Cercopithecus nictitans group which is sometimes divided into two species C. mitis and C. albogularis. The number of subspecies of C. albogularis is also under debate and is based only on differences in pelage colouration and thus far no genetic research has been undertaken on South African samango monkey populations. In this study we aim to further clarify the number of samango monkey subspecies, as well as their respective distributions in South Africa by combining molecular, morphometric and pelage data. Overall, our study provides the most comprehensive view to date into the taxonomic description of samango monkeys in South Africa. Our data supports the identification of three distinct genetic entities namely; C. a. labiatus, C. a. erythrarchus and C. a. schwarzi and argues for separate conservation management of the distinct genetic entities defined by this study.The samango monkey is South Africa's only exclusively forest dwelling primate and represents the southernmost extent of the range of arboreal guenons in Africa. The main threats to South Africa's forests and thus to the samango are linked to increasing land-use pressure and increasing demands for forest resources, resulting in deforestation, degradation and further fragmentation of irreplaceable habitats. The species belongs to the highly polytypic  Cercopithecus albogularis ranges from central and eastern to southern Africa where it occurs in different evergreen forest types including rainforest, Afromontane and riparian forests, as well as swamp and coastal forests [Cercopithecus nictitans group [C. mitis and C. albogularis. Groves [C. albogularis for individuals distributed from Ethiopia to South Africa and occurring in South and East Democratic Republic of Congo and North West Angola. Groves classifies C. mitis as those individuals found from the Congo-Oubangui River System to East African Rift Valley, as well as in Northern Angola and North Western Zambia. The splitting of the group into two separate species by Groves is based on differences in pelage colourations. Napier [C. albogularis as a separate species. Furthermore the International Union for Conservation of Nature (IUCN) has not yet recorded C. albogularis as a separate taxon and C. mitis is listed as least concern [The geographical distribution of the arboreal guenon  forests . The spens group  which is. Groves ,4 recogn. Napier  followed. Napier  and Grub. Napier  do not r concern . No gene concern ,4.C. albogularis recognised in South Africa  is also disputed. Meester [C. a. labiatus [C. a. erythrarchus [C. a. schwarzi [C. a. labiatus and C. a. erythrarchus is closely correlated with the distribution of Afromontane, Scarp and Indian Ocean coastal belt forests in southern Africa and the two subspecies do not overlap in their distribution [Cercopithecus a. labiatus is distributed from the Pirie Forest in the Eastern Cape Province north-eastwards to the midlands of the KwaZulu-Natal Province [Cercopithecus a. erythrarchus occurs from northern KwaZulu Natal through eastern Mpumalanga and central and eastern Limpopo in South Africa, through Zimbabwe and Mozambique up to Malawi  [C. a. labiatus and C. a. erythrarchus are recognised by both the latest national [C. a. labiatus is listed as Endangered as it is considered endemic to South Africa whereas the subspecies C. a. erythrarchus is listed as Vulnerable [C. a. erythrarchus [C. m. erythrarchus as least concern whereas C. m. labiatus is listed as Vulnerable [The number of subspecies of  Meester  followed Meester  recognislabiatus   ,10 . National Threatened or Protected Species (TOPS) permits (Permit no. 0 27520 and 0 27333) as well as provincial permits  were obtained for the capture and anaesthesia of the animals as well as the sample collection, transport and storage. Animals were not removed from the wild. They were captured and released at the same location.C. a. labiatus population in Hogsback , and C. a. erythrarchus from two coastal populations in Cape Vidal  and Sodwana Bay , a population along the Escarpment in Magoebaskloof  and from two troops from the northernmost population in the Soutpansberg mountains . See 1 for the geographic distribution of sampled populations and their acronyms. Trap-door metal cage traps (1.25m x 0.6m x 0.6m) were placed on the ground and were baited with either oranges or apples. Once secured in a trap, the samango monkeys were anesthetised with an intramuscular injection of a tiletamine/zolazepam  combination at an estimated dose of 3.5–5 mg/kg. Three milliliters of blood was collected with a syringe and needle from the caudal saphenous vein and divided into serum and EDTA blood tubes . The blood samples were placed on ice for up to 6 hours, after which they were frozen at-20°C until analysis. Several full length hairs were plucked out from between the shoulder blades of each monkey and placed in plastic Ziploc bags. Small (3mm in diameter) skin and cartilage samples were obtained from the pinna of each monkey using a normal leather punch. The punch was cleaned and disinfected  LTD, South Africa) between samples. The skin and cartilage samples were placed in 1.5ml eppendorf tubes containing 95% ethanol. DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit  following the extraction protocol as outlined by the manufacturer. Each animal was assigned to a gender and placed in one of three age classes, namely juvenile, sub-adult or adult based on tooth eruption where sub-adults have at least one permanent canine and adults have all four permanent third molar. Standard morphological measurements were taken (see below) before the individuals were placed in a darkened cage until they had recovered from the anaesthetic to be released back into the wild. A qualified veterinarian registered with the South African Veterinary Council (registration number D97/4000) and experienced in primate capture and anaesthesia was present at all the capture sites, administered the anaesthetic agents and ensured that animals were fully recovered prior to release.A total of 72 samango monkey blood and tissue samples were collected from five populations in Southern Africa . All indFor 101 individuals, body mass  was obtained using a digital hanging scale , whilst the following standard measurements were obtained (in cm) using both a tape measure and calliper: neck circumference (NC), head and body length from nose-tip to base of tail (HB), tail length (TL), hind foot length, without claw (HF), ear length (EL), ear width at base (EW), canine length (CW) and nipple length (NW). Full body pictures of individuals from different sites were also taken. Measurements made using a tape measure were converted using simple linear regression equations. Apart from nipple length which was only measured in females individuals, variables were log-transformed and analysed statistically for sexual dimorphism and age variation using 2-way Analysis of Variance (ANOVA) in the largest population from Hogsback (n = 67). Owing to significant variation between the three age classes, data from adults were compared between all populations using principal component analysis (PCA). All statistical analyses were carried out using the programme PAST version 1.91 .In order to assess variation in hair characteristics among the different samango populations sampled we analysed individual guard hairs of adult individuals only. As the Hogsback population was sampled at different months of the year  we chosein situ photographs of individuals from the three populations were compared to examine differences in pelage colouration.Hair colour was scored using three different categories: white, light yellow and dark yellow. These categories were established after comparing hairs from all different geographic locations  in order2,200 micro molar (μM) of each 2’-deoxynucleotide triphosphate (dNTP), 10 picomol (pmol) of each of the forward and reverse primer, 1 unit (U) TaqDNA polymerase and 10–20 nano gram (ng) genomic DNA template. The conditions for PCR amplification were as follows; 5 minutes (min) at 95°C initial denaturation, 30 cycles for 30 seconds (sec) at 95°C, 30 sec at 50–65°C and 30 sec at 72°C, followed by extension at 72°C for 20 min. The PCR reaction was carried out in the BOECO TC-PRO Thermal Cycler. PCR products were pooled together and run against Genescan500 LIZ  internal size standard on an ABI 3130 Genetic Analyzer. Samples were genotyped using GeneMapper v. 4.0.All samples collected during the present study were genotyped for polymorphism at 21 microsatellite loci based on previous studies: D15S108, D14S306, D13S765, D18S536, D12S67, D9S922, D8S1106, D4S243, D5S1457, D10S611, D6S311, D5S1466, D1S518, D1S207, D3S1768, D10S1432, D7S503, D2S1326, D11S956, D11S925 and D17S1290 . Amplifi2, 200 M of each dNTP, 5 pmol of each of the forward and reverse primer, 1 unit (U) Taq DNA polymerase and 20 ng genomic DNA template. PCR cycling conditions were as follows: Initial denaturation at 95°C for 5 min, annealing at 52°C for 50 sec and extension at 72°C for 1 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 50°C for 50 sec and extension at 72°C for 1 min. A final extension step of 72°C for 20 min concluded the cycling. After the initial PCR amplification, products were purified according to the Exo/Sap amplicon purification method described by Werle [The purified DNA was used as a template to amplify mitochondrial gene fragments using the Cyt B primers L14724 cgaagcttgatatgaaaaaccatcgttg and H15149 aaactgcagcccctcagaatgatatttgtcctca  and 16S by Werle . Cycle sC. a. labiatus and C.a. erythrarchus), and secondly as four populations, largely reflecting collection localities, that were identified from spatially independent multivariate analyses of the two currently recognised subspecies. The latter populations are Hogsback (HB), Sodwana Bay and Cape Vidal combined (SDBCV), Magoebaskloof (MK) and Soutpansberg Mountains (SM). To estimate the levels of genetic diversity within populations, the mean number of alleles per locus (NA), observed heterozygosities (Ho), expected heterozygosities (HE and uHE) and deviations from Hardy-Weinberg (HW) proportions were calculated using MS TOOLKIT [FST- and RST-based analyses of molecular variance (AMOVA) [MICRO-CHECKER  was used TOOLKIT  and GENA TOOLKIT ,35. Link TOOLKIT ,37. Asso (AMOVA) , that we (AMOVA) ,35. SpatC. a. erythrarchus (n = 35) samples was run with the same settings for K = 1–5. The K with the greatest increase in posterior probability (ΔK) [The level of genetic differentiation was determined among and within the currently recognized subspecies, and the four distinctive populations. The genetic relationship between populations and individual assignments of samples was inferred via a Bayesian clustering analysis using the statistical programme STRUCTURE version 2.3.3 . The proity (ΔK)  was idenity (ΔK) . CLUMPP ity (ΔK)  generateity (ΔK) .ST) were calculated using DNAsp v 5.10.01 [www.fluxus-engineering.com) and Splitstree v 4.10 [Alignment and vetting of sequence data were carried out using Bioedit v7.0.9.0  and Finc 5.10.01  based on 5.10.01  were gene v 4.10   in all variables except for ear length . SignifiWhen analysing variability in hair colour, significant differences between the Soutpansberg Mountains and Magoebaskloof (Inland), coastal populations  and Hogsback populations were found , with Inland showing light yellow bands, Coast showing mostly dark yellow bands and Hogsback showing white bands . The relC. a. labiatus  samples, only two loci significantly deviated from HWE (loci D12S67 and D2S1326), and two loci were monomorphic (loci D11S956 and D6S311). Among C. a. erythrarchus individuals, the pattern was strikingly different, with 11 loci deviating from HWE. Each pair of loci in each population was tested for linkage disequilibrium (420 tests), and 81 of these were significant. However, only eight were significant within C. a. labiatus, and only three locus pairs showed significant evidence of linkage in both populations . Genetic diversity was lower among C. a. labiatus than among C. a. erythrarchus in terms of, among others, the number of alleles, heterozygosity and mtDNA haplotype diversity (C. m. labiatus (HB) samples exhibited at least one of 15 private alleles found at 11 loci in that population. Additionally, at least one of the 50 private alleles found among C. m. erythrarchus samples, i.e. alleles that were not found among C. m labiatus (HB) samples, was exhibited by all individuals (n = 35) from this population. This pattern indicates strong divergence and restricted gene flow between the currently recognized subspecies.In the iversity . The meaST, GST), corrected (standardized) fixation indices (G”ST) and a pure estimate of differentiation (DEST) were calculated to estimate genetic divergence between the two putative subspecies. Pairwise estimates of population differentiation were all high and highly significant  differentiation among the two subspecies (locus s P = 0.479) based on GST. Corrected FST (F’ST) and RST were estimated during AMOVA [ST-based AMOVA showed that 25% of the variance in allele frequencies was explained among populations, and 14% among individuals. FSTmax was 0.467 and FST was estimated at 0.25 (P = 0.0001), giving an F’ST of 0.526. FIS (0.188) and FIT (0.391) were also high and highly significant (P<0.001), indicating strong population structure. The RST-based AMOVA generated a highly significant (P<0.001) RST-value of 0.157, with 16% of variance distributed among the two subspecies. Estimates based on the combined mtDNA sequence data for the two subspecies also indicated significant divergence (Various fixation indices (Fng AMOVA . The FSTvergence . InteresLn) using Bayesian admixture analysis were calculated for K = 1–9, with K = 2 being identified as the most likely true K value based on STRUCTURE HARVESTER results (K = 2 displayed the greatest posterior probability). Results indicated two distinct clusters for C. a. labiatus and C. a. erythrarchus : DSW and Cavalli-Sforza and Edwards Dc  resemble hybrids in that most are equally likely to have originated in either of the aforementioned coastal and inland clusters.The two ulations . HoweverC. a. erythrarchus clusters identified by STRUCTURE, and the Hogsback C.a. labiatus population. Where deviations from HWE remained the same for C. a. labiatus, three loci were monomorphic in the Soutpansberg Mountain  population, and two deviated from HWE (D4S243 and D1S207). Loci D10S1432, D1S207 and D5S1466 deviated from HWE in the MK population, and D11S956 was monomorphic. Loci D2S1326, D10S1432, D5S1466, D10S611, D8S1106 and D12S67 deviated from HWE in the SDCV population. 37 of the 840 pairwise linkage tests were significant, but no locus pair showed significant linkage in all four populations. Over all four populations, GENEPOP identified six pairs of loci as significantly linked , MK had five private alleles . The combined SDB and CV populations exhibited 20 private alleles, and all individuals except one (SDB 3) exhibited at least one of these private alleles.Population genetic analyses were repeated with populations defined as the three y linked . Althougy linked . ObserveHogsback  and the baskloof . All C. Most indices of overall population structure increased compared to the previous analysis comparing only the two subspecies .ST based on AMOVA was 0.29 (P<0.001), FSTmax was 0.50, and corrected FST, therefore, equals 0.58. Also, 29% of variance is explained among the four populations, and 9% among individuals. Pairwise FST values estimated during AMOVA , which is lower than the estimate between these two C. a. erythrarchus populations. Importantly, all populations are significantly different from one another. Overall RST was 0.23 (P<0.001), and pairwise RST-values also indicate high, and highly significant, differentiation among the four predefined populations. The largest differences are again between C. a. labiatus and each of the three C. a. erythrarchus subpopulations , which was assigned to the Soutpansberg Mountain population.C. a. labiatus) share 16S haplotypes with individuals from the SM population (C. a. erythrarchus). The pattern of reticulate evolution reflected in the haplotype networks cannot be represented as a bifurcating phylogenetic tree, but is evident as reduced nodal support in the ML and Bayesian trees  were generated in 28 samples . Each gene region contained five haplotypes, and when analysed together, there were a total of eight haplotypes (haplotype diversity = 0.87). MP-based haplotype networks largely reflect the pattern of divergence estimates from analyses of population differentiation . Haplotyan trees –12 Figs.C. a. labiatus and C. a. erythrarchus described by Meester [C. a. erythrarchus are distinctly heavier and larger in most body metrics than both the Hogsback  population of C. a. labiatus as well as the coastal forest populations of C. a. erythrarchus from Sodwana Bay (SDB) and Cape Vidal (CV) in KwaZulu-Natal. Body size in many animals is known to be strongly influenced by environmental factors resulting in well established “rules” of geographic variation, e.g. towards increasing body size at higher latitudes and elevations (Bergman’s Rule) [C. a. labiatus) show hardly any yellow colouration, neither on the back nor in the ischial region  and clearly setting them apart from Inland  and Coast  individuals. The most pronounced difference between Coast individuals and all the other populations found was their generally lighter pelage colour, the prominent and comparatively extensive yellow wash on the back and the orange ischial region. These results are very similar to those of Roberts [erythrarchus individuals had the most conspicuous reddish colouration in the ischial region. The overall lighter appearance of the pelage in Coast individuals found in our study could be explained by the fact that in 46% of Coast hairs analysed the light bands were equal to the length of the dark bands as opposed to Hogsback and Inland individuals where black bands were either double the length (Hogsback) or longer than light bands (Inland), resulting in an overall darker appearance. The conspicuously black arms found in both Hogsback and Inland individuals further increase the effect of this dark appearance. Roberts [C. a. schwarzi and C. a. erythrarchus is that the ischial region is buffy yellow (Roberts) or reddish (Groves) in schwarzi but not as reddish as it is in coastal C. a. erythrarchus (Roberts). This description compares well to the pelage colouration we observed for the Inland individuals which had a yellow rather than orange (Coast) ischial region. The Inland population was made up of individuals from Magoebaskloof and the Soutpansberg as they showed the same hair characteristics. Morphological results from our study could not be compared to Meester [erythrarchus and labiatus.Morphometric variables in isolation, could not distinguish the two genetic lineages;  Meester  followed Meester . Instead’s Rule) . Whilst ’s Rule)  and as c’s Rule) . Within  Roberts  and Grov Roberts ,4 for C. Roberts  and Grov Roberts  who foun Roberts  and Grov Roberts  found th Meester  and Grub Meester  as they E) of 0.5. The total HE was 0.59. When the dataset was subdivided to reflect the two currently recognised subspecies, C. a. labiatus and C. a. erythrarchus, the former showed lower levels of genetic diversity in terms of number of alleles and heterozygosity (HE = 0.38) and fewer deviations from HWE relative to the latter (HE = 0.62). The HE value for C. a. labiatus falls into the lower range of what has previously been reported for Neotropical primate species [Cebus apella, HE = 0.378) and Squirrel monkeys (HE = 0.239). However, among C. a. erythrarchus, genetic diversity estimates are among the highest described for primate species, for example red howler monkeys  and brown woolly monkeys . The strong deviations from HWE observed among C. a. erythrarchus samples seem to be as a result of population subdivision i.e. the Wahlund effect, as inbreeding and strong genetic drift would have resulted in lower heterozygosity and numbers of alleles [C. a. erythrarchus. In terms of genetic structure and divergence between the currently recognised subspecies, we found strong evidence for isolation based on mutually exclusive private alleles in each subspecies i.e. all the individuals belonging to each taxon exhibited alleles that were not found in the other taxon. This pattern was reflected in the highly significant pairwise estimates of population differentiation , which were as high, or higher, than those reported for currently recognised primate species [Cebus species (GST = 0.08 to 0.11), Ateles species (GST = 0.105 to 0.123) and Aloutta species (GST = 0.189 to 0.259). The RST-value estimated during AMOVA, however, was lower between samango subspecies  than those reported for other primate species e.g. Cebus species pairwise RST-values ranged from 0.26 to 0.33.Across the whole dataset of 72 individuals, the number of alleles detected per microsatellite locus ranged from two to 11, with a mean of four, and a mean expected heterozygosity , and coastal populations in Sodwana Bay and Cape Vidal. Bayesian clustering analysis indicates that the coastal and inland forms are distinctive based on their multi-locus genotypes, but that the Magoebaskloof population that falls geographically between the coastal and Soutpansberg populations, resembles both inland and coastal forms, indicating a possible “hybrid zone” between these two population units.Spatially independent analyses of the nDNA dataset consistently identified strong subdivision between the two currently recognized subspecies, and also within the C. a. erythrarchus sub-populations ranged from 0.34 in the Soutpansberg Mountains to 0.58 in the coastal population . Importantly, population differentiation estimates based on the four predefined populations increased compared to analyses comparing only the two currently recognised subspecies. This, together with the results of the finer scale spatially independent analyses e.g. the assignment test (99% correct assignment) and Bayesian clustering, indicate strong structure among coastal and inland forms of C. a. erythrarchus, and exceptionally strong differentiation between these populations and that of C. a. labiatus.Expected heterozygosity within these C. a. labiatus) and SM (C. a. erythrarchus) populations followed by secondary contact which could occur either via hybridisation, due to human mediated interventions or due to sex-biased dispersal. There is evidence that females are philopatric while males leave their troops before sexual maturity at 6–8 years old [Mitochondrial DNA analysis generated discordant patterns from nuclear DNA in terms of reconstructed relationships among groups. In contrast to both phenotypic descriptions and nuclear DNA, the HB and SM individuals were found to be closely related based on 16S and the SM and coastal populations (SDB and CV) were more similar based on cyt b. The coastal and MK populations were differentiated into two separate groups, and MK was consistently distinctive from all other populations. Discordance between mtDNA and patterns observed for nuclear genomes has been widely reported and has been found to be the highest in mammals . Discordears old –60. HoweCercopithecus a. labiatus is suggested to represent the first dispersal event from East Africa along Afromontane forests down into South Africa before the last glacial maximum (LGM) whereas C. a. erythrarchus is thought to have entered South Africa in a second dispersal event, after the LGM within the last 12.500 years, primarily along the expanding coastal belt forest on the Mozambique seaboard. Cercopithecus a. labiatus was until then in all likelihood the only representative of the species present in southern Africa prior to and during the last glacial maximum [labiatus and erythrarchus diverged about 1.7 Mya (1.6–2.6 Mya) during the mid-Pleistocene. During this period the subtropical African climate periodically oscillated between markedly wetter and drier conditions, with step-like increases of variability and aridity near 2.8 Ma, 1.7 Ma, and 1.0 Ma and also suggesting more varied and open habitats after 1.8 Ma [labiatus was likely isolated by retreating forests during one of the drier Pleistocene periods, indicating that some forest refugia remained south of the Umfolozi system, not only throughout the LGM but also throughout the mid Pleistocene. There is also no genetic indication of secondary hybridisation with later radiations of the species which suggests that a geographic barrier of some sort, such as the Umfolozi system, as suggested by Lawes [labiatus must have, according to our results, occurred prior to the mid-Pleistocene and it thus seems more likely that several older Pleistocene radiation events occurred before the LGM. There is also no reason to assume that gallery forests of river systems (e.g. Limpopo system) connecting forests did not play a role in the radiation of the species. Our results indicate that there are genetic as well as morphological differences between the coastal and inland erythrarchus populations in South Africa. Future sampling of inland and coastal populations from Zimbabwe (inland) and Mozambique (coast and inland) further north will show if this might be a general pattern within the species range and also give additional insights about possible radiation routes and the number of radiation events.Paleoclimatic data of the late Quaternary (last 150.000 years) was used by Lawes  to expla maximum . Our stur 1.8 Ma . These wr 1.8 Ma  suggestsby Lawes , will haby Lawes  is possiC. a. erythrarchus samples. The reticulate evolutionary pathway evident in the mtDNA haplotype networks, discordant from nDNA, likely represents an ancient pattern of hybridisation or introgression among samango monkey populations, and highlights the complex evolutionary history of this group. Differences in morphology and nDNA markers, however, suggest that these populations are on distinct evolutionary trajectories. Our data thus support the two recognised subspecies namely; C. a. labiatus and C. a. erythrarchus. Within C. a. erythrarchus two lineages should be proposed. The Soutpansberg is currently classified as C. a. erythrarchus by Friedmann and Daly [C. a. schwarzi by Roberts and Groves and as the Soutpansberg and Magoebaskloof show the same pelage characteristics, the Soutpansberg population should more accurately be classified as C. a. schwarzi. Whereas, the costal populations (CV and SDB) may be assigned to C. a. erythrarchus . However, the taxonomy of the Magoebaskloof population remains unresolved. This population is highly divergent based on mtDNA sequence data, but is relatively uniform genetically (nDNA) and phenotypically (morphometrics and pelage colour) to the SM population (C. a. schwarzi). However, it is unclear whether this population can be attributed to C. a. schwarzi or if it is a hybrid zone or a distinct lineage. Therefore, the above hypothesis should be tested by further sampling in intermediate localities and adjacent countries to properly ascertain whether clinal variation occurs throughout the range of these taxa, or if they are isolated, as it currently unclear whether these lineages are distinct species or sub-species. Additional markers (such as X- and Y-chromosomes and mobile elements) are also required to fully resolve the taxonomy of this species and test the validity of C. mitis versus C. albogularis. The final taxonomic outcome is dependent on choice of species concept. According to the Phylogenetic Species Concept (PSC), the three lineages are diagnosable (on three independent characters) and therefore represent species. The PSC has gained widespread acceptance although its application to large mammals has been recently questioned by some conservationists and defended by systematists [C. a. erythrarchus and C. a. labiatus) and Management Units  following the definitions of Moritz [Our study gives the most comprehensive insights to date into the taxonomic description of samango monkeys in South Africa. Spatially independent analyses of the nDNA dataset and data based on pelage colour consistently identified strong subdivision between the two currently recognized subspecies, and also within the and Daly  and is gand Daly  or Groveand Daly . Howeverematists –70. Regaf Moritz . DistincS1 Fig(TIFF)Click here for additional data file.S2 Fig(B) Delta K values for real population structures of K = 1–9. Output from STRUCTURE HARVESTER showing (C) Probability (-LnPr) of K = 3 and (D) Delta K values for real population structures of K = 3.(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S5 Fig(a) and (b) are different projections of the same results. Individuals are colour-coded according to their collection locality .(TIFF)Click here for additional data file.S6 Fig(TIFF)Click here for additional data file.S7 FigNumbers at the nodes are bootstrap support.(TIFF)Click here for additional data file.S8 FigNumbers at the nodes are bootstrap support.(TIFF)Click here for additional data file.S9 FigNumbers at the nodes are bootstrap support (<50% not shown).(TIFF)Click here for additional data file.S10 FigNumbers at the nodes are Bayesian posterior probabilities.(TIFF)Click here for additional data file.S11 FigNumbers at the nodes are posterior probabilities.(TIFF)Click here for additional data file.S12 FigNumbers at the nodes are posterior probabilities.(TIFF)Click here for additional data file.S13 Fig(TIFF)Click here for additional data file.S1 Table(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.


Eucalyptus camaldulensis Dehnh.) is a widespread Australian eucalypt inhabiting riverine and floodplain habitats which spans strong environmental gradients. We investigated the effects of adaptation to environment on population level genetic diversity of E. camaldulensis, examining SNP variation in candidate gene loci sampled across 20 climatically diverse populations approximating the species natural distribution. Genetic differentiation among populations was high (FST = 17%), exceeding previous estimates based on neutral markers. Complementary statistical approaches identified 6 SNP loci in four genes  which, after accounting for demographic effects, exhibited higher than expected levels of genetic differentiation among populations and whose allelic variation was associated with local environment. While this study employs but a small proportion of available diversity in the eucalyptus genome, it draws our attention to the potential for application of wide spread eucalypt species to test adaptive hypotheses.As an increasing number of ecosystems face departures from long standing environmental conditions under climate change, our understanding of the capacity of species to adapt will become important for directing conservation and management of biodiversity. Insights into the potential for genetic adaptation might be gained by assessing genomic signatures of adaptation to historic or prevailing environmental conditions. The river red gum ( Trees are foundation species in many terrestrial ecosystems and changes to local environment associated with natural and anthropogenic climate change are projected to impact, or are already impacting, the health of forest tree populations and the ecosystems they service worldwide Reversible plastic responses are important for short term adaptation of natural populations Insights into a species potential for genetic adaptation could be gained through characterisation of the relative abundance of adaptive versus neutral variation in response to historic or prevailing environmental conditions, which might be applied to empirically test hypotheses about environmental adaptation. Forest trees are tractable models for adaptive genetic studies owing to widespread populations traversing environmental gradients, high levels of diversity and low frequency of co-segregation among gene loci (linkage disequilibrium) Eucalyptus camaldulensis Dehnh.) is a large tree found in riparian zones and associated floodplains in arid and semi-arid regions throughout Australia Significant areas of forest and woodland growing in marginal or semi-arid regions of Australia are currently at risk of decline in response to climate change. The river red gum , and 2. xylem cell wall development . The first group have the potential to impact plant water use in response to climate, via directing the movement of water between cellular compartments and out of the leaf stomata, or acting as internal cellular stabilisers in response to dehydration Using single nucleotide polymorphisms (SNPs) we examined the variability of twelve genes in trees sampled across the natural range of E. camaldulensis individuals drawn from Murray-Darling Basin populations in the collection of Butcher et al. Eucalyptus grandis genome sequence  in CLC Genomics Workbench (CLCbio). A twelfth gene, ERECTA, was sequenced in 36 individuals drawn from four Murray-Darling Basin populations of E. camaldulensis , Menindee (8), Wenthworth (8) and Wilcannia (8)), which were sampled from the collection of Butcher et al. Polymorphism data was identified from eleven candidate genes for which amplicons representing the open reading frame had previously been sequenced on a pooled sample of 500 Single nucleotide polymorphisms were selected from DNA sequence alignments based on the following criteria: a base pair change was present, the base position was not an indel and the minor SNP allele occurred in at least three individuals. SNPs were selected to maximise coverage across the gene and avoid redundancy due to linkage disequilibrium, by selecting a SNP at least one to two hundred base pairs apart. Gene sequences were annotated, and for each SNP the base change, gene position (intron/exon) and amino acid substitution were recorded. Ten SNPs were chosen for each gene, with the exception of CAD (8), bZIP (7), Dehydrin (5) and Erecta (5). SNP genotyping was performed on genomic DNA samples using the Sequenom MassARRAY System at the Australian Research Genome Facility (AGRF) . GenotypLinkage disequilibrium (LD) in outcrossing forest tree species is typically low, and in eucalypts such as river red gum co-segregation of SNP loci along a gene decays within several hundred base pairs In total, 76 of the 105 selected SNPs were successfully genotyped. Monomorphic SNPs and trees with >20% missing data were omitted from the data set, resulting in 59 SNPs typed across 191 individuals. Observed and expected heterozygosity and tests for Hardy-Weinberg Equilibrium (HWE) were performed on individual SNPs, and for sets of SNPs representing whole genes, for each population separately and overall using GenAlEx 6.2 ST was also estimated in GenAlEx to obtain both overall and pairwise population estimates of genetic differentiation. Significance of the observed differentiation was tested by performing 1000 random permutations of the data. In the same way, FST was also estimated for these populations (with the exception of Wirrengren Plain) using polymorphism data for the 15 putatively neutral microsatellite loci (nuSSR) applied in Butcher et al.Genetic divergence among populations based on all SNP loci was investigated using several complementary approaches. Firstly, analysis of molecular variance (AMOVA) was performed in GenAlEx to partition genetic diversity residing within and among populations and individuals. Using the AMOVA framework FST were examined via a Mantel test implemented in GenAlEx, and significance was based on 1000 permutations of the data. Divergence was also compared via principal coordinate analysis (PCoA) of genetic differentiation among populations implemented in GenAlEx. Spatial autocorrelation (isolation by distance or IBD) of population pairwise genetic divergence based on SNP loci was assessed using the Mantel function in GenAlEx, and significance was assessed on 1000 permutations. The overall pattern of genetic structure reflected in the SNP dataset was summarised as a reduced set of orthogonal axes following principal component analyses (PCA) in the package StatistiXL. The first 20 principal components, which had an Eigen value ≥1, and cumulatively accounted for ≥50% of the genotypic variance in the SNP data set, were used to describe genetic structure in association tests.To compare patterns of genetic divergence inferred from the different marker systems correlations between matrices of pairwise population FE. camaldulensis. Genetic structure in this species will potentially confound tests aimed to detect adaptation based on co-variation of genetic markers and environmental parameters, in light of the fact that neutral genetic variation and environment are autocorrelated among populations In this study we investigate signatures of genetic adaptation in candidate genes that could underlie adaptive variation in environmentally contrasted populations of ST) at individual loci (genes or SNPs) with respect to a neutral model The SNP markers employed in this study reside within functional genes, and their allele frequencies may be subject to locus-specific effects such as selection. One approach to detect such effects is to assess observed differentiation (FST conditioned on heterozygosity (He) ranging from 0 to 1 was generated via coalescent simulation over 40000 loci applying a Hierarchical Island Model that accounts for user defined population structure ST for SNP loci were subsequently tested. The mean, 97.5% (upper) and 2.5% (lower) confidence intervals for the simulated FST distribution were calculated using ‘cplot’, which is distributed with the Fdist2 package ST and He for 59 SNP loci were generated using ‘datacal’, also distributed with Fdist2. Estimates of FST and He averaged over whole genes were generated using GenAlEx. Significance of marker FST was tested using the ‘pv’ program distributed with Fdist2. Taking a conservative approach, markers which exceeded the upper or lower confidence intervals (P≤0.01) were classed as outlier loci. Additional outliers were also considered between 0.01<P<0.05. To account for multiple testing the false discovery rate (FDR) and q-values for each locus was estimated Firstly, a neutral distribution of FST was interpreted from posterior odds using Jeffrey's scale A second approach based on the multinomical-Dirichlet model was applied to identify SNP loci that may be under selection using the program BayeScan The spatial analysis method (SAM) of Joost et al. E. camaldulensisTests of association between allelic variation and environment were also performed while attempting to account for potentially confounding population genetic structure using two alternative approaches. Firstly associations between 6 component traits and 59 SNP loci were tested across 191 individuals via a least-squares fixed effect general linear model implemented in Tassel The second method was used to validate environmental associations at six outlier loci identified following analyses with both MatSAM and Tassel. This approach utilised a Bayesian framework to test correlations between allele frequencies at these loci and six component environmental variables against a null model specified by the covariance structure of allele frequencies across populations Genetic relationships estimated upon loci that have been targets of local adaptation might be expected to reflect differences in local selection pressure between populations ST) at SNP loci identified as possible targets of diversifying selection  ranging from 0.19 at Normanby River (QLD) to 0.26 at Fortescue (WA). The overall level of LD between SNPs was low with only 0.6% of pairwise correlations between sites (R2) exceeding 0.2. This indicates that the majority of loci screened segregated independently across the 191 individuals sampled. Genetic differentiation (FST) estimated on all SNPs among populations  was greater than the estimate based on 15 nuSSR loci in the same populations (excluding Wirrengren plain) (FST = 0.08) SNP diversity generally conformed to Hardy Weinberg expectations within populations (55 out of 59 loci). When analysed as a combined sample, 76 percent of SNPs departed expectations, reflecting a Wahlund effect due to population structure. When estimated over all SNP loci, genetic diversity was moderate   and refulgens (pilbara) had greater affinity to subspecies minima when relationships were inferred from SNP markers than nuSSRs. Isolation by distance (IBD) was found to be significant when divergence at SNP loci among population pairs was compared with physical distance separating populations   , suggestp<0.001) . Differep<0.001) . This reST) at candidate loci (whole genes and SNPs) was compared to a neutral FST distribution simulated under a hierarchical island model ST outlier tests identified ten SNPs (from: PIP2 (4 SNPs), Dehydrin (3 SNPs), Erecta (1 SNP), COBL4 (1 SNP) and COMT1 (1 SNP)), as well as three genes , whose divergence was greater than expected under neutrality (p<0.01), and whose pattern of allelic diversity among populations might be accounted for by diversifying selection  to very strong (Log10Bayes factor >1<2) (10Bayes factor >0<0.5) based on Jeffrey's scale. Importantly, each of these loci were also identified by the first method employing coalescent simulations. BayeScan is expected to be a more conservative test for outliers than other methods, as it allows for deviations from a simple island model. Model averaging from the posterior distribution in BayeScan also tended to underestimate SNP differentiation (FST) compared to the method-of-moment estimates from allele frequency variance components in Fdist2 and GenAlEx.Outlier testing using an alternative Bayesian analyses identified three loci (from: Erecta (1 SNP) and PIP2 (2 SNPs)) where evidence in support of the model including diversifying selection was substantial (Logor >1<2) . Two adde) and environment among populations. Population level heterozygosity estimated from a reduced data set containing outlier SNP loci only was significantly correlated with environment , ecolPCA1  and geogPCA2 ) ) . CovariaCLIMPCA1 .To investigate further the adaptive evolutionary model suggested for some SNP loci following outlier tests, we looked for evidence of variation in selective constraints among populations that could explain allelic diversity. Tests of association between allelic variation and component environmental variables were performed using several alternative approaches. Firstly, logistic regression of allelic variation and component environmental variables was performed using MatSAM. This revealed associations between environment and allele frequency that were significant for both the LRT and Wald test following Bonferoni correction for number of loci, and supported a hypothesis of adaptive evolution at eleven outlier loci . HoweverST outliers were significantly associated with one or more environmental variable under the GLM , ranged between three and twelve percent, which at the upper end is large compared to those typically observed for quantitative traits in trees To correct for population structure SNP-environment associations were tested via two further approaches, a general linear model (GLM) implemented in the program Tassel to test for covariance of genotype and environmental scores across individuals,  the GLM . The inc10Bayes factor >0.5<1) based on Jeffrey's scale  . In totatructure . We alsoCLIMPCA1 .Eucalyptus camaldulensis across the species range. These populations are differentiated for climate, primarily reflecting variation in rainfall, evaporation, temperature and sunlight which potentially drive differences in water availability, a strong driver of adaptation in diverse plant species Natural populations occurring across environmental gradients offer opportunities to detect signatures of selection resulting from local adaptation, and test specific hypotheses about the spatial, environmental and temporal scales over which adaptive evolution is exerted E. camaldulensis has previously been suggested from variation in adaptive phenotypes which correspond to local environment including: morphological traits  2 assimilation) E. camaldulensis provenances approximating the species natural distribution. This approach provided for the first time a coarse indication of the distribution of potentially adaptive variation in this species at a broad landscape scale.Local adaptation among provenances and populations of ArabidopsisPopulus nigra . In a recent gene expression study in E. camaldulensis both PIP2 and ERECTA were found to be differentially expressed (down regulated) in droughted compared to well watered E. camaldulensis plants We identified six putative examples of adaptive evolution based on variation in SNP allele frequencies among populations and co-variation with environment . These Se) for outliers and climate initially suggested this (CLIMPCA1), was subsequently confirmed for several outlier loci using three different methods while accounting for demographic effects  and exhibited significant adaptive structure  in natural populations of river red gum CLIMPCA1). Tests of association indicate this cline in allele frequency reflects a greater proportion of T:T homozygotes at this locus in the driest populations. The heterozygote at this locus is associated with climates intermediate to the two homozygotes, suggesting an additive mode of gene action on an unknown adaptive trait. Similarly for ERECTA, an increase in the minor (G) allele frequency was associated with reduced values of CLIMPCA1 . The cline in allele frequency at this locus reflects a greater proportion of T:T homozygotes in the driest populations, and both the heterozygote G:T and homozygote G:G are associated with lower temperature and evaporation, suggesting a dominant mode of gene action  from Erecta (a putative leucine-rich repeat receptor-like kinase) and SNP58 (C/T) from PIP2 (an aquaporin), which were both associated with climate  among populations for several putatively adaptive loci was related to population level dissimilarity for CLIMPCA1 and GEOGPCA2 (CLIMPCA1 (2 = 0.008) co-variation of their inferred population genetic structure could point to concerted selection acting upon unlinked loci, both in genes influencing plant water relations, in response to a common selection pressure 2 = 0.41).Genetic divergence  is plotted on the x-axis and PCA casewise scores for populations on the y-axis in each case.(TIF)Click here for additional data file.Figure S2ST estimated on all SNP loci as compared to the 15 nuSSR loci from Butcher et al 2009 .Mantel correlation of pairwise population F(TIF)Click here for additional data file.Figure S3a) 59 SNP and (b) 15 nuSSR markers which indicate grouping by sub species: subsp. minima (•), subsp. obtusa (o), subsp. arida (♦), subsp. refulgens (Δ), subsp. simulata (◊) and subsp. camaldulensis (▴).Genetic relationships among populations, inferred from principal coordinate analysis (PCoA) for ((TIF)Click here for additional data file.Figure S4ST estimated from ANOVA variance components on all SNP loci as compared to physical distance between populations in kilometres (km) identified significant isolation by distance .Mantel correlation of pairwise population F(TIF)Click here for additional data file.Figure S5CLIMPCA1) for: a) Dehydrin , b) PIP2 , c) COMT .Heterozygosity (y-axis) estimated within populations for outlier genes plotted as a function of environment ((TIF)Click here for additional data file.Figure S6Variation in population level allele frequency (x-axis) for the six outlier SNP loci presented in (TIF)Click here for additional data file.Table S1Mean annual estaimtes for environmental variables applied in principal component analyses.(DOCX)Click here for additional data file.Table S2Correlations (loading) between environmental variables and principal components.(DOCX)Click here for additional data file.Table S3Summary of the 59 SNP loci used in this study. Minor allele frequency (MAF) was determined over all populations. Amino acid abbreviations according to IUPAC conventions.(DOCX)Click here for additional data file.Table S4E. camaldulensis individuals used in this study.Biallelic genotype calls for all SNP loci screened across each of the 191 (CSV)Click here for additional data file.Table S5Whole genes and SNP loci identified as having divergence more extreme than expected when compared to the neutral distribution simualted in Arlequin.(DOCX)Click here for additional data file.Table S6SNP loci for which a model including selection was supported following analyses with BayeScan.(DOCX)Click here for additional data file.Table S7SNP alleles exhibiting significant covariation (p≤0.05) with environment following bonferroni correction for both the Wald and likelihood ratio test implemented in SAM.(DOCX)Click here for additional data file.Table S8Significant assocaitions (p≤0.05) identified with component environemntal variables following adjustment for popualtion structure and multiple testing (permutation) in Tassel.(DOCX)Click here for additional data file.


Pinus strobus). We examined the range-wide population genetic structure, tested various hypothetical population history and evolutionary scenarios and inferred the location of glacial refugium and post-glacial recolonization routes. Our hypothesis was that eastern white pine survived in a single glacial refugium and expanded through multiple post-glacial recolonization routes.Knowledge of the historical distribution and postglacial phylogeography and evolution of a species is important to better understand its current distribution and population structure and potential fate in the future, especially under climate change conditions, and conservation of its genetic resources. We have addressed this issue in a wide-ranging and heavily exploited keystone forest tree species of eastern North America, eastern white pine (FST = 0.104) and population structure among eastern white pine populations across its range. A south to north trend of declining genetic diversity existed, consistent with repeated founder effects during post-glaciation migration northwards. We observed broad consensus from nuclear and chloroplast genetic markers supporting the presence of two main post-glacial recolonization routes that originated from a single southern refugium in the mid-Atlantic plain. One route gave rise to populations at the western margin of the species’ range in Minnesota and western Ontario. The second route gave rise to central-eastern populations, which branched into two subgroups: central and eastern. We observed minimal sharing of chloroplast haplotypes between recolonization routes but there was evidence of admixture between the western and west-central populations.We studied the range-wide genetic diversity and population structure of 33 eastern white pine populations using 12 nuclear and 3 chloroplast microsatellite DNA markers. We used Approximate Bayesian Computation approach to test various evolutionary scenarios. We observed high levels of genetic diversity, and significant genetic differentiation  contains supplementary material, which is available to authorized users. Ecological changes and anthropogenic activities over the past 12,000 years have had a profound effect on the distribution of plant populations including that of forest trees in eastern North America . TherefoPicea glauca) [Picea mariana) [Pinus banksiana) [Chamaecyparis thyoides), Mylecraine et al. [In eastern North America , the absence of major geographic barriers to northward dispersal can lead to a presumption that northward post-glacial recolonization simply proceeded uniformly across the longitudinal range of the recolonizing species. Thus the only phylogeographic patterns likely to be introduced during post-glacial recolonization would be south-to-north declines in genetic diversity introduced by repeated founder events . However glauca) , 7, thremariana) , and thrnksiana) . For Atle et al.  argued tPinus strobus) in North America in order to understand how historical processes have shaped the current species’ distribution and population genetic structure. We have chosen eastern white pine because (1) it is a widely distributed, ecologically and economically important key-stone species in eastern North America, (2) it is predicted that this species will expand its range northward under the anticipated climate change, (3) there is no information on range-wide population genetic structure and phylogeography of this species, and its post-glacial migration and evolution is not well-understood, and (4) it is of conservation genetic concerns because it has been heavily exploited for over 150 years [The objective of this study was to examine the range-wide population genetic structure and phylogeography, and infer post-glacial migration and evolution of eastern white pine  if the current eastern white pine arose from a southern refugium, there should be a geographical trend of south to north decreasing genetic diversity as a result of repeated founder effect; (ii) that current eastern white pine populations descended from a single glacial refugium during the LGM and multiple post-glacial recolonization routes; and (iii) the Appalachian mountains and Great Lakes separated the post-glacial colonization routes resulting in longitudinal genetic differentiation of eastern white pine in the north. We take advantage of the fact that the chloroplast DNA is paternally inherited and thus pollen dispersed in conifers, e.g., , and theEastern white pine is a long-lived widely distributed species in eastern North America from Newfoundland in the east to southeastern Manitoba in the west and to parts of Georgia and South Carolina in the southeast . It is hThirty-three natural eastern white pine populations were sampled from throughout the species’ range Table , Fig. 1.RPS1b, RPS2, RPS12, RPS20, RPS25, RPS34b, RPS39, RPS50, RPS6, RPS118b, RPS119 and RPS127) [ [Pinus thunbergii [We isolated total genomic DNA from 300 mg of ground needles from each individual using a modified CTAB method . After ept71936)  to genotpt71936)  in easteunbergii . We used2 (EMD), 1 μL 4 mM dNTPs, 0.1 μL 10 mM untailed primer, 0.1 μL 10 mM fluorescently labelled m13 primers, 0.06 μL 10 mM m13 tailed primer, 0.1 μL 5 U/μL Taq DNA polymerase and 5.09 μL nuclease free water.For each microsatellite marker, we modified one of the two primers to accept a fluorescently labelled m13 tail (700 nm or 800 nm wavelength). Polymerase chain reaction (PCR) reactions for the microsatellite amplification were performed in a volume of 10 μL. The reaction mixtures contained 10–15 ng of DNA, 2 μL 5x PCR Buffer , 0.5 μL 2 mM MgClWe performed PCR amplifications starting with a “touchdown” step from 65 °C to the annealing temperatures according to Echt et al.  and RajoWe used Licor Biosciences IR4300 DNA analyzers  to visualize microsatellite polymorphisms through a 6 % agarose gel matrix , suspended in a TBE buffer. To determine microsatellite fragment lengths, five LiCor 50–350 base pair molecular weight size standards were included in each gel. We used Licor Biosciences Saga Generation 2  to score microsatellite genotype data, which was verified manually. The nuclear microsatellite genotype data was scored as allelic constitution of individuals at a locus. The chloroplast microsatellite data was first scored as allelic constitution of individuals at individual three loci, and then as multilocus haplotypes to account for linkage between the chloroplast regions.N), number of private alleles (AP), and observed and expected heterozygosity (HO and HE) were calculated for the nuclear markers. Number of alleles per locus (AN), Shannon’s Information Index (I) see [E) per locus, rarefaction-based allelic richness (AR), and inbreeding index (FIS) for the nuclear microsatellites using FSTAT v2.9.3.2 [Genetic diversity parameters of individual populations for the nuclear and chloroplast microsatellites were determined using GenAlEx 6 . Number  (I) see , haplotyv2.9.3.2 . Departuv2.9.3.2  and Arlev2.9.3.2 . We calcF-statistics [ST and RST/NST among populations were calculated from the chloroplast markers using 1000 permutations in PermutCpSSR v2.0 [Inter-population genetic differentiation for nuclear microsatellites was determined by using atistics  employinatistics , and AMOatistics  using Aratistics . GST andSSR v2.0 .K) such that Hardy-Weinberg equilibrium is maximized within clusters. By varying the K-values across several runs and inspecting the resulting probabilities for these various K values, one can infer the likely number of groups which best capture the variation present in the data. We performed multiple runs of STRUCTURE to test K values ranging from 1 to 33, over 50 replications, using an admixture model and correlated allele frequencies options [5 burn-in length and 105 MCMC replications for each run. In order to facilitate the selection of the best K value, we used STRUCTURE HARVESTER [K and detecting the number of genetic groups that best fit the data.The population genetic structure resulting from natural barriers and human activities was examined using two Bayesian model-based clustering approaches. First, STRUCTURE  was used options , a 105 bARVESTER ; an onliARVESTER  techniquDue to the large variation in geographical distances among the locations of the sampled populations, we sought to disentangle any artifactual population structure signal caused by populations in close proximity. We did this by performing a second Bayesian population structure analysis using BAPS v5.3 . Unlike FST matrix and individual locus FST pairwise matrices to determine the number of loci that support any inferred barriers.Regions where abrupt genetic differentiation exists over relatively small geographic distances can be indicative of boundaries of population groups and genetic barriers in a species range perhaps where distinct phylogeographic lineages meet. We used Barrier v2.2  to identK-value STRUCTURE runs, Barrier analysis). In order to disentangle these patterns from the present population structure, we employed geographic distribution patterns of chloroplast haplotypes, and tested various phylogeographic hypotheses using the Approximate Bayesian Computation (ABC) analysis.Although we observed varying levels of population differentiation and a significant magnitude of population structure among eastern white pine populations, signals of past phylogeographic patterns were present in nearly all analyses  also showed a trend of decreasing from south to north for the nuclear microsatellite markers: r = −0.3738, p = 0.0326.A geographic trend of decreasing genetic diversity from south to north was observed for both nuclear and chloroplast microsatellite markers Tables  and 3. Prs Table . Generaly Tables  and 3. WFST among the populations was 0.104 from the nuclear microsatellite markers, and it was significantly higher than 0. Likewise, the AMOVA results revealed significant among-population variation of 10.38 %, which was consistent with the FST estimates. As expected for a gymnosperm forest tree species, the majority of genetic variation was explained by within-population variation (~90 %). The inter-population genetic differentiation from chloroplast microsatellites was lower than estimates from the nuclear markers  but still significantly higher than 0.The overall mean delta K peaks, yet the most prominent was at K = 30 genetic groups  were clustered into a distinct group from the rest of the populations  and eastern samples   Fig. , resultiK = 3. We identified two barriers among the 33 sample populations from both the nuclear and chloroplast microsatellite markers that separated the populations into three groups  of population divergence between the southern group (ST) and the western group (WS), the second split (1-t2) between the ST and the ancestral population of the central (CNT) and eastern (EST) groups, and the final split (1-t1) between the central and eastern groups  [Pinus contorta  [N: 6.7, 7.3; HO: 0.47, 0.48; HE: 0.46, 0.49) [E = 0.49) [N = 6.6, HO = 0.74 HE = 0.80) [Pinus [Eastern white pine has relatively high nuclear microsatellite DNA genetic diversity over its range. We observed levels of nuclear microsatellite genetic diversity  in the sampled eastern white pine populations that were on average higher than the microsatellite diversity observed in studies of other widely distributed conifer species, including its sister species western white pine,  = 0.67) , and lodE = 0.43 . The obs = 0.60) , and Har6, 0.49) , Menomin = 0.49)  and in a = 0.80)  based on) [Pinus  and othe) [Pinus  and spatFST using BayeScan ver. 2.1 [http://www.env.gov.nl.ca/env/publications/parks/little_grand_lake_web.pdf), where human impacts have been limited. On the other hand, the New Brunswick and Nova Scotia eastern white populations have been heavily exploited. The number of private alleles did not show any geographical patterns in our study. Private alleles may arise from population-specific new mutations and severely curtailed inter-population gene flow. Geographic patterns for private alleles will be expected if the mutation and gene flow rates were geographically structured among populations within a region: southern, northern, eastern central, and western. Apparently, this is not the case with the eastern white pine populations studied. However, a separate study is required to validate this assumption.Our study clearly demonstrates the existence of south–north patterns in the genetic diversity levels, with the populations in Virginia and North Carolina having higher levels of genetic diversity than the northern populations. This is consistent with the possible repeated founder effects during post-glacial migration northward of eastern white pine from a southern Pleistocene refugium. The lower genetic diversity in the northern eastern white pine populations may also be due to divergent selection in response to south to north gradient in climate factors, such as temperature and moisture regimes, and range marginalization . Howeverver. 2.1 . SomewhaFST and AMOVA analyses, and 26 (BAPS) or 30 (STRUCTURE) groups of populations among the 33 eastern white pine populations. These results clearly suggest that significant population genetic structure and differentiation exist across the range of eastern white pine. The observed levels of genetic differentiation could be considered as low when compared to the plant kingdom as a whole but for the conifer trees, the levels are higher than the average of 0.073 (7.3 %) [Cronartium ribicola) may have also reduced the population size and number of eastern white pine. Encroaching agriculture, grasslands and deciduous forests, and changing precipitation and wind patterns may negatively impact the distances over which seeds and pollen are dispersed between populations. All of the above factors may have reduced the levels of inter-population gene flow and increased inbreeding. However, eastern white pine has strong inbreeding depression [We observed 10.4 % interpopulation genetic differentiation based on the  (7.3 %) . Signifi (7.3 %) . Mortalipression , and selpression . Althougpression ; these fFST = 0.084) [FST = 0.061 [FST = 0.019 [FST: 0.084; Phi variance: 0.071) as previously reported by Mehes et al. [Pinus [The inter-population genetic differentiation of 10.4 % in our study is higher than that reported earlier for eastern white pine from its part of the Canadian range based on microsatellite (= 0.084)  and allo = 0.061 , FST = 0s et al. . Chlorop. [Pinus ) and lonThe phylogeographic patterns emerged from the nuclear and chloroplast genetic markers were consistent between themselves and broadly consistent with the findings from previous fossil pollen studies . The mosIn either scenario, the main northward recolonization route on the eastern seaboard is the source of the four most abundant cpSSR haplotypes observed in the eastern United States, Ontario, Quebec and the Maritime Provinces Fig. . This roUnder our primary hypothesis assuming one glacial refuge, the major population break observed between eastern and western eastern white pine populations was likely the result of two major features of eastern white pine’s geographical context. Initially, high altitude environments in the Appalachian Mountain may have separated the ancestral western migrants from their counterparts to the east of the mountains. Further in north and west, the Great Lakes may have reduced or prevented the dispersal of seeds between populations on the southern and western shores from the populations in Ontario. The cpSSR haplotype and ABC simulations results do not support the possibility that the western (Minnesota and westernmost Ontario) populations are the descendants of populations in Ontario. This inference is supported by the presence of cpSSR haplotypes in the western populations that are not found in any Ontario population  and the populations in central Ontario Fig. . This waPinus banksiana), black spruce (Picea mariana), white spruce (Picea glauca), and red maple (Acer rubrum), three species found throughout the northern United States and Canada, have been reported to have descended from multiple glacial refugia [Chamaecyparis thyoides [The post-glacial phylogeographic patterns and evolutionary history of eastern white pine inferred in our study appear to be unusual as compared with those of other widely distributed tree species in North America, in that eastern white pine appears to have had a single glacial refugium and multiple post-glacial recolonization routes. In particular, populations of jack pine  among the 33 eastern white pine populations sampled. Only when we set the Our results indicate that eastern white pine populations have significant levels of genetic structure and differentiation across the species’ range. We have inferred three postglacial lineages in eastern white pine originating from a southern glacial refugium: eastern, central and western. Localized conservation and management strategies may be required in at least two and perhaps all three regions. The westernmost populations (Minnesota and western Ontario) represent a distinctive lineage and should be the focus of further study to determine if these populations contain adaptive traits for local conditions. As such, this lineage may represent a single Evolutionary Significant Unit (ESU) separated from the central and eastern populations. The divergence observed between the central and eastern coastal populations suggests that these lineages represent at least two additional ESUs. According to Ryder , who gavAs stated earlier in this paper, eastern white pine has been heavily harvested over the past 150 years , and conEastern white pine has also gone through multiple episodes of post-glacial range expansion and retraction , encountEastern white pine has relatively high magnitude of genetic diversity, and significant differentiation and genetic structure across its natural range. Its contemporary population genetic structure shows the signatures of post-glacial migration and evolution as well as effects of natural and human disturbances. The current distribution of eastern white pine is the result of at least two post-glacial recolonization routes from a southern single glacial refugium. The two regions of greatest genetic differentiation corresponding to post-glacial recolonization routes are: (1) west of the Great Lakes and (2) along the eastern seaboard. However, it cannot be determined from the markers used in our study whether any of the geographic patterns in population genetic structure is adaptive. We have identified three ESUs  in eastern white pine which should be taken into account in conserving and managing the species’ genetic resources. If future work also finds evidence for adaptive differentiation among the identified western, central and eastern coastal genetic lineages, eastern white pine conservation and genetic resource management plans should be made specific to each of these three regions, especially under the climate change conditions. In order to better delineate genetic lineages resulting from post-glacial migration, it is necessary to disentangle the confounding genetic signatures of human and natural disturbances on the contemporary genetic structure from that due to post-glacial phylogeography and evolution.The raw nuclear and chloroplast microsatellite data are provided in Additional file


We conducted phylogenetic, population and spatial genetic analyses on 274 individuals of P. schiedeanus to gain insight of the evolutionary history of these populations. Species distribution modeling, isolation with migration and Bayesian inference methods were used to infer the evolutionary transition of mistletoe invasion, in which evolutionary scenarios were compared through posterior probabilities.Ecological adaptation to host taxa is thought to result in mistletoe speciation via race formation. However, historical and ecological factors could also contribute to explain genetic structuring particularly when mistletoe host races are distributed allopatrically. Using sequence data from nuclear (ITS) and chloroplast , with post-glacial population expansions and potentially corresponding to post-glacial invasion types. Similarly, 44 ITS ribotypes suggest phylogeographic structure, despite the fact that most frequent ribotypes are widespread indicating effective nuclear gene flow via pollen. Gene flow estimates, a significant genetic signal of demographic expansion, and range shifts under past climatic conditions predicted by species distribution modeling suggest post-glacial invasion of P. schiedeanus mistletoes to cloud forests. However, Approximate Bayesian Computation (ABC) analyses strongly supported a scenario of simultaneous divergence among the three groups isolated recently.Our analyses revealed shallow levels of population structure with three genetic groups present across the sample area. Nine haplotypes were identified after sequencing the P. schiedeanus populations, accumulating slight phenotypic differences among genetic groups due to recent migration across habitats. Under this scenario, climatic fluctuations throughout the Pleistocene would have altered the distribution of suitable habitat for mistletoes throughout Mesoamerica leading to variation in population continuity and isolation. Our findings add to an understanding of the role of recent isolation and colonization in shaping cloud forest communities in the region.Our results provide support for the predominant role of isolation and environmental factors in driving genetic differentiation of Mesoamerican parrot-flower mistletoes. The ABC results are consistent with a scenario of post-glacial mistletoe invasion, independent of host identity, and that habitat types recently isolated The online version of this article (doi:10.1186/s12862-016-0648-6) contains supplementary material, which is available to authorized users. Most aerial Loranthaceae mistletoes use bird vectors for seed dispersal and, eventually, branch attachment and penetration of woody host plants . Given tdispersal hypothesis), (2) mistletoe speciation by changing host specificity (host-switching hypothesis), and (3) mistletoe speciation without changes in the host . Each of these modes of mistletoe speciation could occur following the allopatric, peripheral isolates or sympatric models of speciation )A phylogeographic approach is appropriate for evolutionary studies of mistletoes, because the assessment of geographic structure and genetic variation within and among populations might reflect underlying spatial and/or temporal discontinuities in available host tree species. Despite the appeal of the speciation model of host race formation by host switching, species formation in mistletoes that are transmitted between hosts by avian vectors is not understood. Speciation via race formation is thought to be the result of ecological adaptation to host taxa, and presumably the evolutionary pathway that eventually lead to mistletoe diversification . HoweverPsittacanthus schiedeanus (Schltdl. & Cham.) G. Don (Loranthaceae) is examined through species distribution modeling and phylogeographic and population genetic analyses of nuclear and chloroplast DNA. We used a broad geographical sampling of populations of P. schiedeanus DNA sequences from the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) in combination with sequences of the rapidly evolving trnL-F intergenic spacer region from the chloroplast genome (cpDNA). This multilocus approach allowed us to explicitly test the set of historical and climatic scenarios in the evolution of P. schiedeanus and identify patterns of population structure that may reflect the evolutionary history of the species. As the chloroplast genome is maternally inherited in most angiosperms, cpDNA sequences may provide a picture of evolutionary haplotype relationships of seed-mediated gene flow compared to the ITS sequences reflecting a combination of both pollen and seed movement   , where atrnL-F sequences. Because three haplotype clusters are hypothesized to be associated with individuals/populations separated by habitat , which corresponded to the plant genetic groups, BREE, SCHI and CALY, respectively; (b) isolation split model 1 (scenario 2), in which Pop1 (BREE) merged with Pop2 (SCHI) at t1 and subsequently with Pop3 (CALY) at t2; (c) isolation split model 2 (scenario 3), in which Pop3 merged with Pop2 at t1, then both populations merged with Pop1 at t2; (d) isolation split model 3 (scenario 4), in which Pop3 merged with Pop1 at t1 and then both populations merged with Pop2 at t2; and (e) isolation with admixture model (scenario 5), in which Pop2 was generated by admixture of Pops 1 and 3 at t1, then Pop1 merged with Pop3 at t2. Although there are numerous possible scenarios of divergence, we considered that these five scenarios represent the close relationships among the groups and the complex patterns of genetic divergence and admixture  and splitting events (100–50 000 generations). Comparison of scenarios was implemented in the DIYABC software . The posP. schiedeanus. The AUC value for the replicate runs was 0.992 ± 0.003  and the trnL-F intergenic spacer (n = 283) for Psittacanthus species were 569 and 321 base pairs (bp) long, respectively. Out of 1018 bp of the concatenated sequences, 588 sites were variable and out of these 464 were parsimony informative. In P. schiedeanus samples, we detected polymorphic sites in nine samples of the ITS region, with four of the 569 aligned sites (0.7 %). These were coded as ambiguous.The aligned sequences of ITS region (trnL-F) and the combined data set all differ in topology and degree of resolution. The trnL-F gene produced the poorest resolved tree  and a clade with moderate support (PP = 0.86) containing samples of the Brazilian species  and samples of P. macrantherus, P. mayanus, P. auriculatus, P. calyculatus and P. schiedeanus. Samples of P. calyculatus and samples of P. schiedeanus from the eastern Mexico cloud forests, central Oaxaca and Chiapas turned out to be monophyletic, but little support (PP = 0.67) and genetic structure was observed within this clade. The Bayes factor tests showed extensive incongruence between the chloroplast and the nuclear partitions. Combining ITS and trnL-F data sets produced a higher likelihood score than those for the alternative ITS or trnL-F data sets .The trees obtained from the Bayesian analyses of the individual molecular markers , the relationships within this clade were poorly resolved  assuming constant population size. Samples of P. schiedeanus formed a well-supported monophyletic group (PP = 0.94), with diversification occurring in the early Pleistocene  and a split between P. schiedeanus and P. auriculatus estimated at 2.80 MYA (95 % HPD 2.08–0.31 MYA) assuming constant population size  yielded 44 ribotypes  yielded 9 haplotypes . Genetic diversity across all populations  was higher than the average within-population . PERMUT analysis showed that NST  was significantly higher (p <0.05) than that for GST, indicating phylogeographical structuring. Similarly, phylogeographical structuring was observed in P. schiedeanus based on trnL-F variation , with genetic diversity across all populations  much higher than the average within-population . Pairwise comparisons of FST values were not significant for ITS when groups of populations separated by habitat type were compared, whereas pairwise comparisons of FST values for trnL-F were high and significant  obtained when populations are grouped by habitat type but differences between groups were not significant in the ITS data set , indicating demographic expansion for SCHI  is estimated to be higher than population sizes of the SCHI and CALY+BREE descendant populations  is the best-supported scenario Fig. , with a ns Table . EstimatIn northern Mesoamerica, cloud forests encompass an extremely heterogeneous mixture of North American temperate tree species that are present in the region since the Tertiary and tropical species with South American origins during the early Miocene –42. The Alsophila firma (Baker) D.S. Conant (Cyatheaceae) [Liquidambar styraciflua L. (Altingiaceae) [Podocarpus matudae Lundell (Podocarpaceae), Palicourea padifolia (Humb. & Bonpl. ex Schult.) C.M. Taylor & Lorence (Rubiaceae) and Rhipsalis baccifera (Sol.) Stearn (Cactaceae) [Begonia heracleifolia Cham. & Schltdl. (Begoniaceae) [Moussonia deppeana (Schltdl. & Cham.) Hanst. (Gesneriaceae) [trnL-F data, only explained 28 % of the total variation according to the AMOVA model, suggesting that low levels of seed flow have occurred across this barrier.Seven studies have explored the phylogeography of widespread, cloud forest-adapted plant species across northern Mesoamerica. High levels of genetic variation and geographical structuring were inferred for wind-dispersed heaceae)  and Liqugiaceae)  or bird-ctaceae) , 98, 100niaceae)  and Mousriaceae) , the genriaceae) , 101, suriaceae) , 34, 98.riaceae) , 96, thoP. schiedeanus, with differentiation between groups of populations separated by habitat type. Despite the genetic differentiation of some P. schiedeanus populations, there are widespread ribotypes and haplotypes in the ITS and trnL-F markers, respectively. In particular, the lower variation found in ITS and the presence of widespread ribotypes in the region are suggestive of effective nuclear gene flow via pollen  has experienced recent demographic expansion, most likely during the LGM.We found population genetic structure in n e.g., , 102). I. IP. schP. schiedeanus (SCHI) populations experienced a range expansion tracking the conditions of the cloud forest distribution and shifted to the lowlands with population connectivity during the LGM, as predicted by the moist forests model [P. schiedeanus expanded to the lowlands, gene flow was extensive, and habitats in which populations had gone extinct during the interglacials were probably re-colonized. Throughout interglacial periods, populations fragmented and contracted as they moved up in elevation with the warming climate and genetic divergence commenced across cloud forest areas dissected by mountains and low-elevation barriers.Predictions of SDMs under past LGM climatic conditions and the significant signal of demographic expansion suggest that ts model , 58. In Liquidambar styraciflua (Altingiaceae), a deciduous tree commonly parasitized by P. schiedeanus with a disjunct distribution between the deciduous forest of the southeastern USA and the Mesoamerican cloud forest, and found signals of demographic expansion of Mesoamerican populations and habitat suitability connected and expanded to lower coastal areas under LGM climatic conditions. However, these patterns of genetic structure are not shared by P. schiedeanus. The observed inconsistency between demographic histories of P. schiedeanus and its most frequent host tree species in cloud forests can be the result of a differential response of species to climate change.Despite the appeal of the contraction/expansion model of populations of cloud forest-adapted species throughout glacial-interglacial climate cycles by shifting elevation, there is little evidence for a common demographic pattern of co-distributed species . Ruiz-SaP. schiedeanus suggests that three lineages occupy geographic regions separated by habitat type, driving isolation and genetic divergence between groups of populations enhanced by environmental differences. Despite the high levels of genetic diversity and genetic structure of cpDNA among groups of populations , the occurrence of widespread plastid haplotypes in most areas  of the range of P. schiedeanus is consistent with the hypothesis of effective seed dispersal episodes. Accordingly, the mobility of bird dispersal agents has erased the genetic structure predicted to groups of populations along the cloud forest distribution , grey silky-flycatchers , and social flycatchers  [P. schiedeanus, and the timing of its arrival coincides with the mistletoe fruiting season [Several bird species consuming fruits of annidae) , 36, 37.annidae) , 36, andannidae) . On theiP. schiedeanus seeds within a short time of ingestion  and the distribution boundaries with phenotypically similar Psittacanthus populations allopatrically distributed in more xeric environments . Genetic differentiation among these groups was strong and significant for the plastid trnL-F marker and non-significant for the nuclear ITS. Collectively, our data suggest that geographical, ecological, historical and environmental factors are the main drivers of genetic differentiation of these Mesoamerican parrot-flower mistletoes. According to SDMs, population expansion tests, skyline plots and divergence time estimations, the invasion of P. schiedeanus to cloud forests in eastern Mexico is relatively recent, and the invasion occurred to host tree species with longer histories in the region . Nonethn . The data sets  supporting the results of this article are available in the Dryad Digital Repository (http://dx.doi.org/10.5061/dryad.t49h6) as Ornelas et al. [Sequence reads can be accessed through GenBank under the Accession Numbers KU922961–KU923047 (ITS), s et al. .


Puma concolor; also known as mountain lions and cougars) in southern California live among a burgeoning human population of roughly 20 million people. Yet little is known of the consequences of attendant habitat loss and fragmentation, and human-caused puma mortality to puma population viability and genetic diversity. We examined genetic status of pumas in coastal mountains within the Peninsular Ranges south of Los Angeles, in San Diego, Riverside, and Orange counties. The Santa Ana Mountains are bounded by urbanization to the west, north, and east, and are separated from the eastern Peninsular Ranges to the southeast by a ten lane interstate highway (I-15). We analyzed DNA samples from 97 pumas sampled between 2001 and 2012. Genotypic data for forty-six microsatellite loci revealed that pumas sampled in the Santa Ana Mountains (n = 42) displayed lower genetic diversity than pumas from nearly every other region in California tested (n = 257), including those living in the Peninsular Ranges immediately to the east across I-15 (n = 55). Santa Ana Mountains pumas had high average pairwise relatedness, high individual internal relatedness, a low estimated effective population size, and strong evidence of a bottleneck and isolation from other populations in California. These and ecological findings provide clear evidence that Santa Ana Mountains pumas have been experiencing genetic impacts related to barriers to gene flow, and are a warning signal to wildlife managers and land use planners that mitigation efforts will be needed to stem further genetic and demographic decay in the Santa Ana Mountains puma population.Pumas ( Puma concolor; also known as mountain lion, cougar, and in Florida, panther) epitomize this dilemma. Although pumas in California have not been subjected to hunting since 1972, and were designated as a Specially Protected Mammal in 1990 Genetic diversity, demography, and abundance – biological characteristics that influence population viability – can vary across a species’ distribution. Species that are generally perceived as wide-ranging and abundant are sometimes relegated to status as “least conservation concern”, in spite of indicators signaling concern and frequently, lack of data. Pumas  has been of conservation concern for decades. Population monitoring and modeling in the 1980s highlighted that urbanization and highways were fragmenting puma habitat e.g., , and thaWe conducted a detailed appraisal of the genetic diversity, relatedness, and population structure of southern California puma populations. Using 97 samples collected over 12 years as part of the UCD study, and a 46-locus microsatellite panel, we evaluated levels of genetic diversity, estimated effective population sizes and tested whether genetic data supported a hypothesis of recent bottleneck in the populations. We assessed whether genetics reflected our telemetry observations of infrequent puma crossings of I-15 between the Santa Ana Mountains and the Peninsular Ranges to the east. Additionally we explored inter-population gene flow at multiple time scales by employing methods that reflect recent (a few generations) and more historical (tens or more generations). Finally, we tested our hypothesis that the Santa Ana population had lower genetic diversity than those sampled from other regions in California.We obtained blood or tissue samples for analysis of nuclear DNA from pumas captured for telemetry studies, and from those found dead or killed by state authorities for livestock depredation or public safety in San Diego, Orange, Riverside, and San Bernardino counties of southern California (n = 97) during 2001–2012 . Pumas cAnimal handling was carried out in strict accordance with the recommendations and approved Protocol 10950/PHS, Animal Welfare Assurance number A3433-01, with capture and sampling procedures approved by the Animal Care and Use Committee at the University of California, Davis (Protocol #17233), and Memoranda of Understanding and Scientific Collecting Permits from the California Department of Fish and Wildlife (CDFW). Permits and permissions for access to conserved lands at puma capture and sampling sites were obtained from CDFW, California Department of Parks and Recreation, The Nature Conservancy, United States (US) Fish and Wildlife Service, US Forest Service, US Bureau of Land Management, US Navy/Marine Corps, Orange County Parks Department, San Diego County Parks Department, San Diego State University, Vista Irrigation District, Rancho Mission Viejo/San Juan Company, Sweetwater Authority, California Department of Transportation , and the City of San Diego Water Department.Whole genomic DNA was extracted using the DNeasy Blood & Tissue Kit . Fifty microsatellite DNA primers were initially screened for this project. Forty-six loci that performed well in multiplex PCR  and conformed to expectations for Hardy-Weinberg and linkage equilibria were selected for ultimate analysis PCR products were separated with an ABI PRISM 3730 DNA Analyzer  with each capillary containing 1 µL of a 1∶10 dilution of PCR product and deionized water, 0.05 µL GeneScan-500 LIZ Size Standard and 9.95 µL of HiDi formamide (both products Applied Biosystems Inc.) that was denatured at 95°C for 3 min. Products were visualized with STRand version 2.3.69 The number of alleles (Na), allelic richness , observed heterozygosity (Ho), expected heterozygosity (He), Shannon’s information index RM) without close relatives in a population SIBS) SIBS is considered conservative since it probably conveys a higher likelihood; however, we recognized that siblings occurred in these populations.The average probability of identity (PID) was calculated two ways using GenAlEx: 1) assuming random mating  via covariance matrices with data standardization ST, was calculated to appraise how genetic diversity was partitioned between populations. As implemented in GenAlEx, we used Nei’s Wright’s F-statistic, FhL, assuming all populations had not been sampled. We then calculated hL/maxL, the ratio of hL to the greatest likelihood among the populations hL or hL/maxL) was determined using the Bayesian approach of Rannala and Mountain We used GENECLASS2 version 2.0.h We tested for evidence of recent population size reductions in Santa Ana Mountains and eastern Peninsular Range regions with one-tailed Wilcoxon sign-rank tests for heterozygote excess in the program BOTTLENECK version 1.2.02 We then estimated contemporary Ne for each of the two regions based on gametic disequilibrium with sampling bias correction Molecular kinship analysis was conducted using a number of software packages. Pairwise relatedness among individuals was evaluated using the algorithm of Lynch and Ritland RM) or mating among sibs (PIDSIBS) across the 42 loci for the eastern Peninsular Ranges were (PIDRM) 6.3×10−22 and (PIDSIBS) 3.1×10−10, and for the Santa Ana Mountains were (PIDRM) 2.8×10−15 and (PIDSIBS) 1.1×10−7 respectively. These very small values indicate that the panel of genetic markers provided very high resolution to distinguish individuals. For example, given this data the probability of seeing the same multi-locus genotype in more than one puma was less than one in nine million for Santa Ana Mountains pumas.Forty-two of the 46 loci that we employed were polymorphic in southern California and selected for the subsequent analyses. The average probabilities of identity with assumptions of either random mating  PCoA;  allowed PCoA analysis of only the samples collected in the Santa Ana and Peninsular Ranges  confirmsST calculations (ST was surprisingly high (0.07) given the very close proximity of the two regions (separated only by an interstate highway). The Santa Monica Mountains pumas and Santa Ana Mountains pumas had the highest FST  of all pairwise comparisons in the state, demonstrating a high level of genetic isolation between these regions.The Santa Monica Mountains and Santa Ana Mountains are less than 100 km direct distance apart, through the center of Los Angeles. However the more likely distance for puma travel between these two mountain ranges, avoiding urban areas and maximizing upland habitat, would likely exceed 300 km .Wright’s Fulations  indicateP<0.01), four with the hL method , and one with the hL/maxL ratio . Pumas F75, M80, and M99 were all captured from the San Bernardino Mountains  estimations using the linkage disequilibrium method (LDNe program) were 5.1 for the Santa Ana Mountains population and 24.3 for mountain lions in the eastern Peninsular Ranges. Statistical confidence intervals for both regions, given the genetic data, were tight .The average pairwise coefficient of relatedness r,  was high−6) than for those sampled in the eastern Peninsular Ranges (0.001). Of a group of six pumas which clustered near one another in PCoA  for internal relatedness as implemented in rHH software was significantly higher  and puma generation time. Luikhart and Cornuet and very low genetic diversity in Santa Ana pumas F95 and M96 may portend manifestations of genetic inbreeding depression similar to those seen in Florida panthers An isolated population of pumas in the Santa Monica Mountains to the north of the Santa Ana Mountains also exhibit low values relative to other western North American populations  across I-15. Despite warnings