LD results have several implications for future implementation of genomic tools in the current farmed Nile tilapia populations. Both GWAS and genomic selection are dependent on LD extent to define the number of SNPs necessary to assure the causative mutation variance  and to a Morgans . Thus, tHaving a number of independent, biallelic and additive QTL affecting the trait we would need a smaller number of markers to achieve a high accuracy. Thus, the minimum number of markers for a high-power genomic analysis should be at least, 9,400, 7,600, and 4,600 for POP A, POP B, and POP C, respectively. Despite the fact that these numbers were slightly lower than those suggested by A recent study tested different marker densities and imputed genotypes to assess genomic prediction accuracies in a farmed Nile tilapia population. The prediction accuracy using genomic information outperformed the estimated breeding values using the classical pedigree-based best linear unbiased prediction, even using a very low-density panel (0.5K) for growth and fillet yield . In addiThe current study revealed similar short-range LD decay for three farmed Nile tilapia populations. The PCA suggested three distinct populations and the admixture analysis confirmed that these three populations are highly admixed. Based on the number of independent chromosome segments, at least 9.4, 7.6, and 4.6 K SNPs for POP A, B, and C, respectively might be required to implement genomic prediction in the current Nile tilapia populations, whereas for GWAs studies more markers may be necessary to achieve higher power and greater precision for QTL detection.The sampling protocol was previously approved by The Comité de Bioética Animal, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile (certificate N° 18179-VET-UCH).GY performed the analysis and wrote the initial version of the manuscript. AB contributed with discussion and writing. GC, MC and AJ performed DNA extraction. KC and JL contributed with study design. JY conceived and designed the study; contributed to the analysis, discussion and writing. All authors have reviewed and approved the manuscript.This work has been funded by Corfo (project number 14EIAT-28667).GY, JPL and KC were hired by a commercial institution (Benchmark Genetics Chile) during the period of the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Abrothrix manni. We aim to assess the genetic structure of this species, testing for the existence of intraspecific lineages, and inferring the recent demographic history of the species. Analyses were based on the first 801 bp of the mitochondrial gene Cytocrhome-b from 49 individuals of A. manni collected at 10 localities that covers most part of its geographic distribution. Genealogical analyses recovered two main intraspecific lineages that are geographically segregated and present an intermediate site of secondary contact. Historical demography shows signal of recent population decrease. Based on these results, we proposed that current genetic diversity of A. manni differentiated in at least two distinct refugial areas in southern Chile. This scenario, in addition to be unique among those uncovered for the so far studied Valdivian forest rodents, is noteworthy because of the reduced geographic scale inhabited by the species.Quaternary climatic oscillations have impacted Patagonian sigmodontine fauna, leaving traceable genetic footprints. In southern Chile, changes in the landscape included transitions to different vegetation formations as well as the extension of ice sheets. In this study, we focus on the Valdivian forest endemic and recently described sigmodontine species  The southern cone of South America has been the setting of major climatic, tectonic and volcanic activity during the past several million years . The QuaEligmodontia morgani and Reithrodon auritus), although more complex processes were also reported, such as within-region differentiation and population persistence at putative refugia  have been recorded in co-distributed species . A patte refugia . Given tAbrothrix manni  presence of well-supported allopatric clades, (b) multimodal mismatch distributions, (c) significant values of demographic indexes  (d) signals of demographic stability in absolute time calibrated skyline plots. Therewith, we hope to contribute filling the gap in the phylogeographic knowledge of one of the least studied areas of southern South America  from 49 individuals of Abrothrix manni collected at 10 localities along most part of its known distributional range , A. jelskii (M35714), A. lanosa (EU683432), A. longipilis (HM167785), A. sanborni (KP666004), and A. olivacea (HM167800) were used to conform the outgroup. Best-fit model of nucleotide substitution for this dataset was determined based on maximum likelihood estimates of model parameters and Bayesian Information Criterion (BIC) using jModeltest2  of the average standard deviation of split frequencies. The first 25% of the samples were discarded as burn-in; remaining trees, sampled well after stationarity was reached, were used to compute a consensus tree with posterior probability (PP) estimates for each clade.Sequences were aligned using Clustal W  implemendeltest2 . The seldeltest2  implemendeltest2 ; branch deltest2 . The BI deltest2 ; four ina priori to test specific geographic and genealogical groupings. In a geographic arrangement, sequences were allocated in two groups, one containing northern sampling sites  and another group containing southern sites . In a second arrangement, haplotypes were grouped according to the results of the genealogical analysis (see below). The amount of genetic variation attributable to differentiation among groups (FCT), among localities within groups (FSC), and among localities relative to the total sample (FST) was estimated.The spatial partition of the observed genetic variation was evaluated using analysis of molecular variance (AMOVA) implemented in Arlequin 3.5 . Severalp-value indicates that the sample is drawn from a recently expanded population  clade A, which considers only the sequences included in this clade (see below); (3) clade B, including only sequences in this clade (see below), (4) northern range, containing all samples from localities 1–6, (5) southern range, with all samples from localities 7–10; and (6) eastern range, containing samples from localities near the Andes, 5–9).Historical demography was assessed on the basis of the distribution of pair-wise differences and related tests using Arlequin 3.5. A multimodal mismatch distribution indicates that samples were drawn from a population in demographic equilibrium, while a unimodal curve is expected for populations that experienced a recent demographic expansion . The smopulation . The fitpulation  In a difpulation  were alspulation . Nucleotpulation . AbsolutCytb of Abrothrix manni contain 46 variable sites that define 22 haplotypic classes , which was implemented in both ML and BI analyses. Resulting topologies congruently recover, although without a strong support  the monophyly of n clades . The fir overlap . HaplotyWhen grouping haplotypes according to a geographical criterion , AMOVA shows that 55.6% of the total variation in the data set is attributable to differences between these two geographic groups. The remaining percentage is due to differences among locality sites (21%) and within localities (23.4%). Fixation indexes were significant at these three levels . When anA. manni, assessed by means of four indexes, recovered contrasting signals for six different grouping of samples. When considering the whole dataset, half of the indexes recovered signals of demographic expansion (SSD and Hr), while the rest failed to do so  and the extension of a glacial ice cap in pre-Andean and Andean areas during the LGM  to assess if the uncovered pattern changes.The existence of Pleistocene refugia in the Valdivian forest of North-Western Patagonia has been proposed in an increasing number of studies, including reptiles , amphibios Andes , some ofos Andes . Sersic micropus . On the refugium . In addirefugium . Whetherrn range  that conrn range . In addirn range –3E show rn range . In thisA. manni from Chiloe, but we observed that island genetic variants are closely related to those recovered in the mainland. This was also observed in the marine otter Lontra felina (Pudu puda (Dromicios gliroides (Oligoryzomys longicaudatus (In addition to the mainland area of Central-Southern Chile, the north-west half of the large Chiloe Island has been also proposed as a refugial area . Due to a felina , the pududu puda , the marliroides , and in caudatus . These rcaudatus .Dromiciops gliroides (referred as clade C in Abrothrix olivacea (Oligoryzomys longicaudatus (A. manni fails to show signals of recent demographic expansion in both northern and southern portions of its distributional range (A. manni have been negatively affected by interspecific competition with other species, likely sigmodontine rodents, which would have expanded in the area after glacial conditions retreated. Also, it is possible that the demographic reduction experienced by A. manni may have been caused by the wildfire that increased in Northern Patagonian forests ca. 11,000–8,500 YA (A. manni is much limited (see In north-western Patagonia, different mammals show signals of demographic expansion within our study area. This is the case of olivacea  and Oligcaudatus . Contraral range . On the al range . This po8,500 YA  and/or e8,500 YA . For theited see ; as suchCytb gene of Abrothrix manni supports a history of persistence during Quaternary oscillations in its restricted distributional area at the Valdivian forest of north-western Patagonia. This, however, does not imply that this mouse was not impacted by climatic changes; on the contrary, forest fragmentation would have caused a differentiation process that led to two divergent intraspecific lineages and the posterior secondary contact between them. These conclusions are noteworthy because of the reduced geographic scale inhabited by the species. They also imply that this small area may have harbored multiple Pleistocene refugia.The genetic variation of the mitochondrial A. manni, we hope to contribute to the understanding of evolutionary history of the local fauna, particularly of the sigmodontine rodents.Finally, with this study, which constitutes the first description of the spatial distribution of the genetic variation and historical demography of the recently described Mann’s soft-haired mouse