Sonneratia caseolaris and a narrowly distributed, closely related species S. lanceolata to infer their divergence histories and related it to historical geological events. We sequenced two chloroplast fragments and five nuclear genes for one population of S. lanceolata and 12 populations of S. caseolaris across the Indo-West Pacific (IWP) region to evaluate genetic differentiation and divergence time among them. Phylogenetic analysis based on sequences of nuclear ribosomal internal transcribed spacer and a nuclear gene rpl9 for all Sonneratia species indicate that S. lanceolata individuals are nested within S. caseolaris. We found strong genetic structure among geographic regions  inhabited by S. caseolaris. We estimated that divergence between the Indo-Malesia and Australasia populations occurred 4.035 million years ago (MYA), prior to the onset of Pleistocene. BARRIERS analysis suggested that complex geographic features in the IWP region had largely shaped the phylogeographic patterns of S. caseolaris. Furthermore, haplotype analyses provided convincing evidence for secondary contact of the South China Sea and the Indian Ocean lineages at the Indo-Pacific boundary. Demographic history inference under isolation and migration (IM) model detected substantial gene flow from the Sri Lanka populations to the populations in the Java Island. Moreover, multi-locus sequence analysis indicated that S. lanceolata was most closely related to the Indian Ocean populations of S. caseolaris and the divergence time between them was 2.057 MYA, coinciding with the onset of the Pleistocene glaciation. Our results suggest that geographic isolation driven by the Pleistocene ice age resulted in the recent origin of S. lanceolata.Glacial vicariance is thought to influence population dynamics and speciation of many marine organisms. Mangroves, a plant group inhabiting intertidal zones, were also profoundly influenced by Pleistocene glaciations. In this study, we investigated phylogeographic patterns of a widespread mangrove species  Pleistocene glaciations have profoundly shaped the biogeography of extant global flora and fauna , 2001. WLumnitzera littorea  and a nuclear gene rpl9. We then sequenced two chloroplast fragments and five nuclear genes in samples from one population of S. lanceolata and 12 populations of S. caseolaris to assess the genetic diversity within S. caseolaris and divergence between it and S. lanceolata. Our results provide new insights into population dynamics and inter-species divergence of mangrove plants under the influence of sea-level fluctuations during the Pleistocene epoch.In this study, we first determined the phylogenetic position of S. lanceolata in Northern Territory, Australia, and five to 20 individuals from 12 populations of S. caseolaris across the IWP region. These 12 populations include four populations around the South China Sea (SCS), two at the Indo-Pacific boundary, two from Java Island, Indonesia, three from the coasts of the Indian Ocean, and one from Queensland, Australia. We collected leaf tissue from each individual and stored it in a plastic bag with silica gel for DNA extraction. The details of sampling locations and sample sizes are presented in Table 1.We sampled 16 individuals from a single population of S. lanceolata within the genus Sonneratia, nrITS and one nuclear gene rpl9 were amplified from two randomly chosen individuals from each population of S. caseolaris and S. lanceolata by polymerase chain reaction (PCR) with LA Taq DNA polymerase . Additionally, two chloroplast fragments (trn L – trn F and trn V – trn M) and five nuclear genes  were amplified from each individual of S. lanceolata and S. caseolaris to assess the patterns of genetic diversity between and within the two species. The primers for the nuclear genes were from Supplementary Table S1.Total genomic DNA of each individual was extracted using the CTAB method . To resoS. lanceolata within the genus, phylogenetic analyses of all six Sonneratia species and the outgroup Duabanga grandiflora were carried out using the combined sequence data of nrITS and the nuclear gene rpl9, as only these two fragments could be successfully amplified in D. grandiflora. The partition-homogeneity test suggests that these two fragments can be combined for phylogenetic analysis in the Sonneratia genus  and retention index (RI) were also employed to estimate homoplasy. Indels were ignored. For ML analysis, TrN + I was selected as the appropriate nucleotide substitution model from a set of 56 models using the Akaike information criterion, as implemented in Modeltest 3.7  and DNA polymorphism  were calculated at each locus using Mega v. 6.06 and DnaSP v. 5.10, respectively. Multidimensional scaling (MDS) analysis was performed based on the pairwise Kimura two-parameter distances in R v. 3.1.31. For both species, only the individuals without any missing data were included in PCA. Starting from KXY estimates, putative geographic barriers in the IWP region were inferred for all 13 populations using Monmonier’s algorithm in the software Barrier v. 2.2 . The divergence time of the two SCS populations CWC and CBA was denoted as t0, while that between the populations from Sri Lanka (SRK) and Bangladesh (BSK and BPF) was represented by t1. For each gene, the longest non-recombining block was extracted from the aligned data set using the Perl script IMgc . Three independent runs were carried out with different, random number seeds to test for convergence. The 5∗106 well-mixed samples were thinned to obtain 100,000 draws from marginal posterior distributions of each parameter. Posterior most likely values were estimated by finding posterior modes , as well as effective population size (Ne) and divergence time (t), among populations from the SCS, Indian Ocean and the Indo-Pacific boundary under the isolation with migration (IM) model. We used data from the five nuclear genes and the IMa2 program . Two popipt IMgc  to avoidor modes . Due to rpl9 were 1,734 bp long after alignment, with 135 parsimoniously informative sites. The MP analysis produced one most parsimonious tree with tree length of 370, CI of 0.911 and RI of 0.933 . The ML analysis yielded the same topology as the MP method. The six Sonneratia species were divided into two clades with strong support, one of which grouped S. lanceolata and S. caseolaris  and the other contained the remaining four species . Surprisingly, S. lanceolata was nested within the clade formed by S. caseolaris samples and was most closely related to the S. caseolaris individuals from Indo-Malesia region with high bootstrap support .The combined sequences of nrITS and the nuclear gene S. caseolaris. We observed two single-nucleotide polymorphisms (SNPs) and one one-bp indel in the combined chloroplast fragments. These sites generated four haplotypes across the 12 populations of S. caseolaris . Only two populations (MSB and ISG) of S. caseolaris were polymorphic with extremely low levels of nucleotide diversity (𝜃π) and polymorphisms .We sequenced 210 individuals from 12 populations of S. caseolaris harbored relatively high levels of genetic diversity, with 𝜃π ranging from 3.660 to 8.850 per kb, and 𝜃w from 2.820 to 5.560 per kb . At the population level, populations of S. caseolaris from its range margins  exhibited lower levels of polymorphism than those from its distribution center , while the population from Sri Lanka (SRK) had the lowest diversity ( = 0.054 per kb).The aligned sequence length of the five nuclear genes ranged from 620 to 1,170 bp, containing 12 to 49 SNPs in the 12 populations. At the species level, S. caseolaris . Four chlorotypes in the 12 populations fell into three major clusters corresponding to three geographic regions. The first cluster comprises populations around the SCS , the second is restricted to the Indian Ocean , and the third is found exclusively in Queensland, Australia . Similar to the chloroplast fragments, haplotype networks of four of the five nuclear genes  showed three distinct groups restricted to the SCS, the Indian Ocean and eastern Australia . Polymorphisms in the cpi gene revealed only two groups, with one predominant in the SCS and the other combining the Indian Ocean and Australian populations . The haplotype networks, in addition to a MDS analysis of the genetic distance matrix, showed that the population from eastern Australia was most highly diverged from the SCS and the Indian Ocean . Within the Indian Ocean group, a relatively weak barrier marked a subdivision between Sri Lanka and Bangladesh, which was in agreement with the Bayesian clustering result with K set to 4. These results indicated that geographic features in the IWP region might have profoundly shaped population structure within S. caseolaris.Bayesian clustering analysis using STRUCTURE suggests that the statistically optimal number of populations is S. caseolaris, the divergence time between the Indo-Malesia and Australasia populations was estimated to be 4.035 million years ago : 2.298–6.318 MYA, Figure 4). Within the Indo-Malesia region, the split between the SCS and the Indian Ocean lineages occurred at 2.561 MYA (95% HPDI: 1.375–3.981 MYA), while the Sri Lanka population diverged from Bangladesh (BSK and BPF) 1.286 MYA (95% HPDI: 0.522–2.174 MYA).Within Figure 3A). When K was set to 2, approximately 74.6% of the nucleotide variations present in the ISG population came from the Indian Ocean lineage, while 25.4% were from the SCS lineage. In the two Malaysian populations, the proportion of SNPs that belong to the SCS lineages was slightly higher .We observed extensive admixture between the SCS and the Indian Ocean groups, as evidenced by the analyses of chloroplast fragments as well as nuclear genes, in the Sangaibuntu population (ISG), located near the Indo-Pacific boundary. Similar patterns were also observed in three other populations that are close to the Malacca Strait . Bayesian clustering likewise showed signals of genetic admixture between the SCS and the Indian Ocean groups in the ISG, MSB, and MSN populations (S. caseolaris populations from the SCS (CWC and CBA), the Indian Ocean  and the Indo-Pacific boundary (ISG) . The effective sample size (ESS) was larger than 70,000, indicating a well-mixed chain. As a result of running the BEAST program on our data, we obtained an estimate of the mutation rate at 1.673 ∗ 10-9 substitutions per site per year . Under this assumed mutation rate, effective size (Ne) of the two SCS populations (CWC and CBA) was estimated to be 107  and 322 , respectively; Ne of the populations from the Indian Ocean (BSK/BPF and SRK) was 537  and 36 (95% HPDI: 0–680), respectively . Ne of the ISG population was estimated at 1,396 , much greater than that in other populations. The migration rate from the Indian Ocean population SRK to the population ISG was estimated to 0.119 (95% HPDI: 0.000–0.452), which was significantly greater than zero .We then used the isolation with migration (IM) model to estimate demographic parameters of S. caseolaris, genetic diversity of the S. lanceolata population (SLA) was extremely low in both chloroplast fragments and the five nuclear genes, with θπ ranging from 0.000 to 0.110 and θw from 0.000 to 0.350 per kb (Supplementary Table S3).In contrast to S. lanceolata within the clade that comprises the S. caseolaris samples suggested by our phylogenetic analysis was verified by the haplotype network reconstruction and Bayesian clustering. No polymorphism was detected in the chloroplast fragments sequenced in S. lanceolata, and this species differs from the Indian Ocean populations of S. caseolaris by only one mutation . At the two of the five nuclear genes (ppi and nhx2), S. lanceolata is most closely related to the Indian Ocean populations of S. caseolaris . At the gene phi, the haplotype of S. lanceolata forms its own clade that differs from the Indian Ocean S. caseolaris samples by 11 mutations . In contrast, the remaining two genes, rpl9 and cpi, contained haplotypes that grouped with the Malaysia population (MSN) of S. caseolaris .The position of K = 2, Supplementary Figure S. lanceolata was assigned to the same cluster as the Indian Ocean and Australian populations of S. caseolaris, distinct from the SCS group of that species . When K = 3, there was a division between the S. caseolaris populations from the Indian Ocean and Australia of S. caseolaris with the S. lanceolata population falling into the Indian Ocean population cluster. Increasing K to 4 results in S. lanceolata samples forming a new cluster with the two Bangladesh populations of S. caseolaris. These results suggest that S. lanceolata is closely related to the Indian Ocean lineage of S. caseolaris. Moreover, based on the pairwise KXY values , two putative barriers to gene flow between S. caseolaris and S. lanceolata were identified. One is located between northern Australia and Java Island, and the other is between the Arafura Sea and the Coral Sea. These two geographic barriers surround the current distribution region of S. lanceolata . This suggests that the isolation of the Arafura Sea from the surrounding oceanic regions might have triggered the S. lanceolata speciation event. Divergence time estimates indicates that S. lanceolata diverged from the Indian Ocean lineage of S. caseolaris at approximately 2.057 MYA with 95% HPDI extending from 1.053 to 3.275 MYA .For the optimal clustering , prior to the Pleistocene. This result suggests a critical role for pre-Pleistocene events in genetic divergence of the two main lineages of S. caseolaris.Strong genetic differentiation among the S. caseolaris during Pleistocene, consistent with the differentiation mechanism suggested for other mangrove species (We further estimated that the SCS lineage diverged from the Indian Ocean lineage 2.561 MYA (95% HPDI: 1.375–3.981 MYA), in the early Pleistocene. Perhaps sea level changes during this epoch drove the genetic differentiation between populations from these geographic areas. Dispersal of seeds via sea currents plays an important role in determining the extant distribution and genetic composition of mangrove species within and among different oceanic regions . During  species  and nume species .S. caseolaris. Analyses of haplotype frequencies at the chloroplast fragments and four of the five nuclear genes showed that the ISG population harbors haplotypes from both the SCS and the Indian Ocean groups . Haplotypes of nuclear genes fell into two highly divergent clusters separated from each other by 5–14 mutational steps and there were no intermediate haplotypes. This pattern is highly unlikely to have arisen within ISG in the face of ancestral polymorphism, strongly suggesting that previously isolated lineages came into contact and mixed in this population. This idea is consistent with our simulation under the isolation with migration (IM) model that identified substantial gene flow from the Sri Lanka population (SRK) into ISG .It is usually difficult to distinguish genetic admixture from incomplete lineage sorting, thus reducing the resolution of signals of gene flow . In thisRhizophora apiculata , subsequent interglacial sea-level rises restored the sea corridors connecting the SCS and the Indian Ocean . It is tpiculata  and Lumnracemosa , as wellS. lanceolata was first described as a new species by S. caseolaris in both morphology and ecology, suggesting a close relationship between these two species . The main differentiating character between these species is stamen color, which is white in S. lanceolata . Bayesian clustering analysis and KXY statistics showed that S. lanceolata individuals  were genetically closer to the Indian Ocean group than either the SCS or Australia groups of S. caseolaris , during early Pleistocene. species . Coincidnceolata , 2006. HS. lanceolata and S. caseolaris. At the boundary between Indo-Malesia and Australasia, over 25,000 volcanic ocean islands and the shallow seas surrounding them are particularly sensitive to sea level fluctuations (S. lanceolata and S. caseolaris.Our results of genetic similarity and divergence time are consistent with an important role of geographic isolation driven by Pleistocene glaciations as a trigger of lineage break between tuations . During tuations , connecttuations . Likewistuations  that spetuations . These tAvicennia integra is a narrowly distributed species confined to the Northern Territory, Australia (A. officinalis has a much wider range extending from South India through Malaysia and Indonesia to New Guinea and eastern Australia (Ceriops, C. tagal and C. australis. Morphologically, C. australis differs from C. tagal by the shorter terete and smooth hypocotyls and longer flowers, calyx lobes and petals (C. australis is restricted to Australia, along the costal lines of North Territory and Queensland, and a part of New Guinea and Indonesia, while C. tagal widely occurs from southern India through Southeast Asia to Australia (C. australis is found more often than C. tagal in Australia (Similar genetic differentiation and speciation might have also occurred in other mangrove genera. ustralia . In contustralia . The divustralia . Anotherd petals . Geograpustralia . Howeverustralia . These fSS and RZ designed the study. YY, FP, and CZ collected materials and performed experiments. YY, FP, JL, and SY analyzed and interpreted the data. YY, ND, FP, RZ, and SS wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer YS declared a shared affiliation, though no other collaboration, with several of the authors YY, FP, JL, SY, RZ, SS to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review.


Cottus hangiongensis and C. koreanus are small bottom-dwelling freshwater sculpin fishes from South Korea that display marked life history divergence but are morphologically nearly indistinguishable. Cottus hangiongensis evolved an ‘amphidromous’ life history with a post-hatching pelagic larval phase. They spawn many small eggs in the low reaches of rivers, and hatched larvae migrate to the sea before returning to grow to maturity in the river mouth. In contrast, C. koreanus evolved a ‘fluvial’ landlocked type with benthic larvae. They release a smaller number of larger eggs, and the larvae undergo direct development, remaining benthic in the upstream rivers throughout their entire lives. We tested whether there were differences in patterns and levels of within-population genetic diversities and spatial population structure between the two closely related Korean sculpins using mitochondrial DNA control region sequences and seven nuclear microsatellite loci.Life history characteristics are considered important factors influencing the evolutionary processes of natural populations, including the patterns of population genetic structure of a species. The sister species C. hangiongensis harboured considerably higher levels of within-population genetic diversities  than C. koreanus. In contrast, the fluvial sculpin exhibited noticeably more spatial population structure than did the amphidromous sculpin, as suggested by pairwise FST statistics. The finding that C. hangiongensis individuals comprised a single random mating population across the east-flowing river basins in the Korean Peninsula, whereas C. koreanus individuals comprised genetically discrete individual populations, was further supported by an individual-based Bayesian population assignment and also factorial correspondence analyses.The combined analyses of both marker sets revealed that Cottus species following speciation from a common ancestor of marine sculpin.The higher genetic diversity, but lower population structure, of the amphidromous sculpin relative to the fluvial sculpin may have resulted from its greater larval dispersal and also possibly, higher fecundity accompanied by an amphidromous life history. Hence, we conclude that contrasting early life histories – including the presence or absence of the pelagic larval phase – may have led to divergent patterns of within-population genetic diversities and spatial population structure between the sister The online version of this article (10.1186/s12862-018-1171-8) contains supplementary material, which is available to authorized users. Life history traits are considered important biological factors affecting the evolutionary processes, as they shape the patterns of population genetic structure of a species –7. An orA number of previous studies suggest that life history characteristics are significantly associated with the degree of population connectivity of freshwater fishes , 13. In Oncorhynchus tshawytscha) is characterized by two distinct life history types with different patterns of geographic distribution as ‘ocean-type’ and ‘stream-type’. These life history types are distinguished based on the duration that juvenile salmon remain in freshwater habitats before migrating to the sea . Similar to the results of STRUCTURE, the FCA of seven microsatellites also revealed a single genetically indistinguishable group within C. hangiongensis, but every population is its own group within C. koreanus  is approximately 8.4 times greater than that in C. koreanus , providing direct evidence supporting our hypothesis.We find that an amphidromous sculpin species ant Fig. . The obsin Japan . The elearger Ne , 60, whius Table . Our Ne C. hangiongensis typically produces many small eggs  in the lower reaches of rivers [C. hangiongensis [C. koreanus produces fewer but larger eggs  in the upstream rivers, and hatched larvae undergo direct development and maintain a benthic life [C. koreanus that leads to restricted gene flow among populations over geographic scales of tens to hundreds of metres. Smaller Ne along with a poorer dispersal ability makes C. koreanus populations more susceptible to genetic drift, resulting in the lower extent of within-population genetic diversity [Rhinogobius sp. in Okinawa Island, Japan, and found much greater genetic diversity in the amphidromous populations. As such, the higher extent of genetic diversity in C. hangiongensis is likely attributable to high levels of gene flow and larger Ne resulting from its amphidromous life history with a pelagic larval phase. In contrast, the low genetic diversity in C. koreanus is presumed to be due to genetic isolation among individual populations resulting from its fluvial landlocked life history with direct development. The greater fecundity (approximately 1.4 times higher fecundity) of C. hangiongensis may also contribute to its higher intraspecific genetic variation [C. hangiongensis may represent a complete picture of the genetic diversity due to high gene flow from surrounding sites, genetic variation in C. koreanus may be partitioned among different localities due to very low gene flow [Amphidromous f rivers . Newly hf rivers , 33. Theongensis . The homongensis . By comphic life , 62. In hic life . These fiversity . A previene flow .C. koreanus and none of the populations in C. hangiongensis showed evidence for population bottleneck (Table S2). Therefore, the observed differences in within-population genetic diversity between these species can be most likely attributed to their differing early life histories. Most populations of both species appear to be in HWE, except for the SCH population of C. hangiongensis and the PC 1, JS, IJ, GP and GG populations of C. koreanus, which suggests non-random mating at the loci tested. Somewhat ironically, the SCH population of amphidromous C. hangiongensis shows a genetic signal of inbreeding, albeit weak, whereas the IJ and GP populations of fluvial C. koreanus show a detectable signal of outbreeding . The magnitude of spatial genetic differentiation, as suggested by pairwise F-statistics, was markedly lower within C. hangiongensis than within C. koreanus  marker [C. koreanus examined, and that this is most likely caused by limited dispersal ability. A recent study of the Manchurian trout Brachymystax lenok tsinlingensis from South Korea inhabiting watersheds that overlap like those of C. koreanus also showed significant genetic divergence among populations [Cottus that differs in the presence/absence of the post-hatching larval stage on Hokkaido Island in Japan, a previous study [C. amblystomopsis populations are less structured than the fluvial C. nozawae. These previous findings also support the hypothesis that the divergent patterns of genetic structure between C. hangiongensis and C. koreanus may have been caused by differences in life history traits.Our results from both mtDNA and microsatellite markers reveal noticeable differences in the magnitude and patterns of population connectivity between the two sculpin species that vary in the presence of a pelagic larval phase. Amphidromous us Table . The FST) marker . These rulations . Examinius study  also demC. hangiongensis, geographic distance among localities does not seem to play a role in shaping the population structure over the geographical scales analysed (9–140 km), perhaps due to the effects of connectivity during larval phases promoted by the oceanic currents. However, a study of short ninespine stickleback Pungitius kaibarae without a larval phase at the east-flowing river basins in South Korea revealed strong inter-population genetic differentiation [C. hangiongensis is highly likely due to the presence of the larval phase. The lack of population structure (i.e. high population connectivity) observed for C. hangiongensis suggests that the juveniles of this species do not necessarily return to their home rivers where they hatched. For fluvial C. koreanus, its very low dispersal ability may mask the role of geographic distance that contributes to the population structure over the geographic scales we tested (1–177 km). Further studies on smaller geographic scales will be required to determine the spatial scales at which C. koreanus populations are structured.IBD analysis reveals no correlation between geographic and genetic distances among the populations in both species, indicating that the geographic proximity of populations is not responsible for the observed spatial population structure of either species. For amphidromous ntiation , which sC. hangiongensis comprises a single random mating population, but fluvial C. koreanus consists of genetically distinct individual populations is further supported by the results of our STRUCTURE and FCA analyses. While genotyped individuals of C. hangiongensis from five localities represent a single population with a high degree of admixture, C. koreanus individuals from thirteen sites were divided into five (or eight) genetically unique clusters , and river basins. Table S2. Statistical tests for a recent bottleneck in each of the five and 10 populations of C. hangiongensis and C. koreanus, respectively from South Korea. P-values are based on the Wilcoxon test. Allelic frequency distribution shape was normal or shifted for mode-shift distortion. Population abbreviations as in text and Additional file Table S1. (ZIP 26 kb)


Oophaga. Here, we explore the phylogeographic pattern among color‐divergent populations of the Little Devil poison frog Oophaga sylvatica by analyzing population structure and genetic differentiation to evaluate which processes could account for color diversity within and among populations. With a combination of PCR amplicons  and genome‐wide markers from a double‐digested RAD (ddRAD) approach, we characterized the phylogenetic and genetic structure of 199 individuals from 13 populations (12 monomorphic and 1 polymorphic) across the O. sylvatica distribution. Individuals segregated into two main lineages by their northern or southern latitudinal distribution. A high level of genetic and phenotypic polymorphism within the northern lineage suggests ongoing gene flow. In contrast, low levels of genetic differentiation were detected among the southern lineage populations and support recent range expansions from populations in the northern lineage. We propose that a combination of climatic gradients and structured landscapes might be promoting gene flow and phylogenetic diversification. Alternatively, we cannot rule out that the observed phenotypic and genomic variations are the result of genetic drift on near or neutral alleles in a small number of genes.Some South American poison frogs (Dendrobatidae) are chemically defended and use bright aposematic colors to warn potential predators of their unpalatability. Aposematic signals are often frequency‐dependent where individuals deviating from a local model are at a higher risk of predation. However, extreme diversity in the aposematic signal has been documented in poison frogs, especially in  The aim of our research was to explore the phylogeographical pattern of O. sylvatica along its distribution range in Ecuador by (1) estimating population structure and genetic differentiation and (2) evaluating which processes could account for color diversity within and among populations of O. sylvatica.Previous research in 22.1O. sylvatica  and O. pumilio  were treated using the same protocol. In order to protect the vulnerable O. sylvatica populations that are highly targeted by illegal poaching, specific GPS coordinates of frog collection sites can be obtained from the corresponding author.We sampled 13 populations of 2.2Liver or skin tissue stored in RNAlater was homogenized using Trizol (Life Technologies) in tubes with 1.5‐mm TriplePure Zirconium Beads . DNA was purified by a standard phenol/chloroform procedure followed by ethanol precipitation according to the Trizol manufacturer's instructions.cox1], 16S ribosomal DNA [16S], and 12S ribosomal DNA [12S] flanked with tRNAVal) and three nuclear  gene regions were PCR‐amplified using the following sets of primers for each gene: CO1 (CO1a‐H: 5′‐AGTATAAGCGTCTGGGTAGTC and CO1f‐L: 5′‐CCTGCAGGAGGAGGAGAYCC)  .Three mitochondrial (cytochrome oxidase subunit 1 [CO1 or Ta) and elongation time (E) for each primer set are as follows: 16S , CO1 , 12S , RAG1 , TYR , NCX . PCR products were analyzed on an agarose gel and purified using the E.Z.N.A Cycle‐Pure Kit following the manufacturer protocol . Purified PCR products were Sanger sequenced by GENEWIZ .DNA was amplified in a 30 μl PCR containing 10 ng of genomic DNA, 200 nmol/L of each primer, and 1× Accustart II PCR SuperMix . The thermocycling profiles comprised an initial denaturation (3 min at 95°C), followed by 40 cycles of denaturation (30 s at 95°C), annealing (30 s) with specific temperature for each primer set (see below), elongation (72°C) with a duration specific to each primer set (see below), and a final elongation step (5 min at 72°C). Specific parameters for annealing temperature  libraries on a subset of 125 samples of Hoekstra . Samples2.4Raw sequence chromatograms for each gene set were edited and trimmed using GENEIOUS 8.0.5  and then aligned using CLUSTALW. Alleles of nuclear genes containing heterozygous sites were inferred using a coalescent‐based Bayesian method developed in PHASE 2.1  and for the concatenated matrix of 2,084 bp were determined with JMODELTEST 2.1 , O. histrionica (N = 2), and O. pumilio (N = 6). Diversity indices were calculated using DNASP, and a mitochondrial haplotype network was inferred under Tajima and Nei model using ARLEQUIN 3.5  and genetic distances based on pairwise difference (ΦST) using ARLEQUIN. p‐Values for FST and ΦST were estimated after 10,000 permutations, and significance threshold level was fixed at p = .05.We calculated population differentiation using conventional F‐statistics from haplotype frequencies  and then corrected for misassembled loci from sequencing errors using the corrections module , which applies population‐based corrections. In order to minimize the effect of allele dropout that generally leads to over‐estimation of genetic variation . The data set used for the subsequent analysis was filtered to retain only one random SNP site per RAD locus to minimize within‐locus linkage and is composed of 3,785 SNPs.Raw fastq reads were demultiplexed, quality filtered for reads with Phred quality score <20, and trimmed to 120 bp using the K populations , and individuals are then assigned probabilistically to one or more populations  with 10 iterations for each value of K. We determined the number of clusters (K) that best described the data using the delta K method  of 0.948  radiate from this central node: Two are composed of private haplotypes from the Durango and San Antonio populations, and the other four have mixed origins belonging to the northern populations of Otokiki, Durango, Lita, and Alto Tambo. These populations show the highest sequence (K) and nucleotide (π) diversity, as well as the highest haplotype diversity  and a mean ΦST of 0.609 (ranging from 0.016 to 0.915), suggesting that northern populations are well differentiated from southern populations  for Otokiki versus Lita and Alto Tambo (both FST and ΦST), and for Durango versus Alto Tambo and Lita versus Alto Tambo (FST only) .In this study, we investigated genetic structure among populations of e Figure . The exie Figure , Bayesiae Figure , DAPC ane Figure , and ddRe Figure , Fig. S5The northern genetic cluster  presents higher mitochondrial diversity with a large amount of unique haplotypes within populations. We observe that four of these populations  have unique haplotypes, and three  share haplotypes with the polymorphic population at Otokiki. These latter populations are weakly differentiated based on genetic data and are geographically close, with distances from Otokiki being, respectively, 4, 5, and 20 km to Alto Tambo, Lita, and Durango Figure . OtokikiFST values from ddRAD data  The Ecuadorian populations of O. sylvatica are composed of two clades that reflect their geographic distribution. Further behavioral, ecological, and morphological information is required to determine whether the two geographical lineages observed in Oophaga sylvatica represent one polymorphic or several species. A combination of climatic gradient and structured landscape generating geographic barriers to gene flow could explain the complex patterns of diversification observed in Oophaga and some other Dendrobatidae  A highly polymorphic population (Otokiki) exists, which provides a unique opportunity for testing hypotheses about the selective pressures shaping aposematic traits. We hypothesize that this polymorphic population arose from either gene flow between phenotypically divergent populations at secondary contact zones or through a range expansion of the polymorphic Otokiki population into surrounding regions. More data are needed to distinguish between these alternative scenarios.By evaluating mitochondrial DNA variation and genome‐wide SNPs, we have gained four important insights about KX553997–KX554204, 16S: KX554205–KX554413, CO1: KX574018–KX574226, NCX: KX785882–KX786090, RAG1: KX785673–KX785881, and TYR: KX785464–KX785672.Sequence data have been submitted to GenBank: accession numbers 12S: Raw data from ddRADseq reads have been submitted to SRA: SRP078453.ABR, JCS, LAC, and LAO designed the research; ABR, JCS, EET, LAC, and LAO collected samples in the field; ABR, BCC, and SNC performed the laboratory research; ABR analyzed data; ABR and LAO wrote the paper with contributions from all authors.The authors declare no conflict of interest. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Genomic information is an important part of the routine evaluation of dairy cattle and provides the wide availability of animals genotyped using single nucleotide polymorphism (SNP) microarrays. We analyzed 2243 Polish and 2294 German Holstein-Friesian bulls genotyped using the Illumina BovineSNP50 BeadChip. For each bull, estimated breeding values (EBVs) calculated from national routine genetic evaluation were available for production traits and for somatic cell score (SCS). Separately for each population, we estimated SNP haplotypes, pairwise linkage disequilibrium (LD), and SNP effects. The SNP genetic covariance between both populations was estimated using a bivariate mixed model. The average LD was lower in the Polish than in the German population and, with increasing genomic distance, LD decays 1.7 times more rapidly in German than in Polish cattle. The comparison of SNP allele frequencies for base populations estimated separately using Polish and German data revealed a very good agreement. The comparison of genetic effects corresponding to various window lengths defined in bp emerged a systematic pattern: regardless of the length of the compared region, few significant differences were found for production traits, while many were observed for SCS. For each trait, the German population had much higher SNP variances than the Polish population and the genetic covariance estimates were all positive. Depending on traits’ inheritance mode, the additive genetic variation can be stored in many genes following the infinitesimal model (like for SCS) or distributed between genes with high effects and the polygenic “background” (like for production traits). Accounting for those differences has implications on the prospective international genomic evaluation. In dairy cattle, many countries have incorporated genomic information into their genetic evaluation systems . As a consequence, several genes with large effects on such traits have been identified as candidate genes  genotyped on well-defined populations with detailed information on phenotypes, environmental factors, and pedigree provides a basis for investigating the genetic background of complex traits. Moreover, a substantial number of genome-wide association studies (GWAS) has been performed on traits routinely measured and selected in dairy cattle, which is best illustrated by 34,754 QTL and association results of 731 studies submitted to the cattleQTLdb  and SNP genotypes to compare the patterns of genetic diversity between German and Polish Holstein-Friesian populations. In particular, the investigated aspects cover: (1) the comparison of LD patterns, (2) the assessment of differences in the effects of genomic regions on the selected traits, (3) the evaluation of differences in SNP allele frequencies, and (4) the estimation of the polygenic (co)variance components for production and udder health traits between German and Polish Holstein-Friesian populations.The analyzed datasets comprised 2243 Polish and 2294 German Holstein-Friesian bulls. Both groups were defined as bulls for which EBVs on a national basis were available only for one of the countries. For each bull, EBVs calculated based on the national routine genetic evaluation models and corresponding effective daughter contributions were available for milk (MY), protein (PY), and fat (FY) yields, as well as for somatic cell score (SCS). Moreover, each individual was genotyped using the Illumina BovineSNP50 BeadChip version 1. Separately for each dataset, SNPs were filtered based on two criteria: the minor allele frequency (MAF) had to be ≥ 0.01 and the call rate ≥ 90%. Only SNPs that were present in both populations after filtering were kept, resulting in a final list comprising 39,557 SNPs representing an intersection of markers in the German and Polish datasets.r2 statistic , . In order to estimate the shape of LD decay in each population, the regression function x represents the Polish (x = PL) or German (x = DE) population, i, and d represents the upper boundary of this interval. Paired t-tests were used to test for differences in the average LD between the Polish and German populations  at a given Mbp interval, with the underlying H0:DPL, DE = 0.SNP haplotypes were estimated separately for both populations using fastPHASE  and the Polish (PL) populations, ith region estimated separately for each population, and NSNPi is the number of SNPs within the ith region. The additive variance of the jth SNP within the ith region  and Iq and Iε are identity matrices with dimensions corresponding to q and ε, respectively, G is a (co)variance matrix of SNP effect estimates between populations, and P and Z are the corresponding incidence matrices. The ASreml software (version 3.0) was used for the estimation of model parameters and (co)variance components  in German than in Polish cattle. For instance, on a short scale between  Mbp, the decrease was by 0.01073 in the German and by 0.00634 in the Polish breeds.At each distance defined in Mbp, a significantly lower LD was observed in the Polish than in the German population. Empirical curves and the corresponding regression functions for the Polish and German populations are visualized in Fig. The comparison of genomic effects of chromosome regions was dependent on the length of the defined window. This situation made it impossible to identify and annotate regions responsible for population-wide differences. On the other hand, comparing results corresponding to different window lengths emerged a systematic pattern: regardless of the length of the compared regions between populations, only a few significant differences were found for production traits, while many were observed for SCS Fig. . For exar2 coefficient amounting to 0.95.The comparison of SNP allele frequencies for base populations estimated separately based on Polish and German data revealed a very good agreement, expressed by high Pearson correlation coefficients of 0.97, and a correspondingly very good fit of a linear regression with the estimated intercept of 0.01, slope of 0.99, and the The pattern of estimated (co)variance components for additive SNP effects was consistent across traits. For each trait, the German population had much higher SNP variances than the Polish population, with the ratio varying between 1.8 for SCS and 2.8 for PY. Genetic covariance estimates were all positive, resulting in genetic correlations of 0.22, 0.24, 0.30, and 0.39 for MY, PY, FY, and SCS, respectively.Our comparisons showed that, although both populations seem to have a very similar genetic background, revealed by the high similarity of allele frequencies in base populations, the differences in selection goals and pressure imposed on Polish and German Holstein-Friesian populations over generations caused a detectable degree of differentiation.r2 depends on allele frequencies, the difference between the studies can be partially attributed to the biased SNPs selection on the genotyping arrays, where SNPs are ascertained non-randomly, aiming at frequent alleles and a comprehensive coverage of the genome, resulting in a uniform allele frequency spectrum. Furthermore, the assembly of large LD blocks that appeared in array-based analyses breaks into series of shorter tracts when LD is assessed from sequence data , the additive genetic effects are very similar in the German and Polish populations, especially for windows up to 1 Mbps in length, while an opposite picture emerges for SCS. This is supported by differences in selection programs between both countries. Production traits have long been in the focus of both Poland and Germany, indicating a similar selection pressure over generations. On the contrary, SCS has been included in the German total merit index already from 1997, while in Poland, the selection index has been enhanced by SCS only relatively recently, in 2008. This results in a strong variation pattern between the two analyzed populations.http://www.interbull.org/ib/maceev_archive, release December 2016) report lower estimates for production (0.84–0.90) traits than for SCS (0.96), which is in agreement with SNP-based correlations estimated in our study. Both models assume a normal distribution of genetic variation across the genome, but, in reality, for production traits, there is an accumulation of high effect SNPs (most remarkably the DGAT1 region on chromosome 14 with effects on MY and FY), which results in lower estimated country correlations despite no significant differences observed between particular genomic regions. In contrast to that, the inheritance mode of SCS appears to be of a purely polygenic nature, i.e., determined by many genes of moderate to small effects, and, thus, meets model assumptions, which is then reflected by a higher country correlation of 0.96. Note that, generally, lower SNP-based (this study) than polygenic-based (Interbull) correlations are due to no common individuals between the German and Polish pedigrees used in this study.Yet another aspect arises in the comparison of estimated genetic correlations between Poland and Germany. Polygenic-based genetic correlations published by Interbull (The comparison of Polish and German Holstein-Friesian populations showed that observed differences in the estimated effects of genomic regions depend on differences in the linkage disequilibrium (LD) pattern between populations and on traits’ inheritance mode. Accounting for such differences has direct implications on the prospective international genomic evaluation based on across-country single nucleotide polymorphism (SNP) effect estimation. Therefore, a proposed option would be the use of a cumulated/averaged effect of SNP groups binned by their genomic location (bp) or, preferentially, by LD, instead of single SNP estimates in the SNP MACE model.


RS) population of Anopheles coluzzii is descended from the preceding late RS generation at the same locality, consistent with dry season (DS) dormancy (aestivation), or from migrants from distant locations. Distinct predictions derived from these two hypotheses were assessed, based on variation in 738 SNPs in eleven A. coluzzii samples, including seven samples spanning 2 years in a Sahelian village. As predicted by the “local origin under aestivation hypothesis,” temporal samples from the late RS and those collected after the first rain of the following RS were clustered together, while larger genetic distances were found among samples spanning the RS. Likewise, multilocus genotype composition of samples from the end of the RS was similar across samples until the following RS, unlike samples that spanned the RS. Consistent with reproductive arrest during the DS, no genetic drift was detected between samples taken over that period, despite encompassing extreme population minima, whereas it was detected between samples spanning the RS. Accordingly, the variance in allele frequency increased with time over the RS, but not over the DS. However, not all the results agreed with aestivation. Large genetic distances separated samples taken a year apart, and during the first year, within‐sample genetic diversity declined and increased back during the late RS, suggesting a bottleneck followed by migration. The decline of genetic diversity followed by a mass distribution of insecticide‐treated nets was accompanied by a reduced mosquito density and a rise in the mutation conferring resistance to pyrethroids, indicating a bottleneck due to insecticidal selection. Overall, our results support aestivation in A. coluzzii during the DS that is accompanied by long‐distance migration in the late RS.In arid environments, the source of the malaria mosquito populations that re‐establish soon after first rains remains a puzzle and alternative explanations have been proposed. Using genetic data, we evaluated whether the early rainy season ( Beyond the equatorial zone, malaria transmission is seasonal and in regions where surface waters required for larval development are absent during the 3‐ to 7‐month‐long dry season (DS), mosquito densities drop dramatically  approximates the number of breeding adults in a population if the population density is stable and parental contributions to the next generation are randomly distributed  and commonly the upper 95% CI included infinity, consistent with a robust aestivating population or with mass migration. This also indicates that the statistical power derived from genotyping specimens at up to 12 microsatellite loci with ~90 alleles is insufficient for the estimation of Ne of these populations. Shallow genetic structure typically characterizes populations of A. gambiae and A. coluzzii  over large distances .Previous genetic studies have aimed to detect seasonal bottlenecks in different populations of the A. coluzzii in the Sahel descend from the preceding late RS generation from the same locality, consistent with aestivation (hypothesis 1), rather than from distinct and presumably distant locations, consistent with migration (hypothesis 2). The former hypothesis was proposed for A. coluzzii and the latter for A. gambiae, respectively  population of 0 Avise,  would re0 Avise, . AlternaA. coluzzii and two of A. gambiae in this study, we focus the tests on the former species with only exploratory analysis of the second. In total, we genotyped 520 mosquitoes at 1,536 single‐nucleotide polymorphisms (SNPs). The specimens represent seven time points of A. coluzzii spanning 2 years from our focal Sahelian village, Thierola, and an additional four samples from three neighbouring villages, as well as two samples of A. gambiae from Thierola spanning 1 year  and by pyrethrum spray collection in Bagadaji  and Kolimana   were removed from the analysis. Following evaluation of A. coluzzii samples only, a further 40 SNPs were excluded because of extreme deviation from Hardy–Weinberg proportions (using a strict Bonferroni‐corrected threshold of α = 0.05/N SNPs), and a further 74 because of extremely low polymorphism . This yielded a final set of 738 SNPs which were included in the present analysis. Similarly 14 mosquitoes , with over 10% genotyping failure rate were also removed, leaving a total sample size of 510 mosquitoes.Quality of SNP scores was carefully evaluated and 619 SNPs were excluded because of poor scoring or a low genotype conversion rate across mosquitoes (<80%). To simplify concurrent analysis of males and females, the relatively few X‐linked loci (He) per locus; and mean number of alleles per locus. To accommodate variation in sample size, these statistics were also computed based on the smallest sample size (n = 40 chromosomes), selecting the first 20 mosquitoes in larger samples. The differences between samples (n = 40 A. coluzzii) in the mean heterozygosity and the mean number of alleles were evaluated using ANOVA, treating locus as a random variable and sample as a fixed parameter and using contrasts to estimate differences between samples according to a priori hypotheses ; expected heterozygosity , where MAF denotes major allele frequency in the pooled A. coluzzii sample, while the subscripts i and j denote different samples) across loci was estimated as distance between samples. Unlike Fst, this genetic distance remains meaningful not only in comparisons between populations but also between samples representing different time points of the same population. The distance matrix among all sample pairs computed by Proc Distance (SAS) was used to generate a dendrogram using an UPGMA tree building algorithms as well as 2D plots produced by subjecting the distance matrix to multidimensional scaling analysis (MDS). Neighbour joining population trees were plotted using MEGA 4  set between 1 and 10 employed 20,000 iterations, the first 10,000 as burn‐in. Probability plots suggested a maximum of K = 6 or 7, and also a rapid plateau in parameter values within the burn‐in period. Consequently, these were followed by test runs of 50,000 iterations, again with a burn‐in of 10,000, with K set between 1 and 7. Each value of K was replicated five times, very low variability in log probability values detected among runs following processing suggesting that additional replicates would not alter the optimum solution. Results were processed with Structure Harvester v0.6.94  the experimenter's sampling variance, genetic drift was not evident, consistent with no reproduction. The genetic component is more difficult to detect the larger Ne is  were computed according to Nei and Tajima (To test whether genetic drift between samples collected during the DS (and immediately after the first rains) has occurred, we partitioned the temporal variation in allele frequency (d Tajima :FT = (xi − yi)2/[(xi + yi)/2 − (xi*yi)] and VT = (1/Sxi) + (1/Syi), where xi and yi are the common allele in locus i  in the first (x) and second (y) samples, and Sxi and Syi are the sample sizes (number of chromosomes) of the first (x) and second (y) samples from that locus (i). The Wilcoxon rank test was used to test whether the median of the distribution of the difference (FTi − VTi) over all loci was equal or larger than zero to determine whether it FT > VT for each pair of samples.Global tests were employed to evaluate significance of multiple tests. The sequential Bonferroni procedure Holm,  was appl3Excluding invariant positions across all samples and applying the stringency criteria explained above see , a total3.1P95 , among samples varied considerably (63%–87%) in the full and in the standardized data set, with sample size fixed at n = 40 chromosomes  and mean number of alleles/locus (A) varied between samples when tested in the standardized data set  and Thierola in August 2008 and in June 2010, as well as A. gambiae in September 2009. A seven‐sample lower diversity cluster consisted of all the remaining samples, except the lowest, single‐sample cluster of A. coluzzii from Thierola in May 2009  to the time when density surged immediately after the first rain (May 2009) of the following year . Populations of A. coluzzii in riparian habitats with year‐round access to surface water (Kolimana and Niono) had absolute values of 0.06 higher A and 1% higher He .Within‐population diversity , while seven other samples presented heterozygote deficiency , whereas the highest and lowest Fis were both observed shortly after the first rain . While heterozygote deficits may represent Wahlund's effect (the pooling of two isolated reproductive units) in small populations, it is difficult to reconcile this explanation with deficits in the very large “perennial” population of Niono (Discussion).Deviations from HWE showed some extreme Fis values across populations Table . The meay Figure . Notably3.3A. coluzzii and A. gambiae populations via multidimensional scaling , surrounded by samples from Niono (October 2011 and August 2010) Bagadaji (September 2010) and Kolimana .Overall genetic distances (Euclidian) between samples were computed across all 738 loci and used to depict the similarities among e Figure . Accordis Figure . Among AK = 2 and K = 5, Fig. K1) and to a similar degree the separation of A. coluzzii into a subpopulation (K2) predominating in Thierola during late May 2009 (Th09Ma) were followed by more subtle subdivisions identifying three additional subpopulations  suggested a hierarchical clustering solution with optima at two and five groups (A. coluzzii sample collected in May 2009 from Thierola (Th09Ma) consisted of a large fraction of cluster #2, which was absent elsewhere or at low frequency , revealed heterogeneity in cluster composition . Clusters #1, #3 and #5 exhibited continuity throughout temporal samples of corresponding species and populations, unlike subpopulations 2 and 4, which were detected during the early RS (May–August), but not in other times. The clusters probably do not represent sympatrically isolated reproductive units, and so the heterogeneous composition of Sahelian A. coluzzii suggests admixture between a local and distant populations, although a stable existence of co‐adapted genes by selection cannot be ruled out altogether. The relatively stable composition of clusters #3 and #5 and the erratic appearance of #2 and #4 are not consistent with seasonal selection. Within each cluster, departures from HWE have mostly persisted  is larger than the experimenter's sampling variance (VT), genetic drift is evident, consistent with reproduction  over the duration of RS and DS that separated them  were small and these samples were clustered together and away from those separated by a RS , probably via long‐distance migration, which would take several generations to dissipate across loci.Although the results above support the main predictions of aestivation, other results did not fit well: (i) The genetic distances separating the two earliest e Figure , contrare Figure . The lari Figure  further i Figure  and S6. A. coluzzii varied markedly over time in Thierola  contrary to prediction (1.1), within‐population diversity in a Figure , but they Figure . This dry Figure . Additionts Fig. , and by A. coluzzii populations were genetically similar, such migration must arrive from a distant area (>150 km). The heterozygote deficits observed in Niono may also attest for admixture with long‐distance migrants and/or with Sahelian populations that surround the narrow (~5 km wide) irrigated area.The departures from HWE Figure  are an uA. coluzzii sample of Thierola from May and to a lower degree by the Kolimana sample from August  in Sahelian A. coluzzii  provides additional compelling evidence for the involvement of long‐distance immigrants. Therefore, we propose that in A. coluzzii (unlike A. gambiae), long‐distance migration is a strategy to contend with dry spells during the Sahelian RS. Our results also indicate an ephemeral bottleneck that accompanied the mass distribution of ITNs in that year (2008) resulting in high variance in within‐ and between‐population diversity. This bottleneck disrupted the typical dynamics of demographic and thus genetic parameters during the first year of our study. A follow‐up study with a longer time series and a greater coverage of the genome would help narrow the uncertainty about the origin of the early RS A. coluzzii in the Sahel and also evaluate the possibility of seasonal selection.In conclusion, our results reveal strong but imperfect agreement with predictions based solely on aestivation in All authors declare no competing financial interests with respect to any aspect of this article.https://doi.org/10.5061/dryad.t5k6f.SNPs data used in the analyses here are available through the Dryad Digital Repository:  Click here for additional data file.


Athene noctua, a widespread species associated with agricultural landscapes. The results show a high risk of population declines over the coming 100 years, especially toward the north of Europe, whereas populations toward the southeastern part of Europe have a greater probability of persistence. In order to be considered genetically viable, individual populations must count 1,000–30,000 individuals. As Little Owl populations of several countries count <30,000, and many isolated populations in northern Europe count <1,000 individuals, management actions resulting in exchange of individuals between populations or even countries are probably necessary to prevent losing <1% genetic diversity over a 100‐year period. At a continental scale, a habitat suitability analysis suggested Little Owl to be affected positively by increasing temperatures and urban areas, whereas an increased tree cover, an increasing annual rainfall, grassland, and sparsely vegetated areas affect the presence of the owl negatively. However, the low predictive power of the habitat suitability model suggests that habitat suitability might be better explained at a smaller scale.The agricultural scene has changed over the past decades, resulting in a declining population trend in many species. It is therefore important to determine the factors that the individual species depend on in order to understand their decline. The landscape changes have also resulted in habitat fragmentation, turning once continuous populations into metapopulations. It is thus increasingly important to estimate both the number of individuals it takes to create a genetically viable population and the population trend. Here, population viability analysis and habitat suitability modeling were used to estimate population viability and future prospects across Europe of the Little Owl  FarmlanHabitat fragmentation might divide populations that were once continuous into either metapopulations, populations that occasionally exchange individuals and genes, or to completely disconnected populations with no gene exchange at all. A metapopulation consists of several populations connected by dispersal. Local populations might go extinct, but the patches remain part of the metapopulation due to the chance of recolonization  simulations, the European populations of Little Owl were modeled as a metapopulation, consisting of populations and subpopulations that to a varying degree are connected as a function of distance.The populations simulated in the VORTEX analysis consisted of several large populations defined by their genetic structure Section . The wesThe MVP is here defined as the minimum population size that maintains at least 95%–99% of the initial genetic diversity over a 100‐year period. In order to find the minimum initial population size that retained 95%–95% genetic diversity, the populations were modeled with a range of different initial population sizes at a constant carrying capacity K. The initial population size was increased/decreased until the minimum initial population size was found. For populations with a positive stochastic growth rate, K was altered in order to find the minimum viable K (MVP(K)) for maintaining 99% and 95% genetic diversity, respectively.2.2.2A theoretical K was estimated for all regions included in the simulations. It was estimated from data on population density and the area of interest Table . This K 2.2.3N) is the mean number of offspring at population size N. MO(0) is the mean number of offspring produced at low densities. K is carrying capacity.Populations of Little Owl breed in a density‐dependent manner, as isolated pairs lay larger clusters  and adult birds, and between populations. Therefore, population‐specific mortality rates were used if available Table . When po2.2.6Genomic data were included in the VORTEX simulations  was performed in R in order to determine the explanatory variables that had a significant influence on the presence–absence of the Little Owl. General linear models (GLM) can be used to determine the environmental factors that shape a species’ distribution and thereby quantify the species niche  were calculated using R 3.1.2. The VIF reports how much of the variability of a given explanatory value is already explained in the model due to correlation  was used to describe presence/absence of the Little Owl, the logit link function (Equation 2) describes the probability (p) that the owl is present  modeling uses information on a species dependency on the habitat to predict the likelihood of occurrence in a given area based on environmental variables , a juvenile (1‐ to 2‐year olds), and an adult stage (2 and older). A total of 83% of all juveniles were assumed to reproduce, while 100% of the adults were assumed to reproduce Møller, .When calculating the mean fecundity on basis of nest surveys, it is important to take the survival rates into account  and an increased survival .2.4.4a, b, and c are input parameters, and Dmax is the maximum recorded dispersal distance . The fao 1 Fig. . Dispers2.4.5K > 100, meaning that at population densities of 100 individuals or less, the fecundity and survival will be halved. The Allee parameter was reduced to 10 if K < 100. Density dependency was set to affect all life stages and to affect both fecundity and survival.The Ceiling density type was chosen  is reached), and Allee effects included. Allee effects describe the situation where individual fitness is correlated with population density  for the northern European population due to the negative growth rate of the population. The initial population would have to count more than 250,000 individuals to secure a loss of <5% genetic diversity, while more than 500,000 individuals are required to maintain 99% of the genetic diversity. As with the northern European population, the population growth rate of the Portuguese population was negative and an MVP(K) could not be estimated. The Portuguese population was very sensitive to changes in the mortality rates, and an increase in mortality from 70% to 75% in juveniles and from 35% to 38% in adults greatly increased the risk of extinction within a 100‐year period. The MVP was only estimated for populations with a positive or stable growth rate. The average MVP (99%) with regard to the carrying capacity for these populations was 4,700 individuals  or 16,625 individuals  was highly correlated with several of the other explanatory variable and was therefore eliminated from further analysis. After eliminating BIO11, all variables had VIF values below 10 and 15 explanatory variables remained. A model with all 15 explanatory variables was chosen for further analysis in R, where 10 were found significant Table a. The spAs illustrated by the habitat suitability map Figure b, the HS3.3Regardless of the initial population size and mortality rate, the abundance of the metapopulation decreased over the course of 100 years. Patch occupancy dropped continuously when simulating low survival and stabilized after 25 years when simulating high survival Fig. . The iniThe number of populations remaining extant for more time steps increased as the survival rates increased Figure . Populat4The already declining population of Little Owls in Europe is likely to continue declining over the next 100 years. The Little Owl prefers warmer areas where the average annual rainfall is low and avoids areas with high tree cover, sparsely vegetated areas, and grassland. Populations further to the north, where the climate is colder, are more likely to decline and disappear than populations further to the south, and the Turkish population will remain the population stronghold over the next 100 years. In order to be genetically viable and minimize the loss of genetic diversity, the individual populations should have a carrying capacity of at least 1,000–30,000 individuals, depending on the actual mortality rates of the populations.4.1The PVA in VORTEX suggested that not all populations in Europe are likely to remain extant and retain 99% of the initial genetic variability over the next 100 years. The Balkan population was genetically viable, as were the Spanish and the Italian populations. The Portuguese and Danish populations were not viable in the long run.The northern European population is estimated to count a maximum of 118,000 individuals. The population is therefore vulnerable to losing a significant amount of genetic diversity. A part of the northern population has already been investigated for loss of genetic diversity, namely the Danish population. A low historic genetic variability was found, and an even lower genetic variability was found in the population at present  required to maintain 95% genetic diversity to be 75 individuals, while 200 individuals are required to maintain 99% genetic diversity , and few are estimated to count more than 30,000 (BirdLife International, None declared.LHA, VL, and CP planned the project and set the aims. IP and CP provided the genomic data used in the analyses. IP performed molecular analyses. LHA conducted the analyses in ArcGIS, RAMAS/GIS, and VORTEX, analyzed the data, and made the first draft to the manuscript, under the supervision of VL and CP. PS provided expert knowledge on the Little Owl and contributed in improving the data analysis and overall manuscript quality. All authors revised and commented on the manuscript and its contents. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Solanum rostratum is a self-compatible annual herb that exhibits a high level of outcrossing in its native populations. However, the mating system of invasive populations of S. rostratum has never been assessed. Here, we investigated the mating system based on 11 microsatellite loci and explored ecological factors that may influence the outcrossing rate among 10 invasive populations of S. rostratum in China. We found that the mean outcrossing rate was 0.69 ± 0.12 (range 0.49 to 0.83) with multiple paternity within progeny arrays (average effective number of sires = 7.86), which suggests a mixed mating system dominated by outcrossing. Combined with the uniformly high outcrossing rate (0.70 ± 0.03) previously reported in its native range, these results indicate that there has been no evolutionary shift in mating system during the invasion in China by S. rostratum. There were no relationships between outcrossing and population size, population density, altitude, latitude or longitude. Furthermore, high outcrossing of S. rostratum in China may be facilitated by enantiostyly and heteranthery.The mating system of flowering plants plays a key role during the process of invasion. Evolution from outcrossing to selfing can allow rapid regeneration of a population after long-distance dispersal by providing reproductive assurance.  As a result, the mating system of invasive plant species plays a fundamental role in determining the genetic diversity and genetic structure of a population and is closely related to adaptive evolution and invasion2. When alien flowering species are introduced to a new region, these species often occur at low density and experience increased pollen limitation because of the reduction of potential mates and suitable pollinators5. Such negative conditions can persist for a long time after the initial founder event resulting in Allee effects7, which can limit the spread of invaders. Therefore, natural selection may favor an association between selfing and colonizing ability8, as selfing can allow a rapid build-up of a population after long-distance dispersal by providing reproductive assurance according to Baker’s Law11. Efforts to explore the mating system of invasive species constitute an important task to better understand the biology of plant invasions and to predict microevolutionary changes in various environments.The mating system of plant is the pattern in which gametes combine in plant populations1. The evolutionary transition from outcrossing to selfing has occurred in some invasive plants, such as Eichhornia paniculata and Eichhornia crassipes15, owing to the stochastic loss of mating types in these species. There are also some cases that have no evolutionary shift in the mating system during invasion, such as those involving Ambrosia artemisiifolia and Senecio inaequidens16, owing to self-incompatibility. However, the evolution of mixed mating strategies, a mixture of self-fertilization and cross-fertilization, may be more complex17. And studies on the maintenance of mixed mating systems in hermaphroditic invasive plants have only been conducted relatively recently.Recent studies have used molecular techniques to evaluate selection on mating systems during biological invasion by comparing outcrossing rates between invasive and natural populations19, but also by environmental conditions, including geographic location and habitat fragmentation21. However, to the best of our knowledge, only a handful of studies18 have assessed the impact of biotic factors and environmental conditions on mating patterns of invasive species. Thus, it is necessary to investigate the factors that influence the mating system of invasive plants.For animal-pollinated plants, there are evidences that the mating system of them is influenced not only by biotic factors, such as floral display, pollinator type and population sizeSolanum rostratum (Solanaceae) is a buzz-pollinated, self-compatible annual herb with hermaphroditic flowers. The species is believed to be native to Mexico and the USA22, but it has spread to China, Europe, Russia, Canada, South Korea, and Australia24. S. rostratum was found for the first time in Liaoning province in 1981, and has spread across a large area in North China during the last 36 years24. Populations of S. rostratum occur in disturbed habitats such as roadsides, riversides, sides of railways and abandoned fields in China, which is as same as the habitats in its origin area. The main pollinators of S. rostratum are bees in the genera Bombus and Xylocopa in both invasive and native populations. The bright yellow flowers of S. rostratum in China exhibit monomorphic enantiostyly and strong herkogamy26 as in its native region. The species has two mirror-image floral morphs alternating along the inflorescence, with flowers that present style and pollinating anther opposite each other, situated either right or left of the floral axis. The stamens of S. rostratum are divided into four small yellow feeding stamens and a single tinged brown large pollinating stamen according to their different functions27. The four centrally located feeding stamens serve to attract and reward bees, and the single pollinating stamen, which is deflected to either the right- or left-hand side of the flower opposite the style, contributes disproportionately to pollen reaching the stigmas of other flowers27. These special floral traits promote outcrossing by reducing selfing between the same flower morph29; in its native Mexican range, S. rostratum is highly outcrossed30. However, the mating system of invasive populations of S. rostratum has never been assessed.S. rostratum among ten invasive populations in China and the effect of population size and other ecological factors on mating in S. rostratum. The present study had three objectives concerning the reproductive and invasive biology of S. rostratum. First, we assessed the mating system of S. rostratum by quantifying its mating system parameters using microsatellite markers. Second, we compared the outcrossing rate of Chinese populations with that of native populations in Mexico. Finally, we evaluated relationships between outcrossing rates and population size and other ecological factors such as altitude and latitude. Our study provides the first detailed evidence of the mating system of S. rostratum in its invasive Asian range.Here, we investigated the mating system of S. rostratum in China showed intermediate to high outcrossing rates (Table tm) across Chinese populations was 0.69 ± 0.12 (mean ± SD), ranging from 0.492 ± 0.225 in population YG to 0.834 ± 0.064 in population CY. The independent samples t-test showed that the outcrossing rates of Chinese populations had no significant difference with the previously reported outcrossing rates of Mexican populations30  were significantly greater than zero (range 0.009 ± 0.027 to 0.137 ± 0.023), except for population NZ (−0.002 ± 0.05), suggesting some degree of biparental inbreeding in most Chinese populations.Estimates for the outcrossing rate between related individuals (tm−ts) were significantly greater than zero in both Chinese populations  and Mexican populations (range 0.034 ± 0.024 to 0.072 ± 0.028)30, suggesting some degree of biparental inbreeding in most populations. The correlation of paternity within sibships for multilocus cases (rpm) was low in all populations (0.13 ± 0.09), suggesting that approximately one in ten maternal siblings were expected to have the same pollen donor parent. Moreover, the effective number of male parents per progeny arrays was 7.86 among the ten studied populations (Nep = 7.86), which was lower than that of native populations in Mexica (Nep = 8.97)30.Estimates for the outcrossing rate between related individuals (P > 0.05). However, the smallest population YQ (population size = 60) had a high outcrossing rate (0.783 ± 0.07). There was no evidence of a relationship between biparental inbreeding and population size (P > 0.05), but relatively high level of biparental inbreeding was found in two small populations .There were no significant relationships between multilocus outcrossing rates and population size, population density, altitude, latitude or longitude (S. rostratum exhibited low to intermediate genetic diversity in China. Table He) ranged from 0.049 to 0.415; and the polymorphism information content (PIC) varied from 0.043 to 0.357. In the ten Chinese populations, the YG population had the lowest average number of alleles per locus, polymorphism information content, expected heterozygosity and observed heterozygosity ; the WSL population had the highest polymorphism information content, expected heterozygosity and observed heterozygosity . We detected more homozygosity than expected across loci within populations, as the inbreeding coefficient (Fis) was positive in all populations (varying from 0.166 in population CY to 0.313 in population NZ). And Chinese populations’ genetic differentiation was high (Fst = 0.216).The ten invasive populations of He = 0.545) and observed heterozygosity (Ho = 0.201) were lower than those of the Mexican populations 30. The inbreeding coefficient of Chinese populations (Fis = 0.225) was lower than Mexican populations (Fis = 0.256)30. While Chinese populations (Fst = 0.216) had higher genetic differentiation than Mexican populations (Fst = 0.159)24.Chinese populations showed relatively lower genetic diversity Table  than Mex20. In many cases, we expect an evolutionary shift towards a higher frequency of self-fertilization, because selfing provides reproductive assurance under the initial founder event, resulting in Allee Effects. Solanum rostratum is a self-compatible annual herb that relies on insect visitation for cross-pollination. Based on our field experiments, in which we excluded pollinators using mesh bags, the capacity for self-fertilization in S. rostratum is very low in North China. Our estimates of mating patterns in S. rostratum indicated a mixed mating system dominated by outcrossing, and the mating system of S. rostratum did not experience an evolutionary transition during its invasion of China. Recent analyses of the accumulated data have highlighted the high frequency of species with mixed mating systems31. However, the evolution of mixed mating strategies of invasive species is complex. On one hand, selfing can produce seeds under conditions of low density or during frequent episodes of colonization with severe inbreeding depression. On the other hand, outcrossing can maintain high genetic diversity within populations. There may be a balance between selfing and outcrossing for S. rostratum under different conditions, and efforts to explore the reproductive success of different populations can help us better understand the maintenance of mixed mating systems of S. rostratum during invasion.Evaluating the mating system of invasive species is an important task to better understand the conditions in which self- or cross-fertilization or a mixed mating strategy is favored in invasive populations33, and the rates of pollinator visitation can directly affect the amount of outcrossing rates in self-compatible plants20. The flowers of S. rostratum exhibit monomorphic enantiostyly and strong herkogamy. In addition, it has been hypothesized that the high outcrossing rate in this species is probably facilitated by its floral morphology30. Moreover, S. rostratum is a buzz-pollinated plant, and insect size is the main determinant of whether a visitor acts as a pollinator or a pollen thief34. During the visit of effective pollinators, heteranthery results in the deposition of pollen on one side of pollinator’s body against the style. Pollen from one floral morph will be deposited on the stigma of the opposite floral morph, thus reducing pollinator-assisted selfing within flowers. However, geitonogamy is unavoidable in large individuals, in which left- and right-styled flowers occur at the same time.There are many ecological factors that can influence outcrossing rates. Variation in flower design and display has been shown to influence pollinator visitation rates35. In our preliminary study, in which we performed pollen addition experiments, we reported that pollen addition significantly increases fruit set and seed production and that smaller populations suffer from more severe pollen limitation36. The ecological context of pollen limitation can negatively impact plant-pollinator interactions and generate diverse selective forces on plant mating systems. In this study, we did not find a significant relationship between multilocus outcrossing rates and population size, altitude, latitude or longitude. However, the smallest population, YQ, had a high outcrossing rate (0.783 ± 0.07), whereas the largest population, CJ, had a relatively low outcrossing rate (0.543 ± 0.069). There is evidence that the outcrossing rate was associated with the floral display17. In our field investigations, plants in the YQ population were small; some individuals only opened one or two flowers per day, and the flowering season lasted for more than four months, which may reduce pollen transfer between the same plants. By contrast, plants in the CJ population were relatively large; and most individuals opened more than five flowers per day during the flowering season, which may promote geitonogamous selfing. Therefore, the floral display may have an impact on the outcrossing rate in this species; however, this phenomenon needs further study.The mating system of flowering plants is influenced not only by its flower design and display but also by population size, geographic location and habitat fragmentation24. This may have given the species enough time to adapt to the environment and co-evolve with local pollinators. Second, population CY is located in riversides without habitat fragmentation, which provides relatively favorable conditions for S. rostratum. Third, the genetic diversity of population CY is very high (PIC = 0.310), which may guarantee the ability to maintain high outcrossing rates13. By contrast, population YG, which had the lowest outcrossing rate (0.492 ± 0.225), is the last population that we found in this study and is located on the side of a railway. The genetic diversity of population YG is also the lowest (PIC = 0.043). The difference in the two populations may indicate that the invasion history, living habitat and genetic background could influence the mating system of S. rostratum. And further studies should be conducted to explain the maintenance of mixed mating system in S. rostratum.In this study, population CY had the highest outcrossing rate (0.834 ± 0.064) in the ten studied populations in China, and population CY also has the highest outcrossing rate in the world based on the present studies. There are reasons explaining why the highest outcrossing rate occurred in population CY. First, population CY has a relatively long invasion history, and it is the first introduced population reported in ChinaS. rostratum. The mean germination rate of the ten populations was high (0.94 ± 0.05), and the germination rate was not correlated with the outcrossing rate. However, in many outcrossing species, the germination rate of self-fertilized seeds is lower than that of outcrossed seeds because of inbreeding depression37. Therefore, inbreeding depression functioning in the offspring of S. rostratum may lead to a higher estimate of the outcrossing rate. Further studies to determine the extent of inbreeding depression in S. rostratum will provide additional reproductive information on this highly outcrossing species.Moreover, the offspring used for mating system analysis were seedlings, and this may overestimate the outcrossing rate of S. rostratum occurs in its invasive Asian range. The average multilocus outcrossing rate was 0.69 ± 0.12 (ranging from 0.49 to 0.83), with an average effective number of 7.86 male parents in Chinese populations. Compared with the high level of outcrossing rates (0.70 ± 0.03) in native populations in Mexico30, these results indicate that there was no evolutionary transition from outcrossing to selfing during S. rostratum’s invasion of China. Moreover, there were no significant differences between the outcrossing rates and population size, altitude, latitude or longitude. We speculate that high outcrossing rates in invasive populations of S. rostratum may be facilitated by precise pollination owing to herkogamy and monomorphic enantiostyly.In conclusion, our results suggest that a mixed mating system dominated by outcrossing of S. rostratum, ten Chinese populations were sampled in a variety of habitats. The ten populations cover a large area of the invasive distribution in China and exhibit a wide range of population size  in plastic seedling-raising dishes in March 2011 and placed the dishes in a greenhouse at Beijing Normal University. Finally, we randomly sampled a total of 1435 seedlings belonging to 120 maternal families  with a Microsatellite PCR kit . The PCR amplifications were performed using a Bio-Rad thermocycler as follows: one cycle of 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 58 °C for 180 s, and 72 °C for 30 s; and a final extension step at 60 °C for 30 min38. The PCR products were sequenced using an ABI 3730xl capillary sequencer with GeneScan 500 LIZ as the internal size standard. The results were analyzed with GeneMapper Software version 3.7 .We extracted genomic DNA from leaves dried in silica gel of the 1435 offspring using a plant genomic DNA kit . We scored the genotypes of the DNA samples using 11 pairs of microsatellite primers, which were previously developed for a), expected heterozygosity (He), observed heterozygosity (Ho), polymorphism information content (PIC) and the combined probability of exclusion of a single parent (PEsp) for each population using the computer programs GENALEX version 6.439 and CERVUS version 3.041. We estimated the inbreeding coefficient (Fis) using FSTAT version 2.9.342. The statistical significance of Fis was tested using 11000 randomizations, and the threshold was adjusted for multiple comparisons using the Bonferroni correction. To test differences in genetic variation between the ten populations in China, we estimated the population differentiation (Fst) among populations using 1000 permutations in FSTAT version 2.9.342. The presence of null alleles was calculated using Micro-Checker software43.We analyzed the number of alleles (N44. We estimated the multilocus outcrossing rate (tm), single-locus outcrossing rate (ts), and outcrossing rate between related individuals (tm−ts). The outcrossing rate between related individuals (tm−ts) can be used to estimate biparental inbreeding. We also calculated the correlation of outcrossing rate (rt), the correlation of paternity within sibships for both multilocus cases and single cases , and the extent of outcrossed paternity by related male parents (rps−rpm). The correlations of paternity (rpm and rps) represent the fraction of siblings that share the same male parents44. We calculated the effective number of male parents per progeny arrays among the ten populations as Nep = 1/45. Standard deviations were computed based on 1000 bootstrap values, with the resampling of entire maternal families. For tests of statistical significance, we examined the distribution of 1000 bootstrap values to determine whether outcrossing rates differed significantly from 1.047. When values were significantly less than 1.0, the species exhibited a mixed mating system. Comparison of outcrossing rates between Chinese populations and Mexican populations was done with independent samples t-test11. The relationships between multilocus estimates of outcrossing rate and population size, altitude, latitude and longitude were analyzed by Spearman rank correlation tests. In addition, the relationship between biparental inbreeding and population size was also analyzed by Spearman rank correlation tests.Mating system parameters for each population were estimated using the expectation-maximization method of the computer program MLTR version 3.2