Prosopis (Leguminosae) individuals are restricted to oases that are unevenly distributed and isolated from each other by large stretches of barren landscape constituting an interesting study model as the degree of connectivity between natural populations depends on their dispersal capacity and the barriers imposed by the landscape. Our goal was to assess the genetic diversity and the degree of differentiation among groups of Prosopis individuals of different species from Section Algarobia and putative hybrids (hereafter populations) co‐occurring in these isolated oases from the Atacama Desert and determine whether genetic patterns are associated with dispersal barriers. Thirteen populations were sampled from oases located on three hydrographic basins . Individuals genotyped by eight SSRs show high levels of genetic diversity  and low but significant genetic differentiation among populations . The AMOVA indicates that most of the variation occurs within individuals (79%) and from the variance among individuals (21%); almost, the same variation can be found between basins and between populations within basins. Differentiation and structure results were not associated with the basins, retrieving up to four genetic clusters and certain admixture in the central populations. Pairwise differentiation comparisons among populations showed inconsistencies considering their distribution throughout the basins. Genetic and geographic distances were significantly correlated at global and within the basins considered (p < .02), but low correlation indices were obtained (r < .37). These results are discussed in relation to the fragmented landscape, considering both natural and non‐natural (humans) dispersal agents that may be moving Prosopis in the Atacama Desert.In the Atacama Desert from northern Chile (19–24°S),  Prosopis individuals of different species from Section Algarobia and putative hybrids co‐occurring in isolated oases from the Atacama Desert (19–24°S) in Chile. Genetic results are discussed in relation to the fragmented landscape, considering both natural and non‐natural (humans) dispersal agents that may be moving Prosopisin the desert.We assess the genetic diversity and the degree of differentiation among groups of  The extreme aridity is primarily caused by the cold water of the Humboldt Current, running parallel to the Chilean and southern Peruvian coasts and preventing precipitation in the coastal areas. Moreover, aridity is intensified by the Andes cord that produces a rain‐shadow effect, blocking moisture from the Amazon basin  includes 44 species distributed in Southwest Asia, Africa, and predominantly America , are considered multipurpose trees. From an economic perspective, humans use it as food as the raw pods can be eaten directly or processed within a variety of culinary preparations , but Muñoz , individuals belonging to different species from Section Algarobia  can be found up to 3,000 masl and are distributed from the Pampa del Tamarugal to the Salar de Atacama basin  co‐occurring in the isolated oases from the Atacama Desert and determine whether genetic patterns are related to dispersal barriers. For this, we characterized by microsatellites the variability, genetic differentiation, and genetic structure among 13 populations considering three distant basins. Through these analyses, we addressed the following questions: (a) Are there differences in the genetic diversity parameters among populations? (b) How is the genetic variation distributed considering the different hierarchical levels ? (c) What is the level of differentiation and gene flow among populations? (d) Is there a genetic pattern compatible with isolation by distance (IBD)? (e) Is there genetic structure evidence? As the Atacama Desert provides habitat fragmentation that could affect the natural gene flow, given by the large stretches of hyperarid landscape, we discuss the genetic results considering the dispersal barriers that separate the populations.The main objective of this study was to assess the genetic diversity and the degree of differentiation among groups of Prosopis populations are restricted to isolated oases, the current hypothesis is that the gene flow restrictions among different populations would determine significant genetic differentiation among populations. Additionally, the dispersal limitation described for Prosopis species . The PCR amplifications were carried out in a 50 µl reaction volume containing 10–30 ng DNA, 0.6 µM each primer, 0.2 mM dNTPs, 0.3 U Taq DNA polymerase (Invitrogen), and 1.5 mM MgCl2. A T100 Thermal Cycler  was used for amplifications with a cycling profile of initial denaturation at 94°C for 5 min followed by 35 cycles at 94° for 45 s denaturation, primer‐specific annealing temperature (56°–59°) for 45 s and at 72°C for 45 s extension, and a final extension step at 72° for 10 min. PCR products were electrophoresed using Macrogen service (www.dna.macrogen.com) and sized using GENEMARKER ver 1.91 . We used a total of eight microsatellites; four developed by Mottura et al.  for P. ca et al.  for P. a2.32.3.1IA) and a slightly modified statistic which is independent of the number of loci (rd) , mean percentage of total alleles observed per locus per population (%TA), allelic richness (Ar), and observed heterozygosity (HO) and expected heterozygosity (HE) under the Hardy–Weinberg equilibrium. Genetic variability estimate mean was compared among populations by the nonparametric ANOVA test . Homozygote excess/deficiency was quantified by the fixation index (FIS) which was considered significant when the zero is not contained in its 95% confidence interval . A, %TA, and HO were estimated using the package diveRsity . In order to avoid bias induced by the presence of null alleles, FST excluding null alleles (FST‐ENA) was also estimated using the software FreeNA  and also as pairwise population genetic differentiation indices. Heat map of FST pairwise comparisons between populations was conducted using the levelplot function of the lattice package  was calculated assuming drift–migration equilibrium using the formula: Nm = (1 − FST)/4FST , and pairwise comparisons were performed by Wilcoxon's pairwise tests using, respectively, the functions Φ statistics by means of the package poppr of R. The molecular differentiation was partitioned into among the sampled basins, among populations within these basins, and among individuals within population components. The significance of Φ statistics was obtained with the function randtest.amova of the ade4 package  was assessed using dapc of the adegenet package  repetitions were 25,000 and 50,000, respectively, and K values were averaged across ten iterations. Considering the range of population distribution, both admixture and no‐admixture models were run considering correlated allele frequencies, no sampling locations as prior information and K from 2 to 14. The optimum K value was determined with STRUCTURE HARVESTER . For a single‐locus analysis, with i‐th, j‐th, k‐th, and i‐th different alleles, a set of squared distances are defined as d2  = 0, d2  = 0, d2  = 1, d2  = 1, d2  = 2, d2  = 3, and d2  = 4 , two pairwise population distance matrices (genetic distances and geographic distances) were constructed and compared through the 33.1P. alba, P. chilensis, P. flexuosa, and P. nigra and just one putative hybrid was identified in ZAPI, QUIN, QUIS, YAYE, TULO, CAMA, and TILO , which are attributable to four populations . In the other nine populations, the association index was nonsignificant , and heterozygosity  were relatively high in all the studied populations  and the lowest in TULO (HE = 0.56). However, KW‐ANOVA indicates that the differences among populations are not significant for all the estimates obtained .The eight SSR loci analyzed in the populations were highly variable showing from two to eight alleles per locus. Percentages of total alleles observed per population (%TA), allelic richness  occur with the same frequency.In five populations, 3.2FST, FST‐ENA, and DJOST indices yielded similar results, indicating that the overall level of genetic differentiation among the thirteen populations was highly significant  but remain highly significant. Based on FST, the gene flow becomes 2.62 and 2.47 migrants per generation in northern and southern basins, respectively. In the central basin, differentiation was significant according to FST index (FST = 0.035) and gene flow was much higher in comparison with northern and southern basins (Nm = 6.95); however, based on DJOST the differentiation among populations within this basin was not significant (DJOST = 0.066). In reference to the global FST‐ENA estimation (FST‐ENA = 0.129), similar decreases in the genetic differentiation indices were obtained within the three basins , but a trend toward higher differentiation between populations from different basins was observed (Tables FST < 0.075) with the exception of ZAPI‐TILI and ZAPI‐TARA comparison that was comparatively higher . In the central group, FST was also very low (FST < 0.078), while the FST between populations of the southern basin was low among almost all the population pairs studied (<0.066). The exception was TULO, which was highly differentiated from all the remaining populations of the southern basin (0.120 > FST < 0.169).The genetic differentiation between populations from the same or different geographical basins showed the same tendency by T Tables , pairwisd Tables . GeneticFST , FST‐ENA , and DJOST . For the three indices, the Wilcoxon pairwise comparisons test was significant among differentiation indices from northern populations with the central and the southern ones . The FST between populations from northern and central basins were lower in comparison with populations from northern and southern basins. Considering the central and northern basins, the most differentiated population pairs were TARA‐QUIS and ZAPI‐QUIS with FST = 0.093 and 0.092, respectively. The largest FST index obtained from comparing populations from northern and southern basins correspond to the pair ZAPI‐TILO (FST = 0.168).The mean pairwise differentiation estimates among populations from northern, central, and southern basins showed significant variation of ΦRT = 0.207), the variation among populations within basins (ΦPR = 0.068) and among individuals within populations (ΦIP = 0.081) were all highly significant (p < .0005). The distribution of molecular variance at different hierarchical levels indicated that most of the variation occurs within individuals (79.32%), and consequently, the variance among individuals resulted in 20.68%. Within this level, the between‐basin component (7.40%) was almost equal to the between‐population within‐basin component (7.47%) followed by the between‐individual within population component (5.81%) representing 35.78%, 36.12%, and 28.10%, respectively.The AMOVA Table  indicateDAPC analysis, three axes were retained and explained 94.2% of total variation . Individuals from TULO are represented by a genetic cluster (blue) that is absent in the southern basin and is represented only in the central population QUIN and QUIS. The individuals from TILI are represented by the red cluster and the individuals from ZAPI by the green one. These genetic groups are absent in the southern basin as they are only represented up to CHIU latitude.For 3.4r = .37, p = .000) and when the analysis was performed considering the northern , central , and southern  basin populations separately , comparable to the results from natural populations of P. alba, P. flexuosa, and P. chilensis (Section Algarobia) inside and outside Chile with different levels of isolation  and is consistent with the differentiation evaluated by the discriminant analysis of the principal components (DAPC), where differentiation is not observed at the uppermost hierarchical level as populations from northern, central, and southern basins were not grouped forming clusters. In agreement, low levels of variation were explained by AMOVA between basins (7.40%). Isolation between the studied basins can be attributed to ancient biogeographical processes linked to aridity fluctuations and geomorphological events that may have driven biological differentiation  were attributed to differentiation dated more than 1 million years ago that has been blurred by more recent gene flow ; however, the paleo‐archeological evidences indicate that Prosopis species from Section Algarobia were not present in the Atacama Desert up to the Formative period  ruling out so ancient possibility . Although Mantel's tests were also significant, indicating that nearby populations tend to be genetically more similar than expected by chance and suggesting gene flow restriction both at short and long distances, the low correlation estimates suggest a high degree of gene flow occurring within basins. According to these results, the geographic isolation that occurs between the populations within each of the basins seems to be able to be crossed by the natural dispersers of Prosopis easier than the large distances that separate the different basins considered here. These results are compatible with endozoic seed dispersal associated with short‐distance spread described for Prosopis  was reported to be comprised up to 69% of plants, and small quantity of P. alba seed plant remains in scats of L. culpaeus in the Atacama Desert  was found at the lowest level , which is expected in outcrossing species as Burkart, . AlthougK = 2 and K = 4) can be considered low. For K = 4, the populations with admixture signals were only six out of thirteen and are mainly located in the central basin  and in less proportion in the south of the northern basin . As predicted by the IBD model, neighbor populations are not expected to be more similar to each other than the distant ones. Here, we detected inconstancies in FST, FST‐ENA, and DJOST differentiation indices among population based on a pairwise analysis considering the basins. Those inconsistencies were found in both the northern and southern basins. In the north, the ZAPI‐TILI and ZAPI‐TARA differentiation indices were comparatively higher than the ones detected within all the northern basin. Similarly, in the south, TULO individuals are much more differentiated from the remaining southern populations. Plausible causes that could explain the inconsistencies in pairwise FST estimations are genetic drift and non‐natural gene flow patterns. Small isolated populations are particularly susceptible to genetic drift and inbreeding, which are thought to reduce genetic diversity. The loss of genetic diversity in turn enhances inbreeding and finally results in inbreeding depression  detected both globally and within each basin can also be discussed in conjunction with the structure and pairwise differentiation results. Since many of the populations studied are constituted by more than one taxonomic species, it is possible to expect a high level of admixture. However, the level of admixture detected by the STRUCTURE analysis  trees . A first approximation could be to link the two genetic groups and the altitude at which the different populations studied are found  located in the oases of the Atacama Desert considering the genetic variability and its distribution, genetic differentiation, gene flow among populations, IBD patterns, and genetic structure. We have discussed the genetic patterns in reference to ecological, historical, and sociocultural characteristics. Based on the analyses of genetic parameters, we propose that the genetic pattern may be the consequence of natural gene flow together with anthropic transport of a wide variety of algarrobo.In summary, we have assessed the genetic diversity of None.Cecilia Bessega: Conceptualization ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Software ; Supervision ; Validation ; Visualization ; Writing‐original draft ; Writing‐review & editing . Carolina Pometti: Formal analysis ; Funding acquisition ; Methodology ; Resources ; Software ; Writing‐original draft ; Writing‐review & editing . Renee H Fortunato: Data curation ; Resources ; Writing‐original draft ; Writing‐review & editing . Fransisca Greene : Resources ; Writing‐original draft ; Writing‐review & editing . Calogero Santoro: Funding acquisition ; Resources ; Supervision ; Writing‐original draft ; Writing‐review & editing . Virginia McRostie: Conceptualization ; Funding acquisition ; Investigation ; Project administration ; Resources ; Supervision ; Writing‐original draft ; Writing‐review & editing .FIGURE S1Click here for additional data file.FIGURE S2Click here for additional data file.TABLES S1‐S3Click here for additional data file.Figure LegendsClick here for additional data file.


Seseli farrenyi (Apiaceae) is an extremely narrow endemic plant, which is considered as one of the species of most conservation concern in Catalonia (NW Mediterranean Basin). Given the accelerated fragmentation and reduction of population size (of over 90%), the environmental agency of Catalonia is currently preparing a recovery plan that includes reinforcements of the extant populations. The present study is aimed at providing the necessary knowledge to carry out genetically-informed translocations, by using microsatellites as genetic markers. Fourteen microsatellites have been specifically developed for S. farrenyi, of which nine have been used. Besides the extant natural populations, the three ex situ collections that are known to exist of this species have also been studied, as they would be the donor sources for translocation activities. Our main finding is that levels of genetic diversity in the natural populations of S. farrenyi are still high (He = 0.605), most likely as a result of a predominantly outcrossing mating system in combination with the limited time elapsed since the population decline. However, population fragmentation is showing the first genetic signs, as the values of genetic differentiation are relatively high, and two well-differentiated genetic lineages have been found even in such a narrow geographic range. These genetic results provide important information when designing conservation management measures. Ne), mating systems, gene flow, clonal structure, colonization history, or the occurrence of evolutionarily significant units, among others. Therefore, genetic patterns should be central in management and recovery plans  is an endemic species with a very small distribution range, being considered by the local environmental authorities as one of the species of most conservation concern in Catalonia (NW Mediterranean Basin). This small Apiaceae was discovered in the late 1970s  have been published to date, developing a set of nSSR has also become a primary aim of the present study.To reverse this trend and to avoid the extinction of Seseli farrenyi; (2) surveying the levels and distribution of the genetic diversity within and among its extant wild populations, as well as of the three ex situ populations; (3) comparing the nSSR genetic variation with the results of Our specific aims are the following: (1) developing specific microsatellite markers for the endangered Seseli farrenyi is a small (up to 30 cm) monocarpic perennial herb with a basal rosette of pinnatisect leaves, and produces abundant compound umbel rays with small white flowers and ovoid seeds of 2.2–3 × 2–2.5 mm  P. Fourn. According to the EU Habitat Directive, it corresponds to Habitat 18.221+ (CORINE) and to HCI (Habitat of Community Interest) no. 1240, which should be object of conservation measures : 38 individuals from the largest population, EBP; 21 individuals from ECM , and three individuals from SES2 (locus classicus (SES1) near the lighthouse , coming from seeds collected by R. Auriault at the locus classicus in 1983. The population (SES1) would have been much larger at that time, as its size was estimated at some 500 individuals in late 1970s  and 12 from the plants grown in the greenhouses of the Sigma-Olot Consortium  under the contract no. PTOP-2017-827. Given the threatened and protected status of this species, no vouchers have been collected this time; however, individuals coming from all sampled populations of this species were extensively vouchered in the past, with good specimens deposited in a high number of herbaria .Given the low population size of rom SES2 ; Table 2rom SES2 , we madeghthouse  could note 1970s ; Table 1® SP Plant DNA Kit , following the manufacturer’s instructions. Microsatellite isolation was carried out by Genetic Marker Services . The protocol for enrichment of genomic library followed S. farrenyi, fifty-eight motif-positive clones were detected and sequenced, of which 22 contained useful repeat motifs sufficiently long as to design pairs of primers. The primer pairs were developed with amplification products that varied between 150 and 280 base pairs (bp) using the software Primer3 ; these 14 loci were selected and universal M13 tails were added for fluorescence labeling .2, 50 μM of each dNTP, 0.45 μM of the reverse primer, 0.012 μM of the extended forward primer, 0.45 μM of the labeled M13 primer  and 1 μL of total genomic DNA.The 14 selected loci were tested with all the collected individuals, both from natural populations and cultivated ones . Amplifications were performed in 12 μL reaction containing 0.5 U AmpliTaq Gold polymerase , 2.0 mM MgClFinally, of the 14 loci selected, only nine loci were amplified for all the individuals sampled. The products of the PCR with fluorescence labeling of these nine primers were separated using an internal standard-size marker in an automatic sequencer ABI 3730xl DNA Analyzer  at the Madrid Science Park (Spain). Fragment analysis was performed using the GeneMarker v. 1.85 .P95); mean number of alleles per locus (A); allelic richness ; number of total alleles per population (TA); number of private alleles (PA); number of rare alleles (those occurring at frequencies below 0.05) (RA); observed heterozygosity (Ho); unbiased expected heterozygosity (He) and FIS). All these parameters were calculated using GenAlEx v. 6.1 ; percentage of polymorphic loci when the most common allele had a frequency of <0.95  iterations in total, INEst was run for each population using two models. The first model (nfb) considered null alleles, inbreeding coefficients and genotyping failures. In the second model (nb) the inbreeding parameter was discarded. According to nfb) fits the data better than the nb model, inbreeding should be considered as the “significant” component of the model and, thus, it explains the observed value of FIS. The INEst program was also used to calculate the parameters Ho and He corrected for the presence of null alleles, and to determine the statistical significance of the FIS values for each locus and population .The programs FreeNA  and INEsNe–4Ne generations, where Ne is the effective population size) in natural populations was explored with two different programs: (1) Bottleneck v. 1.2.02  and the stepwise mutation model (SMM).The occurrence of recent bottlenecks FST values, with and without the ENA  correction for null alleles (K), 20 iterations per K, admixture model, correlated allele frequencies, no locprior, burn-in period of 100,000 and 1,000,000 MCMC replications. The most likely value of K was determined in two ways, as described in K ] and the matrix of the log-transformed geographical distances was carried out with the software NTSYSpc v. 2.2 (m) between natural populations, under the following conditions: 3,000,000 MCMC iterations with a burn-in of 999,999 iterations, sampling frequency of 2000, and delta set at 0.15 .Second, the software GenAlEx was used for performing a Principal Coordinate Analysis (PCoA) at the population level, based on codominant genotypic distances. To assess whether natural populations followed a pattern of isolation-by-distance, a Mantel test  between the matrix of pairwise genetic differentiation [c v. 2.2 . Finallyc v. 2.2  was usedS. farrenyi are the first developed for this species, and nine of them were used for population genetic studies reported in the present article. Properties of the 14 properly amplified polymorphic nSSR markers are summarized in The 14 microsatellite markers characterized for S. farrenyi were considerable  have suffered recent decreases in effective population size . All loci except SFA2 were involved in the detected cases of linkage disequilibrium, and there were no specific pairs of loci overrepresented in this phenomenon. When analyzing population by population, all populations were affected except SES2 and ECM2 (though it has not been possible to apply the test to SES1 because of its insufficient population size), and the SFA2 locus was at least involved in two cases of linkage disequilibrium (one each for ECM and EBP),FST values were similar whether taking the null alleles into account or not . For this reason, the effect of null alleles on the patterns of genetic structure of S. farrenyi could be regarded as negligible. As expected, the matrix of pairwise FST  the EBP population, which showed its own cluster both with K = 2 and K = 3, and (2) the rest of the populations, either assigned to the same cluster or showing admixture of two clusters , suggesting that there is no isolation-by-distance across all populations. Finally, the BayesAss analysis indicated that there were detectable levels of recent gene flow from ECM1 to SES2, and from ECM1 to ECM2  if they are compared with the reference values for plants in general —indeed among the highest values ever found for diploid plants using allozymes. The high levels of genetic diversity detected in S. farrenyi contradict the expectations for Extremely Narrow Endemic species , primarily due to bottlenecks related to the small population size. Reasons explaining the high genetic diversity in S. farrenyi, first revealed with allozymes and confirmed here with nSSR, might include: (1) an ancestor species with an exceptional genetic diversity; (2) a hybrid origin, with potential parents including the taxon for which S. farrenyi has been described as a subspecies—S. elatum L., widely distributed in the NE Iberian Peninsula, and perhaps one of the allopatric species for which S. farrenyi shows clear morphological and ecological affinities—S. bocconei Guss. and S. praecox (Gamisans) Gamisans; (3) the fact that S. farrenyi occurs within one of the main glacial Mediterranean refugia  populations of S. farrenyi are even more evident when observing the total number of MLG, as all the studied individuals except two from the ex situ populations (JBB9 and OLO7) show their own multi-locus genotype , these genetic bottlenecks are in agreement with the demographic bottlenecks that have been observed for the last two decades, with reductions in the population size of up to 90% (locus classicus (SES1 subpopulation), which would have been continuously declining since its discovery, 40 years ago. Moreover, some individuals would have existed in-between SES and ECM populations , probably connecting them. It seems, therefore, that S. farrenyi has not suffered large losses in its genetic diversity in spite of the documented population size declining, at least since the early 2000s when the species was assessed with allozymes.The high levels of genetic variability of genotype ; in othegenotype . Althougp to 90% . The mosS. farrenyi. First, outcrossing may “protect” somewhat against the effects of genetic drift because alleles are less likely to be eliminated; the reason is that in outcrossing species alleles tend to be shared in several individuals/populations thanks to increased gene (pollen) flow compared to selfing species  instead to Ne . As one might anticipate, the JBB and OLO populations show levels of genetic variation close to the donor population, although, unexpectedly, the JBB and OLO populations combined harbor seven exclusive alleles. It is likely that these seven alleles would have been extinguished since 2008, but it is also possible that they were not sampled for the present genetic study; given the threatened status of this species, the smallest individuals of each population were not sampled in the summer of 2017 to ensure their survival.S. farrenyi, including the ex situ ones JBB and OLO, are considerably inbred. One might speculate that both JBB and OLO would have “inherited” the inbreeding pattern characterizing the donor subpopulation over one decade ago, when seeds were collected. Although such a subpopulation does not exist at present (it was placed between the current ECM1 and ECM2 subpopulations), it would have suffered from the strong genetic substructuring of populations  that occurred in all populations at the beginning of this century: about 45% of the genetic diversity was due to differences between the subpopulations within each population , in agreement with the results of the tests for recent bottlenecks provided in FST values should be regarded as unexpectedly high in S. farrenyi, given that (1) populations are separated by just a few km, (2) they reproduce mainly by outcrossing, and (3) the gene flow could be favored by the strong northern winds prevailing in the region (tramuntanades) helping anemochory. This relative genetic isolation could, therefore, be interpreted as a sign of population fragmentation. It can be anticipated that these values could increase over the generations as the demographic bottlenecks are translated into genetic bottlenecks.The S. farrenyi. It is likely that all populations were connected in the past, either forming a macro-population which would include all extant populations and subpopulations, or with the presence of intervening populations or subpopulations, today extinct. The fragmentation between SES and ECM populations is fairly recent . The BayesAss analysis, which has been used to explore the recent gene flow between the four genetic natural populations of S. farrenyi  has revealed that there are still genetic exchanges between the eastern ones. The signs of such population fragmentation in S. farrenyi would have already been detectable in the former allozyme-based study (S. farrenyi (EBP and ECM) are roughly assigned to alternative genetic clusters, while SES1 shows a pattern of admixture , the size of SES1 was still of 90 individuals  is a quite unexpected result, and we believe that it could be an early sign of population fragmentation in ed study , as the ed study . Similardmixture . Today, dmixture ; such “fividuals . The ongS. farrenyi (particularly if the recovery plan is readily implemented), it is important to take into account the existence of these two genetic lineages, already present almost two decades ago. As stated in the “Introduction”, the individuals used in a reintroduction can be inappropriate if the donor population is highly genetically divergent, because this might involve breakdown of the co-adapted gene complexes; such processes would lead to outbreeding depression  and reported in the last 10 years from the same coastal strip  or in close coastal areas  Lassen in Mallorca—As for the restoration of pression  and, finpression . The gen effects , but at  effects ; one migS. farrenyi is a monocarpic species, seed collection should be done with extreme care, ensuring that only a small percentage of the total production of seeds of an individual is taken; the episodic falls on the ratio of reproductive/vegetative individuals  stresses the need to be even more cautious. Collecting all seeds of a given individual would involve most certainly the loss of one of the species’ multi-locus genotypes , which could represent the loss of particular alleles in the case in which they were only present in that individual. Apart from urgent rescue actions, creating an experimental (ex situ) population is also highly recommended, where stocks coming from the two identified genetic lineages would be mixed, and where individual fitness and demographic viability can be compared with those of non-admixture reinforced populations. According to S. farrenyi.Future reinforcements to EBP, in contrast, should be carried out with material obtained from this population and not from other sources. Since S. farrenyi are still high, in spite of its narrow range and its accelerated population decline. Genetic data reported in this article have resulted  in a first contribution to a genetic monitoring program. Our results also demonstrated, again unintentionally, the value of ex situ collections in conservation genetics, as several alleles that are presumably lost in natural populations have been conserved, thanks to the germplasm and living collections of botanical gardens. This being the starting point, a recovery plan could include both wild subpopulations and the new experimentally created mixed population as monitoring subjects, because S. farrenyi has shown enough variation in genetic markers along time to reveal significant changes. An adequate managing of the system of wild/experimental populations can provide the recovery of genetic diversity levels. If genetic indicators are included in recovery success evaluation, the project could thus be considered as a genetic reserve within a protected area (Our main finding is that levels of genetic diversity in the natural populations of ted area  for wild10.7717/peerj.10521/supp-1Supplemental Information 1Raw allozyme data  was geneClick here for additional data file.10.7717/peerj.10521/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.10521/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10521/supp-4Supplemental Information 4Click here for additional data file.


Treptacantha elegans, a large Mediterranean brown macroalga, broadened its depth range to deeper and exposed environments and displayed an unexpected range expansion along the northern coast of Catalonia over the last two decades. Here, we reconstruct the spread of T. elegans in time and space and unravel ecological and demographic traits such as population dynamics and genetic patterns to provide a comprehensive and integrated view of the current status and geographical expansion for this species. Fast-growing dynamics, early fertile maturity, and high turnover rate are the main competitive advantages that allow the exposed populations of T. elegans to colonize available substrata and maintain dense and patchy populations. We also provided evidence that the deeper and exposed populations of T. elegans constitute a single group across the Catalan coast, with little genetic differentiation among populations. This seems to support the hypothesis of a unique source of spread in the last decades from the Medes Islands No-Take Zone towards both southern and northern waters.The widespread decline of canopy-forming macroalgal assemblages has been documented in many regions during the last decades. This pattern is often followed by the replacement of structurally complex algal canopies by more simplified habitats . Against all odds, the fucoid  Species may redistribute to keep their preferred environmental conditions6 or adapt their physiology as a response to global and local stressors7. Macroalgae are dominant primary producers and ecosystem engineers that provide structure and ecosystem services 10 to many temperate and sub-temperate shallow coastal habitats where they play a key role in ecosystem functioning12. As a response to global and local stressors , they may show both geographical expansions, when species colonize new habitats, or contractions, when populations disappear from areas previously inside their distributional range13.How marine populations persist, evolve, and change their geographical distributions as a response to global change is one of the main questions in contemporary ecologyCystoseira sensu lato fucoids  are late successional species and among the major canopy-forming macroalgae. They provide three-dimensional structure, food, and shelter to many associated species15. Decline of these species may drive to a decrease in habitat complexity with important consequences for benthic biodiversity and ecosystem functioning18. All Cystoseira sensu latospecies, except C. compressa, appear in the Annex II of the Barcelona convention (2010) among those taxa that are considered threatened or endangered and need protection measures19. Cystoseira sensu lato species show limited dispersal abilities and propagules often disperse over a few meters20. This leads to the existence of monospecific stands near parent populations21. This trait combined with local environmental factors may explain the lack of connectivity for many populations of T. elegans in different areas of the Mediterranean Sea23, something that seriously jeopardizes their conservation. Nonetheless, the few studies on population genetics suggest that water currents may play a significant role in long-distance dispersal (presumably via drifting of detached reproductive branches) of Cystoseira sensu lato populations26. Thus, despite the well-documented general decline of local populations27, evidences for stability and even expansion have been also reported in some areas29.In the Mediterranean Sea, Treptacantha elegans (Sauvageau) Orellana and Sansón (until very recently Cystoseira elegans14) is a Mediterranean endemic species. The Mediterranean Sea is oligotrophic, with relatively high salinity, high summer temperatures, and extremely reduced tides, which are mainly regulated by the atmospheric pressure30. T. elegans was first described by Sauvageau for Banyuls-sur-Mer (South-Eastern France)31. This species typically inhabits shallow waters down to 2–3 m depth31, where forms extensive stands at the innermost rocky bottoms of coves and other shallow environments characterized by low to medium water movements32. Recent studies have reported a significant regression of T. elegans in shallow and sheltered environments from the N–W Mediterranean Sea23. However, in the past two decades, some populations of T. elegans  have expanded their distribution in the Medes Islands No-Take Zone (NTZ) and the nearby areas (North-Western Mediterranean Sea), where the species can be now found in wave-exposed habitats located between 5 and 15 m depth, with some sparse thalli at 20 m depth. The reasons for this pattern are yet to be determined.T. elegans and the species population ecology; (2) assess its historical and current distribution in the Catalan coast; and (3) provide an estimate of the patterns of genetic diversity and differentiation among populations including ancient and more recently established zones.Here we investigate this recent spread throughout the North Catalan coast. Concretely, our goals are: (1) provide insights about the main traits of deeper and exposed populations of 33, minimum winter temperatures were recorded in February and March  and the maximum in August  just before the gradual drop throughout autumn.The seawater temperature in the north Catalan coast is characterized by strong seasonal variability. In 2018, the mean annual temperature at 5 m depth was 17.27 °C ± 1.26 (Mean ± SE). In accordance with the well-known thermal regime of this areaTreptacantha elegans from deeper and exposed infralittoral habitats (5–15 m depth) began to grow from the perennial basal disc (or holdfast) in early spring when sea water temperature starts to rise  over the three years. No differences were found in density  and mean size of individuals  across the three sites. When grouped into 1 cm size classes, the size distribution of T. elegans individuals mostly ranged from 1 to 23 cm . The mean growth rate within first-year individuals was 8.6 cm ± 0.6 (Mean ± SE). Some specimens could reach relatively large sizes, being 28 cm the maximum growth observed for an individual recruited the previous spring. New, annually appeared recruits were about one-third of the total counts in the permanent plots , 82% of which exceeded the fertile minimum size (4.5 cm). Mean annual mortality rate within the 3 populations was about 36% . Small specimens showed higher annual mortality rates than the larger ones  resulting from heterozygote deficiency  revealed that most genetic variation occurred within populations . Allelic richness was higher in the Medes Islands NTZ population and lowest in the northern population  has been observed during the following 15 years. The southern (Begur) and northernmost (Messina) populations analyzed here have not been described until 2010 and 2011 respectively, even though regular monitoring was carried out in these locations. Hence, we assume that the Medes Islands may represent the source population from which the species expansion across the exposed shores in the Catalan coast begun. The Medes Islands population also exhibited the highest allelic richness, which supports our assumption of it being a mature population thus a reasonable source population. As in other Cystoseira sensu lato species, our results may suggest a common ancestral gene pool but loosely connected populations41. This is supported by the patchy distribution of T. elegans, a pattern likely due to the scattered presence of rocky and suitable habitats in the area and the limited dispersal range of gametes in fucoids42. Unfortunately, our data are not sufficient to detect a clear correlation between distance and genetic differentiation within populations.Our molecular analyses of the three exposed and deeper T. elegans43, here we show the existence of two populations 30 km away from the Medes Islands NTZ. This may be explained by the fact that this species, as other fucoids, is capable to disperse through detached or drifting fertile parts of mature individuals44 or by animals18. This may explain the southern spreading through the Northern Current (NC), which flows with a permanent southwestward circulation in the Northwestern Mediterranean basin45. Although a NC mediated spread in the opposite direction is unlikely, other processes such as wind-induced currents or extreme meteorological conditions play important roles in the water circulation of this complex hydrodynamical region. This may explain the passive transportation of T. elegans from the exposed shores of the Medes Islands to the northern studied exposed areas46.While the dispersal distance of early life stages is less than 10 m from source populations in 7. Morphological differences have been reported in T. elegans individuals between populations from sheltered and exposed infralittoral habitats23 such as the presence of thicker and spinier branches in the individuals from exposed shores, a trait that may be the result of a phenotypic plasticity response (acclimation rather than adaptation) to such environments, as no evidence for genetic differences has been detected in preliminary assays of these morphotypes . The potential source of the first deeper T. elegans population observed in the exposed shores of the Medes Islands is unknown. However, it may have arrived from the original shallow population in Banyuls-sur-Mer (France), where this species was described for the first time31, or from other shallow littoral populations from the north of Catalonia where the species was more abundant in the past23.Increased tolerance and adaptation and/or migration to different locations within their specific niche are the main responses to environmental changes in macroalgaeCystoseira sensu lato populations in the Mediterranean Sea47. Evidence of a general improvement in coastal water quality, particularly during the 1990s48, is a possible explanation for the expansion of Cystoseira sensu lato species in the studied area49. Besides, the Medes Islands NTZ might have worked as a refuge for T. elegans populations during the period of improvement of environmental conditions, mainly because of the lower density of herbivores due to the predatory fish control in the reserve40. In addition to these local stressors, global warming and extreme climatic events (such as storms and heatwaves) are also threatening marine coastal life, especially in shallower waters50. The increase in sea-surface temperature over the last decades52 may have led to species responses in macroalgal assemblages such as the observed here53. While we know that T. elegans may withstand high summer temperatures as an adaptation to the shallow environment23, our results suggest that a shift in the depth-range distribution and a subsequent acclimation to the new environments may be a result of warming.Several environmental drivers and the interaction of some favorable conditions may have promoted the spread observed. Seawater eutrophication is one of the major causes for the disappearance of Cystoseira sensu lato species to persist or even widen their geographical distributions under global change scenarios. For this to happen, however, it is important to improve the water quality and to protect population sources that ensure dispersal like those found in the Medes Islands NTZ, especially in organisms with short-dispersing propagules. Finally, given its fast and stable population dynamics (early fertile maturity and high turnover rate), we highlight that T. elegans represent an ideal organism to conduct habitat restoration actions in previously degraded systems such as sea urchin barrens or turf-dominated habitats43.Our results shed new light on the ability of a Cystoseira sensu lato species54. However, as Treptacantha elegans is characterized by a tiny main axis (1–3 cm length55), this trait hinders accurate measurements in situ unless invasive techniques are used. To discern the best less invasive size indicator for in situ measurements, we collected 27 specimens of Medes Islands NTZ and measured several morphometric parameters. Correlation analyses between the parameters showed high correlation between the maximum length of the longest axis and the rest of the morphometric variables measured  throughout the annual temperature cycle, 20 T. elegans individuals covering a wide size range (2–23 cm) were sampled monthly in the Medes Islands NTZ in 2018. Their fertile maturity status was discerned by pooling the individuals sampled within three stages: (1) immature individuals without receptacles; (2) individuals beginning their reproductive period, with receptacles but still immature; (3) fertile and reproductive individuals. Annual seawater temperatures were assessed by high-resolution hourly temperature recordings at 5 m depth in the Medes Islands obtained from the T-MEDNet platform (https://www.t-mednet.org).To accurately describe the T. elegans from exposed shores (5–15 m depth), we performed a survey at the Medes Islands NTZ in 2017, 2018, and 2019 on three populations. These populations thrive a few hundred meters apart  were placed a few meters apart at around 10 m depth. Thanks to the high density of T. elegans populations in the patches, these tiny 6 permanent plots allowed to monitor 684 specimens. Generalized linear mixed models (GLMMs) with Poisson error distribution were applied to test the similarity of the three selected sites. Density and mean size of T. elegans individuals in 2017 were treated as independent response variables, the site as the fixed response variable and the plots as the random response variable. For the two fitted models we applied a Type II Wald χ2 test to show a non-significant effect of sites. This led us to pool together all specimens from the three sites of the Medes Islands location  fitted with a negative binomial distribution and a logarithm link function after visually checking the most likely distribution of the data and residuals58. GLM analyses were conducted using the package “MASS” for R software59 and GLMMs using the package “lme4”60 with R version 3.3.361.To untangle the population dynamics of Treptacantha elegans in the Catalan coast has been recently reported23. Although descriptive studies have been carried out in the shorelines here investigated since the seventies63, T. elegans was not found in deeper and more exposed environments until the nineties, when Sala reported its presence37 (as Cystoseira spinosa23) for the Medes Islands. All available information about the spatial and temporal distribution of this species along the Catalan coast is reported in Table Past and present distribution of shallow (≤ 1 m depth) and sheltered Treptacantha elegans samples corresponding to branches from 20 individuals haphazardly selected at each location, at a maximum distance among samples of 10 m. The samples were individually preserved by drying them in silica gel until DNA extraction.Samples for molecular analysis were collected in three study locations along the Catalan coast, thus covering the whole distribution range for the study area  were used for genotyping. Polymerase chain reactions (PCRs) were performed in 15 µL total volume containing 1· GoTaq Flexi buffer , 1.5 mm MgCl2, 0.07 mm each dNTP, 0.4 µM of labelled  forward primers, 0.4 µM of reverse primers, 1U GoTaq Flexi DNA Polymerase (Promega), and 5 µL of 1:100 diluted DNA template. In all PCRs, an initial denaturation step  was followed by 30 cycles of 95 °C for 30 s, a primer specific annealing temperature (Ta) for 30 s and 72 °C for 30 s, ending with a final elongation step at 72 °C for 5 min. Amplified fragments were separated using an ABI PRISM 3130 xl  automated capillary sequencer. Alleles were manually scored in STRand64 using the GeneScan 500 LIZ dye size standard (Applied Biosystems).Genomic DNA for each population was extracted from the dried samples using the NucleoSpin Plant II kit  according to the manufacturer’s protocol. Eight microsatellite loci formerly developed for 65. When evidence of null alleles was significant, a correction for null alleles was applied. Estimates of genetic diversity were calculated for each locus and population using the GENETIX software v. 4.0566. This included the mean number of alleles per locus , the non-biased expected heterozygosity (HE), the observed heterozygosity (HO), the number of private alleles, and the inbreeding coefficient (FIS). Pair-wise differentiation, as assessed with FST67, was calculated. Analyses of molecular variance components (AMOVA) were conducted using ARLEQUIN v3.168.All individuals with missing data at three or more loci were excluded from analyses. These corresponded to 4 individuals, 2 from the southern population (Begur) and 2 from the norther one (Messina). Identification of genotyping errors due to null alleles, large allele dropout and the scoring errors were screening with the MICRO-CHECKER software v. 2.2.369 considering admixture and no a priori population assignments. The correlated allele frequency model was run with a burning time of 250,000 repetitions, 1,000,000 iterations and considering a range of genetic clusters (K) from 1 to 5. The model was run 14 times for each K to check the consistency. The best K was estimated using the program Structure Harvester70 which ranks K’s according to Evanno method71.Population structure was inferred using STRUCTURE v2.444Supplementary information.


Much like humans, chimpanzees occupy diverse habitats and exhibit extensive behavioural variability. However, chimpanzees are recognized as a discontinuous species, with four subspecies separated by historical geographic barriers. Nevertheless, their range-wide degree of genetic connectivity remains poorly resolved, mainly due to sampling limitations. By analyzing a geographically comprehensive sample set amplified at microsatellite markers that inform recent population history, we found that isolation by distance explains most of the range-wide genetic structure of chimpanzees. Furthermore, we did not identify spatial discontinuities corresponding with the recognized subspecies, suggesting that some of the subspecies-delineating geographic barriers were recently permeable to gene flow. Substantial range-wide genetic connectivity is consistent with the hypothesis that behavioural flexibility is a salient driver of chimpanzee responses to changing environmental conditions. Finally, our observation of strong local differentiation associated with recent anthropogenic pressures portends future loss of critical genetic diversity if habitat fragmentation and population isolation continue unabated. Lester and colleagues use faecal samples and genetic analyses to investigate the genetic connectivity across chimpanzees. Their results indicate that the global pattern of genetic diversity in chimpanzees is largely characterized by a pattern of isolation by distance with several isolated populations exhibiting strong local differentiation. Our species has largely relied on behavioural flexibility4 to become among the most widely distributed species, inhabiting a diverse range of climates and habitats5. Among our closest living relatives, the chimpanzee (Pan troglodytes)6, also exhibits extensive behavioural variation, both at a local and regional scale, and occupies a broad range of habitats and climates, while displaying little associated morphological variation8. However, to date, their pattern of genetic diversity is equivocal: some studies provide evidence of connectivity among all populations10, while others have concluded that chimpanzees are taxonomically divided into four geographical subspecies15. Given the high degree of behavioural variability and flexibility across chimpanzee populations, characterizing range-wide patterns of genetic diversity in chimpanzees is important for understanding how they adapt to changing environmental conditions16. In particular, a signal of genetic connectivity across their range would suggest that, like in humans, local trait fixation is relatively slow in chimpanzees, and behavioural flexibility allows them to quickly and dynamically respond to ecological challenges.Humans have been characterized as a genetically continuous species18 chimpanzees were nearly continuously distributed across Equatorial Africa7. Rivers appear to be the main barriers to migration7 separating chimpanzees and bonobos (Pan paniscus), as well as three of the four currently recognized chimpanzee subspecies: Nigeria–Cameroon (Pan troglodytes ellioti), central (Pan troglodytes troglodytes) and eastern (Pan troglodytes schweinfurthii) chimpanzees19  and P. t. ellioti, is also dynamic, as this region hosted rainforest as recently as 4 ka BP . Interestingly, discrete geographic clustering of morphological traits along subspecies lines has not been conclusively shown25 and, although chimpanzees do show substantial behavioural variation across populations, to date, there are no universal subspecies-specific behaviours, i.e., accumulative stone throwing is unique to P. t. verus, but has only been observed at a few study sites26.Prior to the recent growth and expansion of human agriculturalist settlements across Africa (ca. 5–2 ka BP)27. Detectable departures from this pattern are evidence of geographic or behavioural barriers that reduce or impede dispersal, thereby leading to increased geographical rates of genetic differentiation. Although chimpanzees are thought to have had a historically continuous geographic distribution across their range7, studies of chimpanzee genetic diversity have tended to show discontinuities consistent with the subspecies classifications13, or some degree of population structuring14. To detect geographic clustering of genetic data, these studies relied on several different ‘spatially agnostic’ approaches not accounting for the spatial distribution of samples. These include Bayesian clustering algorithms , the results of which can be biased when IBD is present in the data31 and principal component analyses, a method specifically intended to maximize between-group differences32. However, to date no studies of species-wide chimpanzee population structure have featured spatially explicit methods, which incorporate the geographic location of the genetic samples into the analysis model and assume IBD in the null model33. Importantly, when using spatially agnostic methods in the presence of IBD, unbalanced sampling will detect discrete stratification of the data, regardless of the actual pattern36, and this cannot be compensated for by analyzing more loci 37. Mostly due to the logistical challenge of obtaining non-invasive samples from chimpanzees’ wide range, which largely encompasses politically unstable or remote regions, previous studies of chimpanzee population structure have relied on small and clustered datasets, and often made use of zoo and sanctuary samples of unknown provenance, which introduced further uncertainty15. Therefore, previous reports of chimpanzee population structure were likely biased by applying spatially agnostic analyses to datasets composed of dispersed and uneven sampling in the presence of IBD33, and were possibly further confounded by incorrect knowledge about populations of origin in some samples. In fact, even the most comprehensive studies of chimpanzee population history, which utilized genome-wide sequence data15, were based on a relatively small number of samples from captive individuals, and focused more on fitting models of divergence among subspecies than investigating recent genetic connectivity in space.In a continuously distributed population, limited dispersal drives a predictable pattern of genetic diversity, known as isolation by distance (IBD), which manifests as a continuous gradient (cline) of declining similarity as geographical distance increases38, over an 8-year period, we non-invasively collected and genotyped >5000 wild chimpanzee faecal samples from 55 localities in 18 countries across the entire species range. We scored individual genotypes  at up to 14 microsatellite markers, as these are cost effective and allow for accurate genotyping of non-invasively collected samples39. Previous studies in other species  have demonstrated that 6–14 microsatellite loci are informative enough to even detect subtle population structure. Furthermore, a previous study of how sampling affects detection of chimpanzee population structure found that eleven loci were sufficient to overcome false signals of population structure caused by sampling bias and uncover a signal of IBD9. As microsatellite markers evolve rapidly and are expected to be selectively neutral, they are highly sensitive to patterns of gene flow, especially within shallow evolutionary timescales. For our analyses, we modeled our georeferenced genetic data in space by employing spatially explicit approaches to inform the distribution, thereby avoiding a priori definitions of the population structure or assumptions of homogeneous sampling, thus minimizing biases associated with previous studies.In this study, we aimed at assessing recent genetic connectivity across the chimpanzees range by analyzing an extensive sample providing an unprecedented level of geographic coverage. As part of the Pan African Programme: The Cultured Chimpanzee (PanAf)n = 69) noticeably reduced differentiation across analyses.The PanAf team and collaborators collected 5397 chimpanzee faecal samples from 55 temporary or long-term research sites spanning the geographic range of chimpanzees from 2010 to 2018 Fig.  and reprD). Rousset47 showed that, in a linear one-dimensional (1d) space, FST/(1 – FST) ~ D and FST/(1 – FST) ~ log(D) in an infinite two-dimensional (2d) space, when mutation is negligible. In principle, one may statistically fit these equations to actual data and interpret deviations from fits as signals of non-uniform spatial patterns of density and gene flow.In an idealized model with uniform population density and dispersal, genetic differentiation among local demes is expected to increase with geographic distance 48, which leads to an overestimation of diversity. While a strict SMM lends itself to unbiased estimation of demographic parameters from microsatellite variation , it rarely accurately describes the actual mutation process at microsatellite loci. Constraints to the maximum and minimum size number of repeats at a given marker50 are especially important, as they determine an upper limit to the genetic differentiation among pairs of populations. With these limitations in mind, modeling genetic differentiation as a function of geographical distance is still a powerful tool to identify discontinuities in the spatial structure of biological populations.Actual geographical spaces are neither 1d nor infinite 2d spaces, and populations inhabiting an elongated 2d space are expected to show relationships of G ~ D) in which subspecies were not defined, we found that D explained >30% (adjusted r2 = 0.314) of G, and as much as 58% (adjusted r2 = 0.579), when three obvious outlier sites were removed (Mt. Sangbé (Côte d’Ivoire), Gashaka (Nigeria) and Issa (Tanzania)). These sites were consistent outliers in all comparisons and analyses ) gave very similar results , as it could be expected given that chimpanzee range’s shape is fairly elongated and somewhat intermediate between a 1d and a 2d space.In a global, unstratified test of our data  and there was no clear IBD pattern within P. t. schweinfurthii . Notably, between-subspecies regressions involving P. t. verus  were essentially flat, suggestive of saturated genetic differentiation at our markers, while the P. t. troglodytes–P. t. schweinfurthii regression was highly similar to within-subspecies regressions, with coefficients in-between P. t. troglodytes and P. t. schweinfurthii in the linear regression and almost identical to within P. t. troglodytes in the log-transformed regression, Fig. Full-range data were then stratified according to whether the two sites in each pair belonged to the same or different subspecies Fig. . Howevernge Fig. , but rat51. This analysis combines spatial and genetic data and relies on MCMC to estimate relative effective migration rates  modeled on a 2d surface, using a mean-centred relative scale. In this model, a ‘flat’ uniform migration surface would indicate perfect IBD across the considered range, and non-uniformity of m in space is interpreted as increased or decreased genetic connectivity. Besides plotting estimated migration surfaces, EEMS results differentiate areas where the probability of m differing from the mean rate is statistically significant . Importantly, the EEMS model assumes that microsatellite markers mutate according to a strict SMM and violations of this assumption may lead to an overestimation of effective migration rates, while homoplasy caused by allelic size saturation may lead to an underestimation. Despite having some deviations from strict SMM and the presence of homoplasy that are apparent in our data, we found the EEMS model to be a good fit to our chimpanzee dataset  softwarem, we did not detect any significant signal of genetic discontinuity, or ‘barriers’ between-subspecies populations  and two patches of significantly high m were identified in the most densely sampled areas of the P. t. verus range. In contrast, significantly low migration was inferred for the region surrounding the Gashaka sampling location within the range of P. t. elliotti and in the mountainous regions of southern Uganda and Rwanda centered around Gishwati in the P. t. schweinfurthii range.Although we observed spatial variation in ons Fig. . Consistels Fig. , m was gm and the considerable contrast between P. t. verus and ETS may subdue more local patterns, we also performed separated analyses for P. t. verus and for the remaining ETS populations , given the high degree of topological, climate and habitat variation in West Africa  from EEMS. At full scale, q in P. t. verus populations were significantly lower than species average  and Côte d’Ivoire (Djouroutou). A focused analysis of ETS  in the P. t. schweinfurthii range also displayed significantly lower q.We visualized spatial patterns of chimpanzee genetic diversity by plotting estimated relative diversity rates  in a geographically clustered subpopulation will appear to have a relatively high migration rate relative to the rest of the sampled populations. Therefore, our observation of relatively high effective migration throughout P. t. verus may indicate higher levels of connectivity in western chimpanzees, but may alternatively or additionally be a signal of a relatively recent (likely northward) population expansion. Indeed, much of the P. t. verus range is savannah mosaic habitat with discontinuous gallery forests53, where chimpanzees tend to have both (1) larger home ranges (over 63 km2)8 than forest-dwelling populations, to seemingly compensate for lower fruit tree density and suitable habitat availability, and (2) a concomitant increase in dispersal distances55, thereby possibly driving a signal of increased migration. However, effective migration was uniform across most of the P. t. verus range, as similar rates were also detected among the forest-dwelling populations in southern Liberia and western Côte d’Ivoire, where home ranges have been reported to be between 6 and 37 km2 in size54, suggesting that dynamics other than ecology are involved. This and, especially, the low diversity in P. t. verus, suggest that the high effective migration we observed in this subspecies should be interpreted as evidence of a population bottleneck followed by a recent and fast range expansion. Indeed, this interpretation is corroborated by among-site comparisons of mean standard deviation of allele sizes  and Garza Williamson’s M , are suggested to have experienced a more recent bottleneck , with strong local effects driving differentiation in a few highly isolated populations. Importantly, we did not find evidence of major discontinuities conforming to the current taxonomic subdivision. However, although effective barriers were not evident in our data, the P. t. ellioti range, between P. t. ellioti and P. t. troglodytes, and in northwestern DRC between P. t. troglodytes and P. t. schweinfurthii. However, these gaps were of moderate extension and spatial homogeneity in sampling is not an assumption of either the EEMS analysis or our IBD regression, so that it is extremely unlikely that sampling gaps represent a main influence on our results. Our analyses, therefore, indicate that previous observations14 of recent genetic discontinuities in chimpanzees likely resulted, at the minimum, from the use of spatially agnostic methods that were biased by geographically clustered genetic samples. Notably, these results do not at all suggest an overall lack of genetic differentiation among chimpanzee populations set apart by geography. Though we found that genetic variation follows a generally clinal pattern across the species, we want to stress that we were specifically focused on patterns occurring on a recent timescale, i.e., within the current interglacial period. Populations are spatially and temporally dynamic, and it is likely that the range of suitable habitat for chimpanzees has fluctuated with the glacial–interglacial cycles, causing repeated population contractions and subsequent expansions58. It is important to remark that our analyses utilized microsatellite markers, which, mainly due to both their high mutation rates and polymorphism, can be very effective at revealing subtle genetic structure. However, fast mutation rate, coupled with constraints on maximum and minimum allele size, restrict the information that microsatellites may provide about deeper demographic events, leading to a shallow effective timescale. In particular, a deep divergence between populations, e.g., chimpanzee subspecies, may appear relatively shallow if there are heavy constraints on allele size (which causes differentiation to become asymptotic). However, if previously isolated populations came into contact in the more recent past, demes that are closer to the interface will exchange more migrants, and thus more alleles. The admixed alleles would diffuse along a gradient, since they would become more dispersed as distance from the interface increases. In such cases, recent gene flow and the relative underestimation of deep divergences by microsatellites may, in principle, create a smooth pattern of IBD, whereas other markers might otherwise reveal steeper gradients. This makes microsatellites highly effective for assessing recent patterns of population structure, as signals of ancient contraction–expansion events are flattened by allele size constraints and relatively high mutation rates. Finally, we wish to stress that gradients are not expected to occur by chance alone. Therefore, observations of IBD in our data represent, at least, clear evidence of recent connectivity among populations.Our estimates of effective migration rates did not detect significant discontinuities corresponding with the Dahomey Gap, the Sanaga or Ubangi Rivers, the three proposed subspecies-delimiting barriers. Although our sampling scheme endeavoured to collect data from as many localities as was practicable, unavoidable sampling gaps occurred in the 62. Therefore, their observed differentiation most likely stems from loss of alleles due to random drift in extremely small populations.The chimpanzee populations that appear considerably more differentiated than expected by IBD alone, e.g., Mt. Sangbé, Gashaka and Issa, are known to have experienced isolation and decline in recent generations as a consequence of local anthropogenic pressures2. Therefore, as humans and chimpanzees are both widely distributed across a variety of habitat types, evolutionary adaptation is expected to be slow in these species, especially given their long life histories. Hominins have long employed behavioural flexibility to mitigate biological limitations, exploit new resources5, inhabit challenging environments  and migrate out of Africa on several occasions before modern times5. Since our results have demonstrated that recent genetic variation in chimpanzees contains little geographic structure on a broad scale, behavioural flexibility may also play an important evolutionary role in their adaptability to environmental heterogeneity63. Chimpanzees have diverse and variable behavioural repertoires65 that often vary among nearby communities66, they utilize learned techniques to harvest otherwise unobtainable foods67 and their bevavioural diversity increases with environmental variability68. Indeed, chimpanzees living in arid habitats employ specialized behaviours to regulate the thermal exposure8 and spontaneously innovate novel behaviours in response to increased environmental complexity arising from anthropogenic pressures71.Our data suggest that, much like humans, chimpanzees likely experienced periods of substantial genetic connectivity across a geographic range encompassing a broad range of habitats, a finding that may point to important evolutionary implications. Environmental heterogeneity and population size act synergistically to influence rates of adaptive evolution. Advantageous mutations are more likely to become fixed in small populations occupying uniform habitats, and are less likely to become fixed in a large population occupying a diverse habitat74. P. t. schweinfurthii, P. t. troglodytes and P. t. ellioti are recognized as endangered19, while P. t. verus is recognized as critically endangered73. Though our results indicate a high level of recent genetic connectivity in chimpanzees, we do not suggest that the distinction among the subspecies populations is irrelevant. Each subspecies range hosts populations that contribute unique behaviours to the species as a whole, and our findings suggest that genetic diversity in chimpanzees is distributed throughout the taxon, underscoring the need to maintain regionally driven conservation approaches. However, these results also highlight the need to preserve and restore corridors to facilitate connectivity among remaining populations to avoid inbreeding depression and the accumulation of deleterious alleles that negatively affect fitness. All populations possess unique traits that cumulatively reflect the full genetic and behavioural diversity observed in chimpanzees. Continued isolation and further population decline will irrevocably erode the overall viability of this critical keystone species, ultimately imperiling their long-term fitness. If connectivity between populations is not restored or deteriorates, the geographic distribution we observe in chimpanzees today will shape the genetic structure of future generations75, which may have negative evolutionary consequences2, and ultimately jeopardizes the long-term survival of wild populations.Although our data point to diffuse genetic connectivity in chimpanzees, this signal originates from recent historical patterns in previous generations and does not represent the current potential for gene flow. Indeed, we found that several local chimpanzee populations are very strongly isolated from a genetic perspective, and many more are known to have become fragmented and/or have undergone precipitous declines in recent decades due to anthropogenic factors, such as habitat fragmentation, hunting and disease transmission76 and stored in the field for up to 2 years. Upon arrival to the lab, samples were stored at −20 °C. DNA extracts were isolated either manually, using the QIAamp Stool Kit (Qiagen), or using an automated process employing the QIAamp 96 PowerFecal QIAcube HT robot (Qiagen), per manufacturer instructions, modified by incorporating a pre-treatment step to improve the DNA quality and yield  was used to calculate allele frequencies, determine the minimum number of loci needed to discriminate individuals (PIDsib)79, and to assess the degree of relatedness of individuals within sites (Supplementary Note 79. In cases where genotypes were unique (not matching to any other genotype), individuals were typed at a minimum of seven loci.Cervus 3.0.7  we examined the relationship between genetic and geographic distances by applying simple linear regression functions to multiple levels of our data, and 2) we used a spatially explicit analysis that identifies significant spatial deviations from clinal variation (genetic discontinuity). We also performed Mantel, partial Mantel and STRUCTURE analyses on the dataset, as they are standard practice in population structure studies; however, reliable inferences could not be drawn as results were severely confounded by biases in the data. Instead, we provide in-depth details about the modeling and results of these analyses in Supplementary Note  we used G ~ D) to assess the relationship between genetic distance (F′ST/(1 − F′ST))80 and geographic distance  at the species, between and within-subspecies and subspecies-comparisons levels. For our analyses, we calculated F′ST as a suitable estimator of FST, which adjusts for differing levels of genetic diversity arising from variation in effective population sizes (Ne) among sampling locations and mutation rate among loci, while not being dependent on within-site diversity80. F′ST/(1 − F′ST) (hereafter G) was modeled as a function of LCP estimators of D, which calculate pairwise distance between sampling localities based on the presence of suitable chimpanzee habitat spanning back 120 ky BP57, and include topological features that could cause resistance to gene flow. We used LCPs specifically to force distance measurements around the presence of large water bodies, such as the Atlantic Ocean, where chimpanzee migration is not possible  and relative effective migration (m) rates, therefore, high levels of genetic differentiation appear as divergent from these means.To test for effective barriers to migration and to plot the distribution of chimpanzee genetic diversity across their range, we used the spatially explicit EEMS program51, demonstrated that a single highly polymorphic locus was able to replicate qualitatively similar effective migration surfaces as when using their entire panel of loci. More importantly, this locus was able to correctly identify the barrier and differentiation between two geographically proximate and closely related species, which is particularly notable as they are known to hybridize where their respective ranges overlap81. In a study of three closely related sympatric beetle species that inhabit forested areas surrounding Kyoto, Japan, the authors used EEMS to examine the spatial distribution of genetic diversity in the presence of habitat discontinuity46. Using a range of nine to ten loci, they detected discrete spatial structuring in two out of the three species, which directly correlated with differing levels of dispersal capability and host species specificity. These results suggest that our use of 14 loci was sufficient for detecting the presence of genetic structure both within subpopulations and between closely related species, especially given the geographic range and depth of sampling in the present study.In a study of both African elephant species Petkova et al.P. t. verus and ETS populations saturated the signal of regional patterns, so it was necessary to perform analyses of these two populations independently. We found several barriers to m that appeared to be associated with individual populations so we performed additional analyses excluding these , P. t. verus (300 demes) and ETS (490 demes) population levels51. For all analyses, to ensure the models reached stability, three independent MCMC chains were run preceded by 500,000 steps of burn-in, at lengths of 6,000,000 repetitions, each beginning with different random seeds.We found that the magnitude of differentiation between P. t. verus (522) and ETS (410). Detailed descriptions of all analyses and p values are provided in the text and Supplementary Note We performed all analyses from a pool of 939 individual genotypes originating from 48 sampling locations with a minimum inter-site distance of 41 km . When necessary, we balanced our dataset by imposing a lower limit of 6 and an upper limit of 20 (using a randomized drawing process) on the number of genotypes originating from the same sampling location (see Supplementary Note Further information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationReporting Summary


Ceratodon purpureus. We applied phylogenetic, population genetic, and molecular dating analyses to a global (n = 147) sampling data set, using three chloroplast loci and one nuclear locus. The plastid data revealed several distinct and geographically structured lineages, with connectivity patterns associated with worldwide, latitudinal “bands.” These imply that connectivity is strongly influenced by global atmospheric circulation patterns, with dispersal and establishment beyond these latitudinal bands less common. Biogeographic patterns were less clear within the nuclear marker, with gene duplication likely hindering the detection of these. Divergence time analyses indicated that the current matrilineal population structure in C. purpureus has developed over the past six million years, with lineages diverging during the late Miocene, Pliocene, and Quaternary. Several colonization events in the Antarctic were apparent, as well as one old and distinct Antarctic clade, possibly isolated on the continent since the Pliocene. As C. purpureus is considered a model organism, the matrilineal biogeographic structure identified here provides a useful framework for future genetic and developmental studies on bryophytes. Our general findings may also be relevant to understanding global environmental influences on the biogeography of other organisms with microscopic propagules  dispersed by wind.Biogeographic patterns of globally widespread species are expected to reflect regional structure, as well as connectivity caused by occasional long-distance dispersal. We assessed the level and drivers of population structure, connectivity, and timescales of population isolation in one of the most widespread and ruderal plants in the world — the common moss  Bryophytes tend to show extensive distributions, often spanning multiple continents and/or both hemispheres . In partSome bryophytes show a truly cosmopolitan or global distribution, similar to some microbial groups . The capCeratodon purpureus (Hedw.) Brid., is one of the most widespread and ruderal moss species known, and can be found in an exceptionally wide geographic range from polar to tropical areas  and phytochrome 2 (phy2). They found two distinct Northern Hemisphere clades, a Southern Hemisphere clade including some Northern Hemisphere specimens, and several more distantly related distinct lineages from equatorial regions, suggesting that migration between Australasian and Holarctic regions was more common than among equatorial regions. Overall, the study found that, while markers differed in implied population structure, the overall global population structure in C. purpureus was sparse, and provided evidence that migration is ongoing.The global genetic diversity in natural populations of C. purpureus and assess the level of connectivity between its globally widespread populations. Building on the previous biogeographic analysis of rps4 gene and trnL-F and atpB-rbcL spacers and, to a lesser degree, one nuclear marker, the Internal Transcribed Spacer (ITS). We tested for global latitudinal and longitudinal population structure, providing an overall assessment of biogeographic patterns in the species. Finally, we assessed the relative divergence time of matrilineal divergent populations, and evaluated timescales over which such widespread populations have been isolated, with a particular interest in those located in isolated, remote regions (e.g. Antarctica).Here, we aim to gain a better understanding of the biogeography of C. purpureus by specialist bryologists. Genomic DNA was extracted using the DNeasy Plant Mini Kit , using a mortar and pestle and liquid nitrogen, following the manufacturer’s instructions. As C. purpureus has very small gametophytes, multiple stems were extracted per sample to ensure sufficient DNA quantity. The trnL-F spacer, the rps4 gene, atpB-rbcL spacer, and ITS were amplified using the primers trnLF-c and trnLF-f (2 and Bovine Serum Albumin (BSA), using annealing temperatures of 60°C, 55°C, 53°C, and 55°C for trnL-F, rps4, atpB-rbcL, and ITS, respectively. PCR products were checked using gel electrophoresis. For ITS, many samples revealed messy or double bands, and for a small selection of samples bands were successfully excised and purified using the Wizard SV Gel and PCR clean up kit , respectively. Forward and reverse sequencing was performed by LGC Genomics  and the University of Wollongong sequencing facility.Moss samples for molecular analyses were obtained from herbaria  and fresh collections , rps4 (589 bp), atpB-rbcL (623–627 bp) and ITS (674–816 bp) were generated, respectively. Sequences were deposited in GenBank as listed in Cheilothela chloropus (Brid.) Lindb. and three Dicranum species from GenBank as outgroups for phylogenetic chloroplast DNA (cpDNA) analyses. CpDNA regions were aligned using the Geneious aligner within GENEIOUS 9.0.4 . ITS was aligned using PRANK . Totals of 74, 94, 80, and 56 concatenated sequences of ng PRANK , using dng PRANK . As McDain MEGA7 .ITS were created; (i) a full PRANK alignment, and with removal of ambiguously aligned or hypervariable regions using (ii) NOISY  NOISY  and (iiii) NOISY , using di) NOISY  to the orps4, with JMODELTEST-2.1.7  a concatenated data set including only samples for which all three cpDNA loci had been sequenced, called the “concatenated cpDNA” data set. This data set was analyzed with and without indels. We also analyzed iv) a data set using all samples and cpDNA loci which had been sequenced, including those samples for which only one or two loci were available.To estimate population and species relationships, phylogenetic analyses were performed using Bayesian and Maximum likelihood (ML) methods. For individual cpDNA data sets indels were coded with simple indel coding SIC;  in SEQST6 generations, sampling every 1.0×103 generations, and discarding the first 25% as burn-in. Convergence was assessed by confirming that split frequencies had an average standard deviation of < 0.01, and by using TRACER v.1.6 (http://tree.bio.ed.ac.uk/software/figtree/).Bayesian analyses were performed using MRBAYES 3.2 . Runs weER v.1.6  to confiER v.1.6 , using tmax) values over a range of 0.001–0.05.To investigate possible species clusters within the “concatenated cpDNA” data set we used the web-based pairwise genetic distance-based Automatic Barcode Gap Discovery approach ABGD; , applyinITS, haplotype networks were constructed using original, NOISY and GBLOCKS alignments.To examine phylogeographic structure, TCS networks were produced for all loci, and for the “concatenated cpDNA” data set within POPART  using deCeratodon, and performed demographic analyses on the overall data set (clades I–VII), and the large ABGD-inferred cluster as defined with Pmax = 0.0017–0.0046 (clades III–VII) and Pmax = 0.001 (clades IV–VII).To evaluate the cpDNA demographic history, we calculated genetic diversity indices, pairwise Kimura-2P distances, demographic and spatial expansion models, Tajima’s D  and Fu’sITS data set into three partitions based on geographical areas. The three a priori latitudinal partitions were based on the general atmospheric circulation cells , 60°E–30°W (Europe/Africa), and 165°W–60°E . We conducted hierarchical AMOVA analysis of partitioning of genetic variation within and between regions in ARLEQUIN v3.5.1.2, and calculated FST and ΦST differentiation, using 10000 permutations. Here, FST estimates reflect differences in composition and frequency between regions, ΦST estimates reflect levels of evolutionary differentiation between different regions.To investigate worldwide latitudinal and longitudinal geographic structure, we divided the “concatenated cpDNA” data set and the GBLOCKS filtered ells see  composed−4 and standard deviation of 1.0 × 10−4 substitutions/site/my, respectively; a rate previously applied in bryophyte studies, and corresponding to the average absolute substitution rate of cpDNA across a wide range of land plants and algae .To estimate the age of splits among populations we investigated divergence times within the “concatenated cpDNA” data set in BEAST v2.6.2 . In the ER v.1.6  was usedER v.1.6  and FIGTatpB-rbcL was most variable, followed by rps4 and trnL-F  sites, respectively). We detected no evidence for positive selection within rps4 (p > 0.05), but note that low numbers of variable sites limits our ability to draw strong conclusions.The final sample set comprised 147 samples with a wide geographic spread across the globe  and PI sites (61), even after treatment with NOISY or GBLOCKS . Many PCR-amplified samples provided a “clean” single band during electrophoresis . We did not investigate the occurrence of ITS copies further (e.g. through cloning) as ITS amplification of other contaminants e.g. fungi, is common in herbarium samples, and this approach would be better pursued through future studies with fresh rather than degraded herbarium material. Recombination tests revealed evidence of recombination signals in five of the seven tests performed  showed very limited resolution by comparison, with most samples falling as a single large polytomy (data not shown).Phylogenetic analyses of the “concatenated cpDNA” data set revealed seven highly supported clades (posterior probability (PP) > 0.97, bootstrap values > 90)  are shown by locus in max = 0.0017–0.0046  and Pmax = 0.001 , respectively  between all latitudinally divided regions, but only the northern (> 30°N) and southern region (> 30°S) showed significant genetic differentiation (ΦST) from one another. When divided based on longitudinal geographic regions, both cpDNA and ITS showed significant haplotypic differentiation (FST) between regions, but no significant evolutionary differentiation (ΦST)  and genetic  differentiation was found within latitudinal bands, respectively, while longitudinal regionalization showed weaker haplotypic differentiation  and no significant genetic differentiation   and genetic  differentiation found among latitudinal geographic regions, while longitudinal regionalization showed a decreased haplotypic differentiation  and no significant genetic differentiation   were removed, suggesting the significant results were mostly influenced by cryptic population structure within C. purpureus. Furthermore, Fu’s Fs was not significant for any data set, suggesting that overall cpDNA does not support a rapid past expansion of C. purpureus on a global scale.The demographic expansion test on the concatenated cpDNA data set of all clades (I–VII) was significant, rejecting a demographic expansion : 6.27–16.56 Mya; see max value, delimited as one species using ABGD; see The divergence time analysis using a coalescent tree prior indicated that the ancestor of the C. purpureus clades originated in the late Miocene ~5.98 Mya . The analysis in BEAST incorporates a relaxed clock, which has a potential to improve phylogenetic accuracy but on the other hand can be less precise than the time-free approach taken in MrBayes, depending on the true underlying evolutionary pattern . Biogeographic patterns were in line with those found by The matrilineal lineages were mainly strongly linked with latitude (ITS marker (C. purpureus sensu lato (s.l.) hereafter.All cpDNA loci  J.S. Burley and Southern Hemisphere clade VI. Although ssp. convolutes was suggested to be found in southern South America, the sub-Antarctic and Australia (C. purpureus ssp. stenocarpus (B.S.G.) Dix., found in equatorial regions of the Old and New World, has a distribution consistent with that of tropical clade II.In their global revision of the ustralia , we alsoC. antarcticus Card. and Arctic C. purpureus ssp. arcticus Kindb., showing an overlap with bipolar clade V; and 2) the distinct European (or Eurasian) C. conicus (Hamp.) Lindb., showing a possible overlap with clade III.Other lineages for which distinct overlap was found in both biogeography and phylogenies of work by Ceratodon between different geographic areas reported by C. purpureus s.l. exists, a pattern that might be similar in other species that are currently assumed to be globally distributed cosmopolitan species.The morphological differences in ITS. The latter revealed clear differentiation of the earlier diverging lineages  in all latitudinal and longitudinal population comparisons. However, while cpDNA showed significant evolutionary differentiation (ΦST) in all latitudinal comparisons , this was only found in one latitudinal population comparison (> 30°N vs. > 30°S) in ITS (ITS showed significant evolutionary differentiation (ΦST) in any of the longitudinal partition comparisons, implying that there is no strong longitudinal biogeographic signal.ST values in latitudinal population comparisons between cpDNA vs. nDNA could have several origins. The different nuclear pattern is unlikely to be attributed to migration of male gametes: the latter (unlike pollen in angiosperms) have an extremely limited dispersal capacity in mosses , genetic interactions among nuclear loci , we highlight the importance of combining nuclear and plastid markers to unravel evolutionary histories, as both are part of a species’ biology. We await future studies on the nuclear genome of C. purpureus (using fresh rather than herbarium samples) to advance knowledge on nDNA biogeographic patterns, as well as the extent, causes and consequences of recombination and genome doubling on the evolution in C. purpureus and other non-vascular plants.Although our genealogical history of C. purpureus s.l. into physically distinct “latitudinal” geographic areas  disturbances could have aided its spread across the globe. The main distribution of C. purpureus s.l., occupying the vast areas of the temperate regions (particularly clades V-VII), became established throughout the late Pliocene and Quaternary. This was a period of high disturbance globally, including global cooling and repeated glacial periods . This reveals that Antarctica is not as isolated as is often assumed for spore-dispersed organisms  Broth.; Bryum argenteum Hedw. in Antarctica, and even the vascular plant Colobanthus quitensis (with much larger seeds than moss spores) was also found to have at least two independent origins in the Antarctic Peninsula  PhD studentship (ref NE/K50094X/1), NERC-CONICYT (NE/P003079/1) and Carlsberg Foundation (CF18-0267) to EB and NERC core funding to the BAS Biodiversity, Evolution, and Adaptation Team, and Australian Research Council (ARC) Discovery Project DP180100113 funding to SR and PC.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.