Subpsaltria yangi the population differentiation, genetic structure and phylogeography of the species were investigated in the Loess Plateau and adjacent areas of northwestern China by analysing mitochondrial and nuclear DNA and comparing the calling song structure of 161 male individuals.Geographical isolation combined with historical climatic fluctuations have been identified as two major factors that contribute to the formation of new species. On the other hand, biotic factors such as competition and predation are also able to drive the evolution and diversification of organisms. To determine whether geographical barriers contributed to population divergence or speciation in the rare endemic cicada The results reveal a low level of genetic differentiation and relatively simple phylogeographic structure for this species, but two independent clades corresponding to geographically isolated populations were recognised. Genetic and geographical distances were significantly correlated among lineages. Results of divergence-time estimation are consistent with a scenario of isolation due to glacial refugia and interglacial climate oscillation in northwestern China. Significant genetic divergence was found between the population occurring in the Helan Mountains and other populations, and recent population expansion has occurred in the Helan Mountains and/or adjacent areas. This population is also significantly different in calling song structure from other populations.Geographical barriers , possibly coupled with related ecological differences, may have driven population divergence and allopatric speciation. This provides a possible example of incipient speciation in Cicadidae, improves understanding of population differentiation, acoustic signal diversification and phylogeographic relationships of this rare cicada species of conservation concern, and informs future studies on population differentiation, speciation and phylogeography of other insects with a high degree of endemism in the Helan Mountains and adjacent areas.The online version of this article (10.1186/s12862-018-1317-8) contains supplementary material, which is available to authorized users. Bactrocera cucurbitae (Diptera: Tephritidae) [Apocheima cinerarius (Lepidoptera: Geometridae) [Panesthia cockroaches (Blattaria: Blaberidae) [Halyomorpha halys (Hemiptera: Pentatomidae) [Geographic isolation is essential to most speciation events, because biogeographic barriers  separate populations, impede gene flow, and drive genetic differentiation, which may lead to allopatric speciation –3. The mritidae) , Apocheietridae) , Panesthberidae) , and Haltomidae) . During tomidae) , and subtomidae) , 13. Durtomidae) , 15. Suctomidae) , 17.-deserts which may serve as geographical barriers to species with limited dispersal ability [-deserts in this region may promote the differentiation of populations and increase the likelihood of speciation.The increasing desertification of northern China caused by the uplift of the QTP effectively changed the biodiversity of this area , 19. The ability , and the ability , 26. TheSubpsaltria yangi Chen is the only known species of the tribe Tibicininii in China. The species was previously placed in the subfamily Tettigadinae [S. yangi was rediscovered in June of 2011 during a survey of the insect fauna of the Helan Mountains which is surrounded by deserts and semi-desert. This provided an opportunity to study this little-known species, and led to the discovery of the positive phonotaxis and acoustical sexual mimicry in males [S. yangi since 2011, a few more populations were discovered from the Loess Plateau in Shaanxi, Shanxi and Gansu provinces, which were all attracted and sampled using our special acoustic playback method [S. yangi occurring in the Helan Mountains mainly feeds on Ephedra lepidosperma C.Y. Cheng (Ephedraceae), a shrub endemic to the Helan Mountains, but populations distributed in the Loess Plateau mainly feed on Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow (Rhamnaceae) [The large body size and low dispersal ability of cicadas has led to their being used as model organisms for phylogeographic and speciation studies in different regions of the world including New Zealand , 28, Souigadinae , 33 but igadinae . This raigadinae  is unusuigadinae , 33. Thein males –37. Durik method . Interesmnaceae) –37. The S. yangi, we explored the pattern of genetic divergence among populations of this species using phylogeographic analyses based on mitochondrial and nuclear gene sequences. The genetic distance and divergence times were estimated to assess the potential influence of historical climatic fluctuations on population differentiation and niche expansion. The calling song structures of males of four representative populations were also investigated and compared to further clarify the extent of population differentiation or speciation.To determine whether geographical barriers are important factors driving population divergence or allopatric speciation of S. yangi, including Shaanxi Province, Shanxi Province, Gansu Province, and Ningxia Hui Autonomous Region. In total, 161 specimens, representing ten natural populations, were collected from 2013 to 2017. Sweep netting and acoustical trap sampling were used to collected individuals from different drought-tolerant dwarf shrubs or herbaceous plants, which generally do not exceed 1 m in height in the dry habitats sampled. Sampling site and sample-size information are summarized in Table Samples were collected throughout the formerly documented range and potential range of COI), cytochrome c oxidase subunit II (COII), cytochrome b (Cytb), and ATPase subunits (A6A8)) and two nuclear gene segments  and the elongation factor-1 alpha gene (EF-1α)) from representative samples. The primer pairs used in this study for these six genes are presented in Additional file COI, 44.9 °C for COII, 54.4 °C for Cytb, 47.7 °C for A6A8, 59.3 °C for ITS1 and 54.4 °C for EF-1α), and an extension at 72 °C for 60 s, and a final extension step of 72 °C for 7 min. PCR products were visualized on a 1% agarose gel with ethidium bromide staining. PCR products were then purified and sequenced in both directions by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. All sequences gathered in this study have been deposited in GenBank .A Biospin Insect Genomic DNA Extraction Kit  was used to extract total genomic DNA from leg muscle following the manufacturer’s instructions. Standard polymerase chain reaction (PCR) methods were used to amplify partial sequences of four mtDNA regions . Multiple sequence alignment was carried out in CLUSTAL X v1.81 [-parameter (K2P) model.The chromatograms of each sequence were proofread and then assembled using SEQMAN PRO . Each protein X v1.81 , with ga X v1.81 . To conf X v1.81  was used X v1.81 , as no r X v1.81  was cond X v1.81 . We test X v1.81 . Mean se-reconnection (TBR) branch swapping on ten replicates. Bootstraps [COI + COII + Cytb + A6A8 + EF-1α + ITS1), and performed subsequent analyses on the (COI + COII + Cytb + A6A8) and (EF-1α + ITS1) datasets, respectively. Sequences from two closely related species, Okanagana utahensis and O. canadensis, obtained from Sueur et al. [The program PARTITIONFINDER v1.1.1  was usedotstraps  were conr et al. , were usBI analysis was conducted using MrBayes v3.1.2. The Markov chain Monte Carlo (MCMC) algorithm was run for 2,000,000 generations, with four incrementally heated chains. The analysis involved starting from a random tree and sampling every 100 generations. The average standard deviation of split frequency among runs was lower than 0.01, indicating that the sampling of posterior distribution was adequate. Convergence was examined using Potential Scale Reduction Factor (PSRF) and the average standard deviation of split frequencies. Stationarity was determined using TRACER v1.5  by plottThe numbers of haplotypes (H), the values of haplotype diversity (h) and nucleotide diversity (π)  for everS. yangi [. The genetic clusters probabilities (from K = 1 to K = 20) were surveyed with COI gene under “mixture analysis” and “spatial clustering of individuals” models [The model-based Bayesian Analysis of Population Structure (BAPS) v6.0 was used to study the genetic structure of S. yangi . The gen” models . In addiS. yangi. Pairwise Fst [COI + COII + Cytb + A6A8) calculated with ZT v1.1 [Pairwise mismatch distribution analyses were carried on to detect evidence of past demographic expansions by DNASP v5.0. Multiple methods were performed to understand the population genetic structure of wise Fst  was examwise Fst  for each ZT v1.1  were use-coding genes with substitution model HKY + I + G and the other containing the ribosomal RNA gene with HKY + G. Coalescent tree priors were set to the constant size model. Because no fossils are known for this lineage, it was not possible to calibrate the molecular clock using fossil-based minimum ages [COI gene 2.3%  and 3.54%  per million years [The divergence times for the haplotype lineages were estimated using BEAST v1.8.1  based onmum ages , 61, 62.on years , 64. Twoon years . TREEANNon years  was applon years .We obtained 23 acoustic samples of the male calling song in the field on 06 June, 2017 at the Chunshugou Valley in the Helan (HL) Mountains, Ningxia Hui Nationality Autonomous Region; 21 on 01 June, 2014 at Hancheng (HC), Shaanxi Province; 19 on 05 June, 2014 at Tongchuan (TC), Shaanxi Province; and 15 on 19 June, 2016 at Pingliang (PL), Gansu Province, China, at an average temperature ~ 30 °C. We used a linear PCM recorder  for all acoustic recordings using two stereo microphones. The original stereo format (WAV) was converted to mono at a sampling rate of 44.1 kHz with 16 bits resolution. The analysis of acoustic properties was conducted using RAVEN PRO v1.4  and SEEWAVE v2.0.4  which isS. yangi males produce both timbal and stridulatory sounds for intraspecific communication [P < 0.05 was considered significant.Previous study has indicated that nication . TherefoCOI, 699 bp of COII, 561 bp of Cytb, 657 bp of A6A8, 727 bp of ITS1, and 711 bp of EF-1α. The Chi-square test revealed no significant base composition heterogeneity among the populations for any gene fragment (P > 0.05). COII is the most variable gene, and most of the substitutions occurred in the third codon positions of this gene. The ITS1 gene is more conserved than the mitochondrial genes. Within S. yangi, 5.46% of the sites in COII were parsimony-informative, compared to 0.18% in EF-1α.In total, 790 sequences of the six genes were obtained. The final alignment included 561 bp of COI + COII + Cytb + A6A8), (EF-1α + ITS1) and (COI + COII + Cytb + A6A8 + EF-1α + ITS1), respectively. Both the phylogenetic trees and the network reveal low levels of genetic differentiation and a relatively simple phylogeographic structure for this species  was 89.04% , but only 6.42%  among populations within groups, and 4.54%  within populations  of genetic variation occurs within populations, but only 8.11%  among groups (Table EF-1α + ITS1), AMOVA indicates 85.46%  of genetic variation occurs within populations . Similarly, the BAPS clustering analysis for the COI gene supported four main groups within the ten populations ; group 4: SS2 ; Additional file ITS1 revealed two groups  and Tajima’s D (p < 0.05) statistics. These results significantly reject the hypothesis of neutral evolution in this population, indicating that recent population expansion has occurred in the Helan Mountains and/or adjacent areas. The unimodal curve of the mismatch distribution supports the same inference of a population expansion   to 0.0735 (HL population) Table . SignifiS. yangi and outgroup species are shown in Additional file S. yangi (0.001–0.018) are distinctly lower than distances between S. yangi and the outgroup species (0.108–0.148), without overlap. Except for comparisons between the HL population and other populations, most pairwise comparisons show very low genetic distance values, suggesting these populations have a low level of genetic diversity , when the conventional insect mtDNA divergence rate of 3.54% per million years is used   (N = 23) , and each timbal sound is made up of ~ 2–4 echemes   from those of the remaining populations .The frequency domain of F1 of each timbal sound among the four representative populations  shows no significant difference (05) Fig. a, which 05) Fig. a. The duons Fig. b. The HLSubpsaltria yangi is the only species of the subfamily Tettigadinae known from China [S. yangi from the Helan Mountains in June 2011, a few more populations were discovered using our special acoustic playback method [S. yangi suggest that this species is restricted to the Loess Plateau and adjacent areas of northwestern China. Previous studies show that S. yangi is similar to cicadas of the genus Paharia Distant in morphology and habitat  [Paharia cicadas are patchily distributed primarily in the Middle East [S. yangi occurs locally in patches of suitable habitat. The distribution patterns of Subpsaltria, Paharia and their relatives suggest that the evolution of this tribe has been closely tied to geographic range contraction resulting from historical climatic change [om China . This raom China . It was om China . After tk method . Our disdeserts) –71. Pahadle East . Similarc change .S. yangi. The population structure of S. yangi was revealed to comprise one large unit with low genetic diversity hierarchy but obvious differentiation was revealed between the HL population and other populations based on analyses of genetic distance, haplotype networks, and the calling song structure of males. The deserts and semi-deserts surrounding the Helan Mountains were previously shown to represent a major dispersal and climatic barrier [S. yangi. Subsaltria yangi may, therefore, represent a new example of the early stage of speciation in insects. Cases such as this, where speciation occurs through geographic isolation and genetic differentiation of a peripheral population have been referred to as “budding speciation” [Our study based on combined maternally inherited mitochondrial DNA and bi-parentally inherited nuclear genes provided a comprehensive framework to analyse the phylogeography and speciation of  barrier . Such baciation” .S. yangi are consistent with the scenario of glacial refugia and interglacial climate oscillation in northwestern China. Our estimates , an endemic plant in the Helan Mountains, but other populations except the PL population occur in the Loess Plateau and adjacent areas and feed on Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow. Hence, we hypothesise that ecological differences among habitats, coupled with the geographical isolation, might have reinforced genetic and behavioral divergence of the HL population.Quaternary glaciations are often regarded as a major factor in forming the biodiversity of various extant species . Our divtes Fig.  suggest tes Fig. . The HelCarabus solieri [Parnassius mnemosyne [Apocheima cinerarius [Acromyrmex striatus [S. yangi. Our phylogeny, genetic distances, BAPS and haplotype networks of S. yangi all indicate that a major genetic gap exists between the PL population and remaining individuals  sequence data [Estimated divergence time between nce data . Preference data . FurtherS. yangi haplogroups is well supported   analysesed Table , and 89.ns Table . The divon Figs.  and 3, wS. yangi. Ecological differences in habitats, coupled with the geographical isolation and diet shifts might also have contributed to the divergence of the HL and PL populations from other populations and may represent early stages of speciation. Further analyses on divergence time of this species based on more molecular loci are required to test this hypothesis. Our results improve the understanding of genetic differentiation, acoustic signal diversification and phylogeographic relationships of this rare species of conservation concern, and inform future studies on population divergence, speciation and phylogeography of other insects with a high degree of endemism in the Helan Mountains and adjacent areas.Habitat fragmentation and desertification resulting from Quaternary glaciations and human activities are the most likely factors that yielded the observed genetic diversity and population differentiation in Additional file 1:Table S1. Primer names, sequences used in PCR reactions of genes sequenced. Table S2. List of grouping and haplotypes about populations with mtDNA and nuDNA genes. Table S3. Population clusters of S. hilpa found by BAPS. Table S4. Intraspecific and interspecific genetic distance of S. yangi and other related species based on (COI + COII + Cytb + A6A8) gene. (DOCX 27 kb)Additional file 2:Figure S1. Phylogram reconstructed based (EF-1α + ITS1) genes. Bayesian posterior probabilities and ML bootstrap values are indicated near tree branches. (TIF 1020 kb)Additional file 3:Figure S2. Phylogram reconstructed based (COI + COII + Cytb + A6A8 + EF-1α + ITS1) genes. Bayesian posterior probabilities and ML bootstrap values are indicated near tree branches. (TIF 1513 kb)Additional file 4:Figure S3. Pairwise mismatch distribution of HL population. X axis: Pairwise Differences. Y axis: Frequency. Obs means the observed distribution of pairwise difference. Exp means the expected equilibrium distributions. (TIF 1187 kb)Additional file 5:Figure S4. Scatter plots of genetic distance vs. geographical distance for pairwise population comparisons . (TIF 206 kb)Additional file 6:Figure S5. The divergence time analysis of S. yangi based mtDNA gene, using the rate of 2.3% per million years. Estimates of divergence time are shown at nodes above branches. The divergence times occurred at approximately 0.01 Ma and under 0.01 Ma are not shown. (TIF 1010 kb)Additional file 7:Figure S6. Acoustic analyses of the male calling song structure of S. yangi from Pingliang (PL). A, oscillogram and spectrogram of the timbal and stridulatory sounds were produced alternately . B, detailed oscillogram and spectrogram of timbal and stridulatory sounds (marked by the red box in A). C, D, power frequency spectrum of the signal showing dominant frequencies marked by F1. (TIF 2804 kb)Additional file 8:Figure S7. Acoustic analyses of the male calling song structure of S. yangi from Tongchuan (TC). A, oscillogram and spectrogram of the timbal and stridulatory sounds were produced alternately . B, detailed oscillogram and spectrogram of timbal and stridulatory sounds (marked by the red box in A). C, D, power frequency spectrum of the signal showing dominant frequencies marked by F1. (TIF 3031 kb)


Sistrurus catenatus). First, we use SNP data to confirm previous reports that these populations exhibit high levels of genetic structure . Second, we show that most populations have contemporary Ne estimates <50. Heterozygosity–fitness correlations in these populations provided no evidence for a genetic cost to living in small populations, though these tests may lack power. Third, model‐based demographic analyses of individual populations indicate that all have experienced declines, with the onset of many of these declines occurring over timescales consistent with anthropogenic impacts (<200 years). Finally, forward simulations of the expected loss of variation in relatively large (Ne = 50) and small (Ne = 10) populations indicate they will lose a substantial amount of their current standing neutral variation  over the next 100 years. Our results argue that drift has a significant and increasing impact on levels of genetic variation in isolated populations of this snake, and efforts to assess and mitigate associated impacts on adaptive variation should be components of the management of this endangered reptile.Endangered species that exist in small isolated populations are at elevated risk of losing adaptive variation due to genetic drift. Analyses that estimate short‐term effective population sizes, characterize historical demographic processes, and project the trajectory of genetic variation into the future are useful for predicting how levels of genetic diversity may change. Here, we use data from two independent types of genetic markers (single nucleotide polymorphisms [SNPs] and microsatellites) to evaluate genetic diversity in 17 populations spanning the geographic range of the endangered eastern massasauga rattlesnake ( In the United States, it primarily exists in relatively small, isolated patches of habitat surrounded by heavily modified landscapes .Here, we use two independent genetic datasets to make conservation‐relevant inferences about genetic and demographic characteristics of individual populations across the range of this species. Our specific goals are to (a) evaluate the degree of genetic isolation among populations using a new genome‐scale dataset; (b) estimate contemporary effective population sizes to generate a better understanding of the present and future importance of drift in these populations; (c) re‐evaluate previous results  using two multilocus genetic datasets: One based on DNA microsatellite loci previously analyzed by Chiucchi and Gibbs (n = 229) of individuals were characterized using both genetic markers. Both datasets were used for estimating contemporary effective population sizes. Only the RADSeq dataset was used for the historical demographic analyses, which are based on modeling site frequency spectra (SFS) (see below). For the microsatellites, we used previously published data from 17 loci reported in Chiucchi and Gibbs  of the individuals genotyped for RAD variation. These included samples from SSSP (N = 5), BERG (N = 7), CCRO (N = 18), GRL‐2 (N = 7), GRL‐3 (13), SPVY (N = 6), WLRD (N = 5), GLAD (N = 5), JENN (N = 8), VNGO (N = 7), and PRDF (N = 10). We log‐transformed (ln) these data and performed a major axis regression in the R package lmodel2  and body mass data were available for a subset  in New York City. Prior ranges for the three parameters “NCURR,” “NANCES,” and “TBOT” were 10–50,000, 10–50,000, and 1–10,000, respectively, and were not bounded on the upper end.We used historical demographic analyses to test between models of population history. Our goal was to assess whether evidence exists for declines in population size within each population. For declining populations, we assessed whether the declines occurred over a timescale consistent with anthropogenic impacts (arbitrarily defined as <200 years—see Discussion) or were better explained by historical factors occurring over longer timescales. To avoid problems associated with model overparameterization, we analyzed the simplest models possible that captured the key features of the process (population size change) we were interested in exploring. Specifically, we tested between two simple demographic models Figure  used a sWe tested among the competing historical demographic models with fastsimcoal 2.5.2 .−8 per site/generation as estimated for humans  may represent loci under divergent selection. We explored the sensitivity of our demographic analyses to such loci in two ways. First, we used Outflank version 0.2 . The average numbers of total non‐paralogous loci and polymorphic loci per population were 52,815 and 641, respectively Table , while t3.2r = 0.74; p < 0.05) with microsatellite‐based values averaging ~22% less than matched RADSeq‐based values. The variation in pairwise values is similar, with coefficients of variation of 28.1% and 31.6% for pairwise Fst values based on RADSeq and microsatellite data, respectively. We note that direct comparisons of Fst values based on multi‐allelic microsatellite loci and bi‐allelic SNP loci are problematic because differences in Fst based on each type of marker do not scale in the same way  of 17.3 ± 3.5. Microsatellite‐based estimates of LDNe ranged from 3 (GRL‐1) to 64 (JENN) with an overall mean that is slightly higher (20.6 ± 3.6 SE). For estimates based on RADSeq data, all populations had values <50, whereas for microsatellite data, 13 of 15 (87%) populations were below this threshold. These summaries include all populations including those where estimates were not available for one type of data. When paired estimates for single populations are compared, mean values based on RADSeq data are slightly lower . Paired values based on each type of data are not significantly correlated with each other .Point estimates of the effective population sizes for individual populations vary depending on the type of genetic data and method used to generate the estimates Figure . A smallr = 0.89; p < 0.05). In general, although estimates of contemporary Ne vary depending on method and marker type, most populations have small effective sizes of <50 with only a few populations having larger sizes.For sibship frequency‐based measures of Ne, point estimates range from 8 (SPVY) to 140 (BPNP) (mean: 36.38 ± 10.2) for RADSeq data, and 14 (GRL‐1) to 95 (BPNP) (mean: 44.06 + 5.8) for microsatellite data. Proportionally fewer populations  had sibship‐based values <50. Paired estimates show similar patterns  but, in contrast to LDNe estimates, are highly correlated with each other . We also performed regression analyses on individual populations with a minimum of five samples, but as for the full dataset, no significant positive relationships were detected (results not shown).There was no significant relationship between our estimate of individual fitness (standardized mass index−SMI) and genome‐wide individual heterozygosity estimates based on RADSeq data . These findings indicate that even if our datasets contain subsets of loci under divergent selection not detected by our Fst‐outlier tests due to a lack of power, it is unlikely such loci are having an impact on our demographic inferences.3.7All simulated populations lost variation over time, with the greatest loss occurring in the simulation based on the smallest fixed value of Ne Figure . Based o44.1Sistrurus catenatus. For example, estimates based on LDNe analysis of RADSeq data show that mean Ne is ~22 individuals (range: 2–48), although the confidence estimates for individual populations can be large. This result of small Ne values holds regardless of the method used to generate the estimates , or the type of genetic marker (microsatellites vs. RADSeq), and suggests that genetic drift is poised to have a significant impact on the maintenance of genetic variation in these populations.A major result is that point estimates of contemporary Ne are small (<50) for the majority of populations sampled from across the range of fastsimcoal generated by Sovic et al. (S. catenatus with the closely related but non‐threatened Western Massasauga Rattlesnake (S. tergeminus). The demographic patterns they detected were consistent with differences in the conservation status of each species. The long‐term effective population size was much smaller for the representative S. catenatus population than for the S. tergeminus population, and the S. tergeminus population was best modeled as a growing population, whereas S. catenatus showed evidence of a long‐term population decline.These estimates are much smaller than the effective sizes based on microsatellite variation reported by Gibbs and Chiucchi  for manyc et al.  and herec et al.  or for ec et al. . Long‐tec et al.  comparedIn contrast, the short‐term estimates of Ne based on single sample estimators reported here measure effective population size over much more recent timescales—approximately the period of sample collection, and hence are useful for different conservation purposes  or similar  timescales for declines. This lack of a pattern suggests that local conditions may play a primary role in determining the timescale of population declines. This hypothesis could be explored by incorporating a historical dimension to evaluating habitat suitability in the local area where each population is found (e.g., McClusky et al., These results are at odds with previous work that showed limited evidence for population declines in these populations using different methods of analyses and microsatellite genetic data. Specifically, Chiucchi and Gibbs  used allOther studies using similar approaches have found evidence for either recent or historical bottlenecks in other endangered vertebrates (Dussex, Rawlence, & Robertson, 4.4Sistrurus catenatus populations will lose 20% or more of their existing variation over the next 100 years. These conclusions are based on several assumptions. First, they assume populations will remain no larger than their currently estimated effective size with no increase in migration. This is reasonable given that trends over time are for populations to remain isolated with the same or declining numbers Szymanski et al.., Sistrurus, studies of adaptive variation within populations are scarce, but Jaeger et al. (Sisturus and that high levels of drift have yet to purge this variation. However, whether the rate at which it is expected to be lost parallels the results of our simulations is unclear. Because selection generally acts to retard the impact of drift on the loss of variation within populations, we suspect that the timeline for loss of variation established by our simulations may be conservative.Our simulations suggest that if there are no changes in key population parameters like size and levels of migration, most r et al.  have docSistrurus may not yet have paid the true genetic cost of living in their current size population, but that this cost could be “paid” in the near future. As such, despite their history of living in small populations over historical timescales (Chiucchi & Gibbs, Sistrurus, especially given the fact that this species represents a taxon where outbreeding depression is not expected to be a significant issue (Frankham, These results have conservation implications. They suggest that existing populations of None declared. Click here for additional data file.


Plecotus austriacus. We use ecological niche modelling and environmental dissimilarity analysis to locate areas at high risk of exposure to future changes. Combining outlier tests with genotype–environment association analysis, we identify potential climate‐adaptive SNPs in our genomic data set and differences in the frequency of adaptive and neutral variation between populations. We assess landscape connectivity and show that changing environmental suitability may limit the future movement of individuals, thus affecting both the ability of populations to shift their distribution to climatically suitable areas and the probability of evolutionary rescue through the spread of adaptive genetic variation among populations. Therefore, a better understanding of movement ecology and landscape connectivity is needed for predicting population persistence under climate change. Our study highlights the importance of incorporating genomic data to determine sensitivity, adaptive potential and range shift potential, instead of relying solely on exposure to guide species vulnerability assessments and conservation planning.Climate change is a major threat to global biodiversity that will produce a range of new selection pressures. Understanding species responses to climate change requires an interdisciplinary perspective, combining ecological, molecular and environmental approaches. We propose an applied integrated framework to identify populations under threat from climate change based on their extent of exposure, inherent sensitivity due to adaptive and neutral genetic variation and range shift potential. We consider intraspecific vulnerability and population‐level responses, an important but often neglected conservation research priority. We demonstrate how this framework can be applied to vertebrates with limited dispersal abilities using empirical data for the bat  IncreasEcological niche models (ENMs), also known as species distribution models, offer an effective tool for forecasting how climate change may alter future species distributions and patterns of diversity  and the south of England. These areas represent the species’ southern and northern range limits, as well as the centre and margin of the species’ ecological niche, respectively. We included 10 populations  that had at least eight individuals with sufficiently high DNA quantity and quality (N = 94). These populations represent different geographic areas and combinations of climatic conditions  from s Figure .2.2maxent v3.3.3  and the IPCC5 + 8.5 W/m2 Representative Concentration Pathways scenario  criteria. Climatic suitability was determined based on predicted relative probability of occurrence and was averaged across the four cells adjacent to the population location to cover the colony home range  were used to test for environmental dissimilarity between current and future (2070) conditions. These variables represent climatic conditions that are predicted to change under future projections for Iberia  in more than two populations. Population‐level analyses were carried out on populations containing a minimum of seven individuals to ensure an adequate representation of allele frequencies . The significance of genetic differences between populations and geographic regions (England vs. Iberia) was determined based on a multilocus analysis of molecular variance (amova) implemented in the r package gstudio , as described by Schoville, Widmer, Deschamps‐Cottin, and Manel  Frichot, Schoville, Bouchard, and Francois (2013) approach, implemented in the r package LEA  identified by structure assignment tests as latent factors in the models. We performed five lfmm repetition runs with 1,000,000 iterations and 500,000 iterations for burn‐in. Z‐scores of multiple runs were combined using the median value, and p‐values were adjusted for expected FDR of 0.05 . SNPs that were found to be both under directional selection based on outlier tests and statistically associated with climatic variables based on the genotype–environment association analysis were classified as potentially associated with climate‐adaptive genetic variation, that is with adaptations to local climatic conditions. However, it is important to note that these SNPs may represent genomic regions linked to genes under selection rather than specific climate‐adaptive genes.We carried out a genotype–environment association analysis to test for associations between allele frequencies and local environmental variables . We used the latent factor mixed model  and within Iberia, to account for clines in allele frequencies at neutral loci due to genetic drift and allele surfing during population expansion , F being the coefficient estimation of observed (Obs) versus expected (Exp) homozygosity (Hom):Neutral genetic diversity was estimated based on levels of heterozygosity in the population after excluding SNPs identified as outliers (under selection) by Bayescan. We used the –het function in Plink to compare observed and expected individual levels of homozygosity. Heterozygosity was calculated as 1 − . The analysis included landscape variables and resistance costs that were previously shown to affect functional connectivity in P. austriacus  because they can lead to the identification of spurious inferences  to 100 (strong barrier to movement). The sea was assigned a resistance cost of 200 to reflect the lower likelihood of bats crossing large expanses of water than land because previous studies have found limited gene flow across seas in this species . MRDM was also used to test whether genetic differentiation (Fst) between populations in climate‐adaptive SNPs was a function of environmental dissimilarity  between locations (isolation by environment).We used multiple regressions on distance matrices  to high (4) based on changes in climatic suitability as predicted by the ENMs (reduction in relative probability of occurrence and changes from suitable to unsuitable conditions) and the extent of environmental dissimilarity between present and future conditions Table .Sensitivity was determined based on the frequency of alleles in SNPs identified as potentially associated with warmer and drier climatic conditions (adaptive sensitivity), as well as overall levels of neutral genetic diversity . Levels of adaptive sensitivity were determined based on the frequency of potential climate‐adaptive alleles in the population, looking at both overall mean frequencies across all loci , and number of adaptive alleles present at particularly low frequencies (<0.25) in the population Table . Levels Range shift potential was determined according to the degree of connectivity to other populations under future conditions or to areas predicted to be climatically suitable, as estimated based on Circuitscape movement density (cumulative current) maps. Populations were deemed to have low range shift potential if they were predicted to become isolated (low connectivity) under future conditions, or only connected to adjacent populations and climatically unsuitable areas.P. austriacus, ranging from low to high risk. When combining the three components, we placed higher weights on exposure to future changes in climatic conditions . The main variables affecting habitat suitability for Plecotus austriacus were winter and summer temperatures, summer rainfall and land cover  or in small isolated fragments of suitable habitats . Habitat suitability was predicted to increase slightly in England and around the north Iberian coast  will experience the greatest increase in maximum temperatures (>7°C) and the greatest proportional reduction in summer rainfall  of all individuals screened and contained a maximum of three SNPs . After excluding individuals and SNPs with low coverage and removing SNPs with low minor allele frequencies, the final genotype data set contained 6067 SNPs scored from 83 individuals, belonging to 10 populations, with a total genotyping rate of 0.919. All populations had an average coverage >70% (Table structure and lfmm).From the high‐throughput sequencing, we identified 39,825,843 demultiplexed, paired‐end reads, from which Stacks resolved 11,116 RAD tags that were present in at least 70%  or summer rainfall (10 SNPs). Significant correlations were also identified within Iberia between five SNPs and maximum temperatures and seven SNPs and summer rainfall Table .structure assignment tests divided the full data set into two main genetic clusters, separating the English and Iberian samples. The Iberian cluster was further divided into two clusters, separating the two northern populations, Bizkaia and Girona , but was substantially lower among the north and eastern Iberian populations and among the southern and western populations. Highest levels of differentiation were found between one English population (Devon) and most other populations, apart from the north Iberian populations  and summer rainfall , while within Iberia it was correlated with summer rainfall , with highest values between the English populations and all Iberian populations  and regions  were highly significant. Differences between populations remained significant within Iberia .Genetic differentiation between populations based on the neutral data set ranged between 0.024 and 0.106 . Iberian populations had generally high levels of heterozygosity, with relatively lower levels in Granada (0.844) and Lisboa (0.885), and highest levels in Valencia, Bizkaia and Valladolid . Relative neutral genetic diversity was ranked from low (heterozygosity<0.75) to medium (0.75–0.9) and high (>0.9) Table .3.3R2 = .649, F = 63.1, p = .0001) and to landscape resistance due to decreasing habitat suitability, as measured by the ENM , decreasing forest cover , increasing altitude  and increasing slope . The ENM showed the strongest correlations with genetic differentiation and was the only landscape variable that remained significant after accounting for geographic distance , central  and southern (Granada) populations were predicted to become isolated and were therefore assigned low range shift potential. However, landscape connectivity was predicted to increase across the Pyrenees between the two northern populations and along the north Atlantic coast. Movement out of Iberia appears to be limited both under present and future conditions, but Iberia is predicted to become isolated under future conditions due to decreased habitat suitability in southern France Figure .3.4When combining the effect of the three framework components, we identified one Iberian population  at high risk due to high changes in climatic suitability , low frequency of SNPs identified as associated with climate‐adaptive genetic variation and limited future landscape connectivity. We identified two additional populations in the central regions  that are of medium–high risk because despite high exposure to future changes and limited future connectivity, they have a relatively high frequency of adaptive genetic variation and high levels of neutral genetic diversity. In contrast, populations along the Atlantic coast (north and northwest of the peninsula) are likely to be of lower risk due to more limited changes in climatic suitability and either high future landscape connectivity or lower sensitivity Table .4We propose an applied integrated framework to identify wildlife populations under threat from future climate change based on their extent of exposure to changing climatic conditions, inherent sensitivity due to identified signatures of adaptive and neutral genetic variation and range shift potential Figure . Our fra4.1P. austriacus across the southern part of its range, accompanied by expansion into more northern latitudes. The inclusion of land cover variables and finer‐scale resolution in the ENMs resulted in less severe projections of future range losses in Iberia, but greater projected losses in France, which will isolate the Iberian Peninsula.To assess exposure to future climate change, we combined ENMs with a comparison of environmental dissimilarity between current and future conditions in key climatic variables that are likely to affect bat survival and reproductive success. This helped identify priority areas that are predicted to experience the greatest magnitude of change, the central regions and the Mediterranean coast. However, it is important to note that apart from north and northwest Atlantic coast areas, all Iberian populations are projected to experience maximum temperatures outside the current thermal range of the species. Indeed, the entire Iberian Peninsula is recognized as being under high threat from the effects of future climate change, and Mediterranean ecosystems are predicted to experience the greatest biodiversity changes in Europe due to the combined effect of climate and land use changes  or increasing landscape connectivity to facilitate range shifts and the spread of adaptive genetic variation to reduce threats to medium‐ and medium–high‐risk populations. As such, our framework can contribute to transforming conservation management under climate change from a crisis‐driven response to more anticipatory and predictive measures (Gillson, Dawson, Jack, & McGeoch, O.R. conceived and designed the study. K.P. and G.J. advised on study design. O.R., J.J., C.I., H.R. and A.A. collected the genetic samples. O.R. and J.B.T. carried out the molecular laboratory work and performed the data analysis. S.M. advised on data analysis. O.R. wrote the first draft of the manuscript, and all authors contributed to revisions. Click here for additional data file.


Fontainea rostrata, and its potential requirements for population enhancement and translocation to withstand the effects of anthropogenic fragmentation and climate change. We used microsatellite markers to gain an understanding of the way genetic diversity is partitioned within and among the nine extant populations of F. rostrata identified in this study. We combined the results with species distribution modelling to identify populations vulnerable to possible future range shifts based on climate change projections. We found regional differences between the species’ main distribution in the south and a disjunct northern population cluster , in mean allelic richness , expected heterozygosity (HE = 0.376 vs 0.328), and inbreeding (F = 0.116 vs 0.219). Species distribution models predicted that while southern populations of F. rostrata are likely to persist for the next 50 years under the RCP6.0 climate change scenario, with potential for a small-scale expansion to the south-east, the more highly inbred and less genetically diverse northern populations will come under increasing pressure to expand southwards as habitat suitability declines. Given the species’ genetic structure and with the aim to enhance genetic diversity and maximise the likelihood of reproductive success, we recommend that plant reintroductions to supplement existing populations should be prioritised over translocation of the species to new sites. However, future conservation efforts should be directed at translocation to establish new sites to increase population connectivity, focussing particularly on habitat areas identified as persisting under conditions of climate change.Assisted migration can aid in the conservation of narrowly endemic species affected by habitat loss, fragmentation and climate change. Here, we employ a multidisciplinary approach by examining the population genetic structure of a threatened, dioecious rainforest tree of the subtropical notophyll vine forests of eastern Australia,  The subtropical rainforest communities of eastern Australia have been significantly reduced and highly fragmented following intensive land clearing for agriculture and urban development, leading to decreased population sizes and increased population isolation for many species . Taxa coin-situ or became extinct [et al. [In Australia, the climatic fluctuations of the Plio-Pleistocene have led to significant changes in the distributions of rainforest communities , 8. Clim extinct . Current extinct . Rossett [et al. .Fontainea rostrata Jessup & Guymer (Euphorbiaceae) is a dioecious tree known from only a small number of the remaining fragmented pockets of subtropical rainforests of the Burnett-Mary region of southeast Queensland. These remnants occur across a latitudinal range of approximately 80 km, between Maryborough and Gympie  to classify current and future habitat suitability under projected climate change, can effectively identify and evaluate the potential of translocation and enhancement strategies in the mitigation of threats currently facing F. rostrata’s more vulnerable populations. As the species occurs in a variety of rainforest types comprising several hundred taxa, findings are likely to be of broader relevance to species sharing similar life-history characteristics, while the multidisciplinary methodology reported here provides a novel benchmark for conservation management.In this study, we employed a multidisciplinary approach to determine whether assisted migration is a suitable and necessary conservation management strategy for F. rostrata were identified from which a total of 211 individuals covering the geographical range of the species were sampled , observed heterozygosity (HO), expected heterozygosity under conditions of Hardy-Weinberg equilibrium (HE), and the fixation index (F) as a measure of past inbreeding [PIC) was calculated in CERVUS version 3.0.3 [AR) and private allelic richness (PAR) for each population were obtained via rarefaction using the program HP-RARE [PAR), where a Mann-Whitney U-test was used due to a skewed distribution.GenAlEx version 6.5  was usedbreeding . Polymoron 3.0.3 . Measure HP-RARE , based o HP-RARE  was used HP-RARE , 41, witF. rostrata’s distribution. The average pair-wise level of genetic differentiation (FST) [K (number of clusters) between 2 and 9 were performed, employing a burn-in of 100,000 followed by 500,000 Markov Chain Monte Carlo (MCMC) steps for each run. The geographic location of samples was omitted from the cluster analysis, with results across each run summarised to infer the optimal value of K [We used several methods to analyse population structure across on (FST)  among poon (FST) . To lookon (FST) . Correlalue of K  as implelue of K , processlue of K , and vislue of K . Nei’s ulue of K  was usedlue of K . Mantel’lue of K  was run lue of K , to examF. rostrata presence were compiled from herbarium voucher records [F. rostrata model , whereupon the variables that made the least contribution to model performance were omitted. The final baseline model was run with three-fold cross validation. Ten thousand background points were randomly selected by the model from the study region and the model was run with 500 iterations using the logistic output format, which represents the habitat suitability probability values within the range of 0–1 for each grid cell in the model [We used MaxEnt v3.3.3k –61to modlgorithm  Whilst tlgorithm –67 and hlgorithm . To idenlgorithm  and MaxEst century, prior to stabilising shortly after 2100. The RCP6.0 scenario is also an intermediate scenario, with the GHG emissions peaking in the late-21st century then stabilising [th, 50th and 90th percentile projections. The 50th percentile climate projection surfaces were ultimately used for the final model. An assumption was made that the geological/geomorphological variables would remain constant for the entire modelling period of 50 years, thus current condition datasets were used for all projections.Two future climate projections were derived using SimCLIM v 3.0.0.5  for one bilising , 72, meaF. rostrata by applying the minimum habitat suitability threshold value based on the mean 10th percentile training presence value across cross-validated models, which uses the 10th percentile of the probability threshold range of the species presence records [F. rostrata currently occurs, and to assess the extent of vegetation in each community type within high probability habitat areas using ArcGIS version 10.2 [Model performance was evaluated by the area under the curve (AUC) in receiver operating characteristic analysis (ROC) of the cross validated model output. An AUC score of 1.0 indicates a statistically valid, perfect model fitting, while an AUC value of <0.5 indicates a model performing poorly and no better than random . The bas records . The rec records  were theion 10.2 .F. rostrata individuals with a mean number of alleles per locus at the species level (NA) of 4.417  within respective populations was 2.657 (AR) was 2.622 .A total of 53 alleles were resolved across the 12 microsatellite loci assayed in 211 of 4.417 . The numas 2.657 , althougas 2.622 . HoweverPAR) of 0.044  across populations ranged from 0.247 to 0.432 (mean HO = 0.312), with levels of expected heterozygosity (HE) calculated under conditions of Hardy-Weinberg Equilibrium between 0.307 and 0.426 (mean HE = 0.360)  compared to the southern populations  (F) indicated a moderate level of inbreeding (F = 0.148) when averaged across all populations of the species, with individual population values ranging from -0.025 to 0.269 (F = 0.116) than the northern populations . These trends were confirmed via three sub-analyses where three separate sets of three loci  were removed from the analysis (A), observed heterozygosity (HO) and expected heterozygosity (HE) were consistently higher in the southern versus northern populations, while the inbreeding values were consistently lower in the southern populations (Rxy = 0.174) but significant (p = 0.010) correlation between genetic and geographic distances over the species range indicating a genetic cline between distributional extremes (p > 0.05) of either homo- or heterozygotes at any of the loci tested, suggesting the downstream populations have been established for a considerable time.The observed heterozygosity (= 0.360) . Once ag > 0.05) . The fixto 0.269 . Howeveranalysis . The meaulations . Togetheextremes . The BOTFST) between populations was 0.155, translating to a low level of historical gene flow, barely sufficient to combat genetic drift; FRT = 0.074) between the three population clusters, with another 8% (FSR = 0.088) of genetic variation due to differences among populations within regions. An additional 14% of variation was due to differences between individuals, leaving 71% of the genetic variation contained within individuals within populations  and ranged from a minimal level of differentiation between two relatively proximate populations  providing membership proportions to each value of K among regions ; F. rostrata has not been previously reported, leaving approximately 62% of ‘high-quality habitat’, which currently exists as non-remnant, degraded areas , from the rainforests of northern New South Wales is comprised of 12 populations totalling 350 individuals and has a small distributional range of less than 100 km [Ocotea catharinensis (HE = 0.730) and O. odorifera (HE = 0.782) from Brazil’s Atlantic Rainforest with a range of 800–1000 km were found to display correspondingly higher levels of diversity [F. rostrata (mean HE = 0.360) was somewhat depauperate in comparison to the widespread rainforest tree, Toona ciliata var. pubescens (HE = 0.644) [F. picrosperma (HE = 0.407) [HE) [PIC) of the loci employed, the microsatellite markers used for this study were the product of rigorous screening, with approximately 75 monomorphic primer pairs  being rejected because their non-informative nature [HE of 0.360 is likely an appropriate representation of the genetic diversity for F. rostrata.Many Australian rainforest endemics belonging to ancient Gondwanan lineages, such as aternary , 34, 75.aternary , 76, 77.aternary , who useaternary  than refaternary –82. For n 100 km , whereasiversity . Similar= 0.644) , but was07) [HE)  are necee nature  and therFontainea rostrata is a dioecious, obligate outbreeder, although relative to other outbreeding genera such as Eucalyptus , with potential detrimental effects to the genetic integrity of populations of many large-seeded species. In fact, they suggest that the loss of such dispersal agents may have had a greater influence on the distributions and population genetic structure of susceptible taxa than decreased habitat availability. Given the current lack of dispersal agents, it is probable that habitat fragmentation and degradation since European settlement may be exacerbating adverse genetic and demographic effects in F. rostrata populations through the accelerated loss of connectivity among populations, decreasing gene flow and increasing the risk of inbreeding depression and localised extinctions in the future, particularly in the less diverse and more highly inbred northern populations (F = 0.239). In contrast, the combined  fixation indices for the two southern populations was low (F = 0.029), a level similar to that recorded for widespread eucalypt species (F = 0.016) [F. rostrata continues to increase. = 0.863 ; E. nite = 0.911 ; E. glob = 0.851 ), the spopagules  and pollopagules . Similar= 0.016) –89, althFIT = 0.291; F. rostrata at the species level. Such information can be used to optimise strategies to cover the predicted effects of climate change. Short-distance seed dispersal in F. rostrata is primarily by gravity, however hydrochory provides a means of secondary dispersal over greater distances. Both processes are apparent in the clumped population structure of the species not only within populations, but also at a landscape level.This study found evidence of genetic differentiation  and south-western  clusters based on watershed characteristics ; an obseF. rostrata have been found along Tinana Creek between the south-eastern and northern population clusters. Therefore, with suitable habitat predicted to move southwards under the climatic conditions predicted over the coming century, management of the three isolated northern populations at Tahiti Rd, Weir Rd and Weir Rd 2 should be prioritised regarding assisted migration and/or population enhancement.In its more elevated upper reaches (150–200 m asl), Tinana Creek is steep-sided and fast-flowing when in flood. Any rainforest species that manage to recruit on the terraces between flood events appear to be mostly removed by the next flow. It is only on the lower elevation floodplains (50 m asl), now extensively cleared for the cultivation of sugar cane, that flow rates decrease enough to allow more permanent establishment of propagules from upstream. In fact, despite a considerable search effort spanning two decades (including surveys not part of this study), no populations or even individuals of F. rostrata, are known to exhibit high habitat specificity and narrow thermal tolerances [F. rostrata are currently confined to seven subtropical rainforest community types, which comprise only 8% of the high-probability habitat under present environmental conditions, indicating the potential vulnerability of the species’ and its core habitat into the near future. Added to this, under RCP6.0 conditions, a relatively moderate southward range shift of F. rostrata was predicted by the model, with high-probability areas in the northern region projected to contract southwards into new areas of suitable habitat predicted to emerge between the current northern and southern population clusters, and to the south of the existing southern populations. However, the unassisted migration of F. rostrata propagules may not be an option due to extensive habitat-loss, -fragmentation and -degradation of these areas over the last 150 years, not to mention the absence of actual physical avenues for hydrochorous dispersal from source populations. The species’ medium to large-sized seeds, and possibly the seeds of co-occurring taxa with similar biology and life histories, will therefore need to be manually introduced into suitable pockets of remaining habitat as predicted by the SDM.Many narrowly endemic rainforest species, such as lerances . Howeverlerances . PopulatF. rostrata in this study, and the predictive power of our models could be further improved by exploring several other modelling algorithms to assess and compare the full amount of model predictive uncertainties. Additionally, a relatively low number of presence records exist for F. rostrata, and our SDM was restricted to the southeast Queensland region, which may not fully represent the species theoretical niche. However, F. rostrata is a highly endemic species confined to small, fragmented habitat remnants within the modelled region, and despite extensive field surveys to bolster existing historical occurrence records, the species only occurs in relatively few locations, even within suitable habitat. Thus, as with other studies that utilise a relatively low number of occurrence records by necessity [In this study SDM provided a useful tool to evaluate general trends in habitat distribution, as required for the aims of this study. However, it does not always represent the uncertainty associated with model selection, variable selection and future climate projections . For insecessity –67, MaxEF. rostrata at a regional scale under current and future climatic conditions. This approach gives advanced warning of the conservation and genetic management actions required for F. rostrata, and perhaps similar restricted endemic taxa, over the next 50 years and beyond.Another important consideration when appraising model efficacy is that climate projections are subject to varying levels of uncertainty dependent on the combination of downscaling methods and choice of GCMs and emission scenarios . The futF. rostrata, likely due to differences in multilocus composition arising from unidirectional hydrochorous dispersal and founder effects within the downstream (northern) populations, overall allelic diversity is relatively limited. Therefore, considering the low availability of suitable habitat within appropriate ecosystems, set amongst a landscape matrix that generally comprises non-remnant, degraded land, the implementation of a genetic rescue program to increase local genetic diversity, reduce inbreeding and potentially improve reproductive success is recommended. While the use of neutral markers means that low genetic diversity does not automatically equate to reduced population fitness, and in some instances less genetically diverse populations may be better adapted to local environmental conditions, the impacts of climate change and continuing degradation of landscape scale ecological processes via anthropogenic causes means that the adaptive potential for F. rostrata is likely to be compromised. Mixing genetic stock through population enhancement has the potential to increase adaptive capacity, maintain species-level genetic structure [F. rostrata’s core habitat as a result of climate change.Maintenance of genetic diversity is considered an essential determinant of long-term persistence for many threatened plant species , 93. Alttructure  and enhatructure  particulF. rostrata populations are sufficiently low that the species would likely benefit from a controlled program of genetic supplementation among populations, as a means of increasing allelic diversity and heterozygosity, and mitigating further inbreeding [F. rostrata are likely to persist for the next 50 years under the RCP6.0 climate change scenarios. This suggests that the consolidation of existing populations should be prioritized over translocation of the species to newly established sites, with reintroduction efforts focusing on increasing population sizes, and increasing allelic diversity, especially in the more isolated populations, as these are most likely to suffer the deleterious effects associated with genetic erosion. Translocation efforts in this manner should be in cognizance of local adaptations being important to the survival of low diversity populations, and of the fact that in some instances, populations with low measured diversity may be well adapted to localised conditions and hence provide valuable genetic source material [Although there is potential for hybridization, genetic swamping, and outbreeding depression when mixing genetically dissimilar stocks , the levbreeding . The SDMmaterial , 98. TraF. rostrata without intervention. As the number of alleles per locus contained within individual populations (A = 2.167–3.083) was less than the total across populations (A = 4.417), we recommend that F. rostrata seedlings and cuttings should be exchanged amongst existing populations predicted to maintain suitable habitat under the climate change scenarios modelled in this study. Therefore, as a priority, reintroduction efforts should focus initially on using genetic material from the south to enrich the isolated and genetically depauperate populations in the north, which are predicted to come under increasing stress over the next 50 years due to climate change. However, as each of the two regions contain unique alleles, reciprocal augmentation to increase the diversity among the southern population cluster, in addition to using propagules from the north to a much lesser degree, is advised in the medium term. Future research examining several of F. rostrata’s co-occurring species with similar life-history and habitat preferences is required to confirm the trends found in this study and further define the genetic architecture and long-term needs of these little-studied ecosystem remnants.This project validates the importance of a multidisciplinary approach to guide conservation management of restricted, threatened taxa. We have demonstrated the value of combining SDMs alongside an understanding of genetic diversity of source populations and the population genetic structure of a species, to provide a balanced viewpoint for optimal management of endemic taxa under threat of climate-induced range shifts. Given the prospect of continuing habitat deterioration, there are likely to be eventual negative genetic and demographic consequences for S1 TableSourced from herbarium voucher records , online (DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 TablePercentage contributions of each predictor variable to the final models and minimum and maximum values or selected class codes of each predictor variable across the 20 presence records are given.(DOCX)Click here for additional data file.S7 Table2 with percentage contribution to the total in parentheses. Area abundance of vegetation communities other than the seven RE types within the species’ preferred habitat envelope is also given (other RE types and non-remnant).Approximate area abundance of RE vegetation types within the species’ high-quality habitat is given in km(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 TableMeans are shown between horizontal lines.(DOCX)Click here for additional data file.S1 FigThe probability of *statistical significance (p) based on 999 random permutations is given.(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.


Genetic isolation of breeds may result in a significant loss of diversity and have consequences on health and performance. In this study, we examined the effect of geographic isolation on caprine genetic diversity patterns by genotyping 480 individuals from 25 European and African breeds with the Goat SNP50 BeadChip and comparing patterns of homozygosity of insular and nearby continental breeds.FROH values . Goats from Mediterranean islands represent a more complex case because certain populations displayed a significantly increased level of homozygosity (e.g. Girgentana) and others did not (e.g. Corse and Sarda). Correlations of number and total length of ROH for insular goat populations with the distance between islands and the nearest continental locations revealed an effect of extremely long distances on the patterns of homozygosity.Among the breeds analysed, number and total length of ROH varied considerably and depending on breeds, ROH could cover a substantial fraction of the genome (up to 1.6 Gb in Icelandic goats). When compared with their continental counterparts, goats from Iceland, Madagascar, La Palma and Ireland (Bilberry and Arran) displayed a significant increase in ROH coverage, ROH number and These results indicate that the effects of insularization on the patterns of homozygosity are variable. Goats raised in Madagascar, Iceland, Ireland (Bilberry and Arran) and La Palma, show high levels of homozygosity, whereas those bred in Mediterranean islands display patterns of homozygosity that are similar to those found in continental populations. These results indicate that the diversity of insular goat populations is modulated by multiple factors such as geographic distribution, population size, demographic history, trading and breed management.The online version of this article (10.1186/s12711-018-0425-7) contains supplementary material, which is available to authorized users. The advent of next-generation sequencing and high throughput genotyping techniques has made it possible to identify, in the genomes of multiple species, continuous homozygous stretches of sequence, which are named runs of homozygosity (ROH) . The genSUCLA2 gene [Geographic isolation of populations may lead to a considerable loss of diversity, an increase in inbreeding and vulnerability to stochastic events . For insLA2 gene . In cattLA2 gene  reportedLA2 gene , 13, whiLA2 gene .Recently, genome-wide single nucleotide polymorphism (SNP) data for a comprehensive panel of goats breeds has become available . For theGoats were sampled and genotyped as part of the AdaptMap project  and see The Zanardi software  was usedFROH) was calculated by dividing total ROH length per individual by total genome length across all 29 autosomes (2456.50 Mb). Inbreeding coefficients i.e. Fhet, Fhat1, Fhat2, and Fhat3 were calculated with the PLINK software [Fhet) and on the other hand, we used the—ibc command of PLINK [Fhat, Fhat2 and Fhat3 parameters. Observed heterozygosity (Ho) and effective size (Ne) parameters were retrieved from estimates provided by Colli and coworkers [Genomic inbreeding derived from ROH coverage  of number and total length of ROH for insular goat populations with the distance between each island and the nearest continental location.Statistical analyses were performed by using the R software v.2.15.3 . The valnlme package . We did not perform statistical comparisons between groups or between breeds belonging to different groups. The following groups of breeds were taken into consideration: (1) Icelandic goats, Danish, Dutch and Finnish Landrace; (2) Arran, Bilberry, Mulranny, and Fosses; (3) Argentata, Aspromontana, Ciociara Grigia, Corse, Girgentana, Maltese Sarda and Sarda; (4) Mallorquina and Blanca de Rasquera; (5) Moroccan goat and Palmera; and (6) Androy, Diana, Menabe, Matebele, Sofia and SudOuest. On the basis of exploratory analyses of the data (results not shown), we used a model which assumes inequality of the variances (heteroscedasticity) that are associated with each of the three parameters taken into consideration  and each population:We used a generalized least squares model implemented in the .2.15.3, ) to infend Bates . The leand Bates  and multOur panel of goat breeds included insular breeds from different regions  were observed between goats from Iceland, Madagascar, La Palma and Ireland (Bilberry and Arran) and their continental counterparts , and Malagasy goats (FROH = 0.19–0.35). The genetic isolation of the Dutch goats [FROH = 0.25) rather than by the number of ROH (41–102). Inbreeding coefficients are also high for the Palmeran (FROH = 0.23), Bilberry (FROH = 0.22) and Arran (FROH = 0.30) populations  and significant , which reflects that populations with smaller Ne undergo more genetic drift than larger populations [FROH and the four inbreeding coefficients calculated with PLINK [P value < 0.01) or very significant . Previous studies have reported similar trends for correlations between FROH and a wide range of inbreeding coefficients [In most breeds, we found an inverse relationship between ROH size and frequency Fig. . Howeverth PLINK  i.e.  and total length  were positive and significant.We also calculated Spearman correlations coefficients of ROH length and number with the distance of insular goat populations to the nearest continental coastal site. For goats raised in distant islands, such as Iceland (1277 km from Norway) or Madagascar (1051 km from Mozambique), ROH number and total length had the largest values. Spearman correlation coefficients of the distances with ROH number  and a very high  Mb and aFROH  [The above results are in strong contrast with the majority of the Mediterranean insular goat breeds showing ROH patterns and ns Table , 3, 4, 5viduals) . The Girviduals) . Thus, fAs illustrated by the Finnish, Danish and Dutch Landrace breeds, genetic isolation may also occur in continental populations that are raised in remote locations from Finland and Denmark or subject to a strict breeding management and selection in combination with the occurrence of founder effects (Netherlands). The Dutch goat population has been revived since 1958: it started with two remaining individuals, involved undocumented crossbreeding and resulted in the current population of ca. 2000 animals . GeneticA common finding in several studies of ROH , 28, 38 Our data show that insularization generally involves increased levels of homozygosity. At the same time, patterns of ROH are highly divergent among insular  goat breeds: whereas goats that are raised in Madagascar, Iceland and La Palma, show high levels of homozygosity, those bred in Mediterranean islands display homozygosity patterns that are comparable to those found in continental populations. These results indicate that the effects of isolation are modulated by a complex network of factors including population size, breed history and demography, geographic distribution, trading and breed management, which either maintain strict isolation or allow cross-breeding.Additional file 1: Figure S1. Geographic locations of the insular and continental breeds considered in our study. Red and dark blue indicate insular and continental breeds, respectively, with high homozygosity. Pink and light blue indicate insular and continental breeds, respectively, with low or modest homozygosity.Additional file 2: Figure S2. PCA plot of 25 insular and continental goats for coordinate PC1 against PC2, PC3 and PC4 averaged across individuals. Red and dark blue indicate insular and continental breeds, respectively, with high homozygosity. Pink and light blue indicate insular and continental breeds, respectively, with low or modest homozygosity.Additional file 3: Figure S3. Relationship between observed heterozygosity versus (left) ROH coverage or (right) within-population allele-sharing distances. Red and dark blue indicate insular and continental breeds, respectively, with high homozygosity. Pink and light blue indicate insular and continental breeds, respectively, with low or modest homozygosity.Additional file 4: Table S1. Spearman correlation analysis between genomic inbreeding derived from ROH coverage (FROH) and observed heterozygosity (Ho), effective size (Ne) and inbreeding coefficients calculated with PLINK i.e. Fhet, Fhat1, Fhat2 and Fhat3. The data represent the statistical analysis between genomic inbreeding and inbreeding coefficients.Additional file 5: Table S2. Inbreeding coefficients  for populations with at least 20 individuals calculated with the PLINK software. The data represent the inbreeding coefficients calculated with PLINK software for different populations analysed in this work.


BoLA-DRB3 genetic diversity in Spanish Morucha and Colombian Normande cattle was analyzed and an approach to estimate functional diversity was performed. Sequence-based typing was used for identifying 29, 23, 27, and 28 alleles in Spanish Morucha, Nariño-, Boyacá-, and Cundinamarca-Normande cattle, respectively. These breeds had remarkably low heterozygosity levels and the Hardy-Weinberg principle revealed significant heterozygote deficiency. STF and AD genetic distance showed that Colombian Normande populations had greater variability than other phenotypically homogeneous breeds, such as Holstein. It was also found that Spanish Morucha cattle were strongly differentiated from other cattle breeds. Spanish Morucha had greater divergence in the peptide-binding region regarding other cattle breeds. However, peptide-binding region covariation indicated that the potential peptide repertoire seemed equivalent among cattle breeds. Despite the genetic divergence observed, the extent of the potential peptide repertoire in the cattle populations studied appears to be similar and thus their pathogen recognition potential should be equivalent, suggesting that functional diversity might persist in the face of bottlenecks imposed by domestication and breeding.Bovine leukocyte antigens (BoLA) have been used as disease markers and immunological traits in cattle due to their primary role in pathogen recognition by the immune system. A higher MHC allele diversity in a population will allow presenting a broader peptide repertoire. However, loss of overall diversity due to domestication process can decrease a population’s peptide repertoire. Within the context of zebu and taurine cattle populations,  The MHC consists of a group of loci in jawed vertebrates encoding molecules which are fundamental for regulating the immune response . Class Iathogens .+ T-cells. The BoLA-DQ region comprises DQA and DQB loci, which may vary in number depending on the haplotype; this enables additional diversity by means of intrahaplotype and interhaplotype pairing of DQA and DQB molecules  is located on chromosome 23 and has the overall structure characterizing other mammals’ MHC . DR and olecules . BoLA-DRnctional .BoLA-DRB3 is the most polymorphic bovine MHC gene; 136 different alleles have been reported to date  and drift within and between populations . SeveralBoLA-DRB3 genetic diversity in cattle, information about allele frequency is not available for some breeds (only for a few out of over 800 breeds of cattle recognized worldwide via sequence-based typing) (Despite advances in characterizing  typing) . The few typing) . However typing) .BoLA-DRB3 genetic diversity, structure, differentiation, and selective pressure in Colombian Normande and Spanish Morucha breeds was characterized and compared to worldwide taurine and zebu breeds. Each cattle breed’s BoLA-DRB3 PBR variability and covariation was then analyzed as an approach to compare potential peptide-binding repertoire size and indication of functional diversity. Genetic divergence patterns based on population genetics and PBR variability analysis were similar to that observed for functional divergence; however, the potential peptide-binding repertoire was very similar for all breeds. These results provide an insight into MHC evolution and contribute toward efforts at describing bovine MHC diversity according to breed and location.This study has thus been aimed at determining whether domestic cattle breeding has decreased actual MHC variability. MHC diversity of two new breeds has been characterized. The Normande breed was chosen out of convenience. Spanish Morucha, a breed having relatively low human intervention, has been used here as an approach to what could be considered a natural cattle population. Whole blood was collected aseptically from the coccygeal vein of 165 Normande and Morucha cattle. Considering that Colombian and Spanish cattle farming is characterized by extensive livestock production systems having a relatively few animals per herd, phenotypically purebred animals were sampled from the regions having the greatest amount of purebred cattle in Colombia (Normande) and Spain (Morucha), sampling the most representative amount of farms from each region . Table 1BoLA-DRB3 alleles. The BoLA-DRB3 gene’s exon 2 was amplified by polymerase chain reaction (PCR), using a high-fidelity polymerase along with DRB3F and DRB3R primers , and 50 ng gDNA in a 50 μl final volume. Two independent reactions were performed for each sample, following Lenz and Becker’s recommendations ; colony PCR, with the DRB3F and DRB3R primers, was used for screening the resulting colonies for positive recombinant clones. The colony PCR reaction mixture contained 1X NH4 reaction buffer, 250 μM of each dNTP, 0.25 μM of each primer, 1.5 mM MgCl2, 1 U BIOLASE DNA polymerase  in a final 10 μl volume. A Zyppy Plasmid Miniprep Kit  was used for plasmid extraction, following the manufacturer’s instructions. A BigDye Terminator kit (Macrogen) was used for sequencing clones in both directions, using ccdBsec-F and ccdBsec-R external primers  was used for assembling each independent PCR and clone sequences  and heterozygous positions were identified for producing a consensus containing IUPAC ambiguity codes. The genotype for each animal was assigned by comparing these sequences with database  using HAdatabase , accordia) were obtained by direct counting; 95% confidence intervals (95%CI) were calculated for allele frequencies, according to Fung and Keenan (oh) and unbiased expected heterozygosity (eh) according to Nei    includedulation) .dS) and nonsynonymous (dN) substitutions per site by Nei-Gojobori’s method with Jukes-Cantor correction and Z-test was used for assessing dN/dS ratio significance , nucleotide diversity (π), and the average amount of pairwise nucleotide differences between populations . MEGAX sificance .STF statistics , as described by Weir and Cockerham (STF) and Genepop v.4.7.0. . POPTREE2  analysis, based on AD genetic distances, and principal component analysis (PCA), based on allele frequencies.Normande population structure was evaluated first because samples from this breed were obtained from three geographically defined regions having local breeding practices which could have differentiated cattle populations. Population structure and genetic differentiation between populations were evaluated by estimating ockerham  using ArPOPTREE2  was usedREE2  high-frequency alleles (> 5%) were identified in Normande cattle, accounting for 55.6% of all alleles. Six of these alleles were found to be high-frequency alleles in at least two out of three regions. BoLA-DRB3*001:01 and BoLA-DRB3*002:01 (16.1% cumulative frequency) occurred with high frequency in all three Colombian regions.Sequence-based typing identified 29 alleles in Spanish Morucha, 23 in Nariño, 27 in Boyacá, and 28 in Cundinamarca Normande cattle  occurred with high frequency, accounting for 51.9% of all alleles. Only BoLA-DRB3*005:01 and BoLA-DRB3*048:02 were shared by both breeds as high-frequency alleles. Regarding Morucha cattle, only four alleles (STF (STF = 0.022) and pairwise STF calculated within the Normande breed indicated that the three populations were differentiated at a similar degree to that seen between breeds, and therefore these three Colombian Normande populations were considered separately for diversity analysis.Overall oh) and expected (eh) heterozygosity and the fixation index (ISF) in Morucha and Normande cattle populations and compared them to those reported for other cattle breeds to examine BoLA-DRB3 locus variability and potential departures from Hardy-Weinberg equilibrium. No relation with the original sample size was detected when oh was calculated from randomized datasets of 30 animals, indicating that this estimate (and its derivatives) can be used to compare genetic diversity in populations having unequal sized samples. For the three Normande populations as well as the Morucha population, eh was higher than oh with a significant heterozygote deficiency .We determined the observed (The amount of polymorphic sites (S) and nucleotide diversity (π) were estimated to evaluate nucleotide genetic variability within populations . The remaining comparisons had intermediate values. By contrast with Normande populations, Spanish Morucha had strong differentiation from Holstein and zebu populations, similar to that observed in Japanese Jersey. Boyacá Normande was more differentiated from Cundinamarca Normande than from Nariño Normande. Nariño Normande had the highest differentiation from zebu populations.dN/dS ratio. Selection pattern was very similar for Normande and Spanish Morucha populations, as well as between all populations (dN) was 0.10, in both Spanish Morucha and Normande cattle and the synonymous substitutions rate (dS) ranged from 0.04 in Cundinamarca Normande to 0.09 in Spanish Morucha, with a significant dN/dS ratio being found in most populations. The difference between dN and dS was more prominent when only considering PBR codons and all populations had a significant dN/dS ratio. Spanish Morucha had the lowest dN value of all populations (0.22) while sharing the lowest dS value (0.07) with other populations.Codon selection was evaluated using the STF values were used for assessing genetic differentiation between populations . Nariño Normande was also observed to be most divergent from zebu populations and Boyacá Normande was less differentiated from taurine populations than Boyacá Normande and Nariño Normande. The highest differentiation was observed between Bolivian Nellore with Holstein populations and when comparing a group consisting of Japanese Jersey, Spanish Morucha, and Chilean Hereford to Bolivian Nellore and Peruvian Nellore-Brahman. The remaining comparisons had an intermediate level of differentiation.Pairwise AD genetic distance matrix was used to construct a dendrogram, using the NJ algorithm . The zebu group , the Holstein group , a group formed by Cundinamarca Normande and Nariño Normande and a group formed by Spanish Morucha and Japanese Jersey. Boyacá Normande was noted to be closer to the other taurine populations than Cundinamarca Normande and Nariño Normande.The Allele frequencies were used for PCA and these results were compared to MDS to identify which alleles contributed most to differentiate or cluster cattle populations. Groups clearly identified in both MDS and in the first two PC were depicted as having the major alleles contributing to such grouping  , contributed to differentiating these populations  . The Bonferroni test gave significant differences between Spanish Morucha and Holsteins populations  for all randomizations. Holstein populations’ percentage similarity was lower than for Spanish Morucha (81.61%), indicating that Spanish Morucha had less diversity in the PBR. When identity in PBR positions was assessed, we found that 129 out of 140 alleles had a different PBR.Five randomized sets of 30 animals were constructed to reduce possible sampling effects, considering the clustering observed in diversity analysis results. The groups compared were Holsteins, zebu populations, Nariño Normande, Cundinamarca Normande, Boyacá Normande, and Spanish Morucha. Significant differences between the evaluated groups were found for all randomized datasets . Three groups could be discriminated  allele distribution would have been expected to be that of balancing selection with excess heterozygotes . Neverthractions . HoweverBoLA-DRB3 divergence between Colombian Normande populations was even higher in some cases than the divergence observed between breeds on different continents . BoLA-DRB3 divergence between Normande populations can be highlighted compared to that for Holstein populations which are not so differentiated, despite also being phenotypically homogeneous. Comparing French and Colombian Normande cattle might provide insights into Normande populations’ regional divergence because the French Normande breed is slightly variable and is considered a completely closed breed  and susceptibility (BoLA-DRB3*001:01 and BoLA-DRB3*015:01) to mastitis  does not mean loss of functional diversity. Genetic improvement regarding breeding and domestication appears to have produced three different Colombian Normande populations based on BoLA-DRB3 genetic diversity. When analysing these populations regarding the diversity described for other cattle, it was noted that the forces which have contributed toward moulding BoLA-DRB3 locus diversity in Colombian Normande have produced remarkable differentiation compared to other phenotypically homogeneous breeds, such as Holstein. It is also remarkable that Spanish Morucha was shown to be strongly differentiated in all types of analysis.Despite genetic divergence being observed between cattle populations, their peptide repertoires seem to be functionally equivalent. The great similarities and covariation among populations regarding the All datasets generated for this study are included in the article/The animal study was reviewed and approved by The Universidad de Ciencias Aplicadas y Ambientales  Research Ethics Committee (Minute No. 201901) approved this study.MB, DO, CS, and MP designed the study. MB, DO, and CV carried out experimental procedures. MB, DO, CS, BV, CV, JL-A, AM, IO, and MP analyzed the data and wrote the manuscript. AM and MP supervised the study. All authors approved the final version of the manuscript.www.isciii.es) under grants: RICET RD16/0027/0018 and PI16/01784. MB was partially financed trough a scholarship from the Microbiology Postgraduate Program, Universidad Nacional de Colombia. DO was financed through a scholarship from the PhD Program in Biomedical and Biological Sciences, Universidad del Rosario.This study was partially supported by the Diputación de Salamanca, Caja Rural de Salamanca and the Instituto de Salud Carlos III, ISCIII, Spain (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.