Haloxylon salicornicum moq. Bunge ex Boiss (Rimth) is one of the native plants of Kuwait, extensively depleting through the anthropogenic activities. It is important to conserve Haloxylon community in Kuwait as it can tolerate extreme adverse conditions of drought and salinity to be potentially used in the desert and urban revegetation and greenery national programs. Therefore, a set of 16 inter simple sequence repeat (ISSR) markers were used to assess genetic diversity and population structure of 108 genotypes from six locations in Kuwait. The ISSR primers produced 195 unambiguous and reproducible bands out of which 167 bands were polymorphic (86.5%) with a mean PIC value of 0.31. The overall average values of Nei’s gene diversity (h') and Shannon’s diversity index (I) were 0.254 and 0.375, respectively. Results of AMOVA revealed high genetic variations within populations (77.8%) and low among populations (22%). The values of Fixation index , Genetic differentiation  were indicative of heterozygous populations. The results of STRUCTURE and split decomposition analysis suggested that the Rimth accessions of Kuwait can be grouped into five and six subpopulations, respectively. Principal coordinate analysis (PCoA) grouped them into three clusters. The pairwise Nei’s genetic distances (DS) among populations demonstrated a narrow range from 0.047 to 0.187 . The Mantel’s test revealed a weak correlation  between the genetic distance and geographic distances. Our results suggest that the narrowly distributed Haloxylon community in Kuwait demonstrated a high genetic diversity within the populations however the overall population structure was weak. Kuwait is an arid country with smallest land area. The native plants have a narrow distribution. The species that exist in the ecosystem are unique as they offer a valuable genetic pool for drought, heat and salt-tolerance . In addiHaloxylon salicornicum Moq. Bunge ex Boiss (Rimth) is one of the main structural elements in Eastern Arabian vegetation associations [H. salicornicum community is under threat from overgrazing leading to reduction in percentage of distribution from 22.7% to 2.2% in Kuwait [Haloxylon communities demonstrated considerable variations due to wind erosion and land degradation [ciations ,5 belongciations ,7. The pciations . It is cn Kuwait ,9. The pradation . Informaradation .To formulate effective conservation and restoration strategies, the understanding of a population’s genetic diversity is imperative. A wide range of molecular methods are now being applied to measure the genetic diversity of a population. The long-term survival of a species is challenging particularly if it has reduced variability. These species suffer from homozygosity and inbreeding depression causing reduction in reproduction rates indirectly leading towards extinction . A narroAgave victoriae [Acacia raddiana [Breonadia salicina [In recent years, PCR based molecular markers has allowed the use of DNA sequences in genetic analyses and has provided, a better understanding of the genetic diversity and differentiation of natural populations . Moleculictoriae , Acacia raddiana  and Breosalicina .H. salicornicum. This would aid in defining conservation studies for the declining Haloxylon in Kuwait.In view of the aforementioned the aim of this study was to assess the genetic diversity and population structure of 2. Located in the north eastern corner of the Arabian peninsula, it shares border with Iraq and Saudi Arabia. A survey was conducted to locate the populations of H. salicornicum through direct searching as the species is retreating from Kuwait. Consultations were done with botanical experts to pinpoint the habitats where healthy populations of the plants could be found. Six areas from the Central, Northern and Southern part of Kuwait such as Al-Subiya, Om-Qaser, Al-Ritqa, Al-Shagaya, Al-Abraq and Al-Abdally, were identified for collection , as per the manufacturer’s instructions. One hundred mg of leaf tissue was weighed and ground to a fine powder in liquid nitrogen using a pre-chilled mortar and pestle. Cell lysis followed by protein precipitation and washing was carried out using the different reagents supplied in the kit. DNA purity (Absorbance ratio A260/A280) and quantity (Absorbance at 260 nm) were measured by the Nanodrop  and Qubit fluorometer . All the samples of 2 and HOT FIREPol DNA polymerase. Primers (2 μl) were added at a concentration of 10 μM to yield a final concentration of 1.5 μM in the final mix. Ten nanograms of DNA per microlitre was added to the PCR reaction mix. Reaction was carried out in Veriti Thermal Cyclers  at specific annealing temperatures depending on the melting temperature (Tm) of each primer. The steps for PCR reaction were, initial enzyme activation at 95°C for 12 min followed by 45 cycles of denaturation at 95°C for 45 s, annealing at 45–60°C for 45 s and extension at 72°C for 1.5 mins. Final extension step was carried out for 7 min at 72°C. DNA bands were visualized on 1.8% agarose gels (13 cm long and 15 cm wide) ran at 5V/cm for 2 h. Gel images were documented using the gel documentation system . One Kb and 100 bp ladder were used as reference. ISSR primers that produced polymorphic bands were repeated twice independently with three technical replicates for each sample  resolving power (RP), mean resolving power (MRP), marker index (MI) and Shannon’s index (H′). PIC is the probability in detecting polymorphism by a primer PIC  =  1-Σ(pi)h allele ; RP is th allele : RP  =  h allele . Shannonh allele .Ne), mean expected heterozygosity (HE), mean Shannon’s Information Index (I), Nei’s gene diversity (h'), and Nei’s pairwise genetic distances by using the GenAlEx 6.5 software [ST = HT-HS/HT). The GST was corrected according to Hedrick [ST and D, respectively. We also performed an analysis of molecular variance (AMOVA) to examine the distribution of genetic variability within and among populations using the Arlequin software (version 3.5) [ST (Fixation index). Based on the FST the Gene flow (NM = 1(1/FST-1)/4 was also calculated [ST from the Arlequin software were used to derive the Slatkins relationship (FST/1-FST) to create an isolation by distance plot through the Mantel’s test employing 10,000 permutations [Genetic diversity within each population was estimated through percentage of polymorphic loci (P), mean effective number of alleles   and estilculated . The paiutations .ST between the populations [Population structure was determined by the neighbour-net split decomposition network generated by SPLITS Tree v4.6 and bootstrapping runs of 1000 replicates , a PCoA ulations  and Bayeulations . The admulations . The valulations  was usedulations . The useH. salicornicum, 16 primers produced 195 unambiguous and reproducible bands. Out of them167 were polymorphic (86.5%). Smeared and faint bands were not considered for analysis. The bands ranged from 135–1729 bp. Number of bands per primer ranged from minimum 8  to maximum 21 (ISSR 826) with an average count of 10.5 bands per primer. All the primers had a high discriminating power as observed by their PIC (mean-0.31) values. Highest PIC was obtained with ISSR 7 (0.46) and the lowest (0.18) with ISSR 13 and 23  and PIC and RP .For 3 and 23 . Other pH. salicornicum, an average number of observed alleles (Na) ranged from 1.144  to 1.610 (Al-Subiya). Relatively higher levels of allelic diversity (Ne) were observed in the populations of Al-Subiya, Om Qaser and Al-Ritqa, while moderate levels were recorded in Al-Shagaya and Al-Abraq and a lower level in Al-Abdally (I) and Nei’s gene diversity (h') with highest values in Al-Subiya  and lowest in Al-Abdally . Al-Shagaya and Al-Ritqa showed I and h' values comparable to Al-Subiya and Om Qaser. The overall average values of h' and I were 0.200 and 0.303 respectively. The percentage values of polymorphic loci were ≥ 60% for Al-Subiya, Om Qaser, and Al-Ritqa; ≥ 45% for Al-Shagaya, and ≤ 27% in Al-Abraq with an average polymorphism above 50% in six populations of H. salicornicum. The populations within Al-Subiya, Om Qaser, Al-Ritqa, and Al-Shagaya showed relatively high values of He and uHe (> 0.200) as compared to Al-Abraq and Al-Abdally (< 0.200). The genetic diversity was highest in the populations of Al-Subiya, followed by Om Qaser, Al-Ritqa, Al-Shagaya, Al-Abraq, and Al-Abdally.In -Abdally . SimilarST = 0.221, P = 0.000) in H. salicornicum to prevent inbreeding.The Analysis of molecular variance (AMOVA) test was applied to the binary matrix data file to obtain information on the variation within and among populations by the Arlequin software. The results of the AMOVA indicated that most (77.83%) genetic variation was within populations, with a lesser variation (22.17%) among the populations . the FixDS) among populations were low ranging from 0.047 between Al-Ritqa and Al-Shagaya to 0.187 between Al-Abdally and Al-Subiya populations of H. salicornicum  based on the fixation index (FST) among the populations was ~ 1 (0.88).Values of genetic differentiation GH. salicornicum. The approaches used were the split decomposition analysis, the Bayesian clustering and the principal coordinate analysis (PCoA).Clustering analysis was performed to study the population structure of H. salicorncum populations into six weakly separate sub-populations (The neighbour-net based split decomposition returned a Split tree dividing the ulations . Group-AK) showed a visible peak at 5 and a smaller peak at 8  among the genetic and geographic distance  under the current study was approximately twice of that of the rare plants. According to Cole’s observations, He (expected heterozygosity) for a rare plant was recorded as 0.113, whereas, for a common plant, it was 0.150 [Haloxylon populations refers to the occurrence of rare microsatellite alleles imparting much of the diversity in the species. Higher levels of expected observed and expected heterozygosities were recorded in H. aphyllum [The level of genetic diversity is often correlated with its range of distribution. In the present study, as 0.150 . The numaphyllum .I = 0.330; h’ 0.200) among the Haloxylon populations were observed in our study. Similar findings were reported for Haloxylon distributed in China [H. salicornicum are small grayish in color and attached to a perianth, which gives a wing-like appearance assisting in their long distance dispersal by wind [Haloxylon.The level of heterozygosities obtained are indicative of more genetic variation and thus leaving behind a scope for population expansion. Consequently higher diversity indices . In addition, corrected G’ST and Jost’s D between 0 to 1 further supported the fact that the Haloxylon populations under study were not completely homozygous and considerably differentiated genetically [H. ammodendron in China [Relative to the low genetic diversity among the populations the overall fixation index was high enough to prevent allele fixation or inbreeding. For plant populations values of Ftegories , 57. Thietically ,35,37. Cin China ,54.M > 1 as an indicator of sound gene flow necessary to maintain genetic variation when population size is small. Overall gene flow ≤ 1 (0.880) was recorded in the present study. An increased geneflow is suggestive of decreased inbreeding. The gene flow (NM) observed in the present investigation less than 1 is deemed to be sufficient to maintain genetic variation among the Rimth populations. Comparable levels of gene flow were also recorded in H. ammodendron in China [Genetic differentiation is inversely proportional to the gene flow. Slatkin  posited in China .Haloxylon in Kuwait were weakly isolated by distance. The clustering pattern revealed that the groupings were largely in agreement with the geographical distribution of the accessions. Al Abraq always clustered with Al Shagaya owing to their geographic proximity. The populations of Om Qaser, Al Ritqa and Al Shagaya formed overlapping clusters with each other in the Splits tree and the Structure Plot. This could be explained by the admixture events detected from the sampling of hybrid individuals as subpopulations come into contact via dispersal or from sharing a border [ST values were also recorded to be relatively lower among the populations in close vicinity. The PCoA was suggestive of interbreeding between the specimens of Al Abraq and Al Shagaya as well as the populations of Al Subiya, Al Ritqa and Om Qaser. Al Abdally was the farest and most distinct group in all the cases. This would be attributed to, an unknown sympatric speciation event occurring in the area restricting the gene flow with neighboring populations. However, more information on phenotypic variations needed to be gained for species in Al Abdally region.The correlation between the genetic and geographical distance (Mantels test r = 0.188 p< 0.05) revealed that the populations of a border . The paiHaloxylon in Kuwait. Another explanation given for the weak population structure is the effectively maintained geneflow. In absence of any topographical barriers the genes among the populations effectively communicate with each other. In the present case the number of migrants among the populations almost equaled to 1 (NM = 0.880). Migration is a common mechanism that erodes population structure [Haloxylon species is observed in Kuwait. Sewell Wright [We observed that the individual clusters were not far away from each other. The weak site differentiation among the clusters can be explained due to the low genetic distance among the populations . From antructure . The absl Wright  describeH. salicornicum and the significant genetic differentiation within its populations are encouraging because these characteristics are advantageous for preserving genetic resources and broadening the genetic basis of these native plants. However, we detected a recent bottleneck event occurring in population structure, suggesting that individual populations may suffer from a dramatic decline in population size. Given the small number of existing populations and the low, genetic distances among the populations, these populations should be conserved as much as possible to maintain their current status, especially the populations with higher diversity, such as populations in Al-Subiya for Haloxylon, should be protected as a priority for in situ conservation.From a conservation standpoint, the high genetic diversity maintained within S1 FigLanes 1–3 represent 3 technical replicates in first set of PCR (Day 1), Lanes 4–6 represent 3 technical replicates in second set of PCR (Day 2); M—Marker (100bp + 1Kb ladder).(TIF)Click here for additional data file.S2 FigH. salicornicum samples, Marker (100bp + 1Kb ladder).Generated by ISSR primer 2 in (TIF)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(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Arthropodium cirratum; Asparagaceae), an endemic New Zealand coastal herb, using nuclear microsatellite markers. This species was brought into cultivation by Māori within the last 700–800 years for its edible roots and was transplanted beyond its natural distribution as part of its cultivation. We found very high levels of genetic structuring in the natural populations (FST = 0.84), indicating low levels of gene flow. Reduced genetic diversity was found in the translocated populations, suggesting a large loss of genetic diversity early in the domestication process. The data indicates that rengarenga was brought into cultivation independently at least three times, with the sources of these introductions located within a narrow area encompassing about 250km of coastline. Hybridization was inferred between A. cirratum and the closely related A. bifurcatum, despite A. birfucatum not occurring in the vicinity.We examined the genetic structuring of rengarenga ( New Zealand was the last substantial landmass to be colonised , about 7Arthropodium cirratum (G.Forst) R.Br.  (Asparagaceae) is a perennial lily-like herb endemic to New Zealand. It is primarily a coastal species and its natural distribution is thought to be north of around 38°S . Permission to collect samples from the Otari Wilton's Bush botanic gardens was provided under permit 145 and the permission of individual land owners was obtained prior to the collection of samples from private land.Sampling included the specimens for which chloroplast loci had been sequenced , with ad sampled . Many poA. cirratum [For samples that did not already have DNA available, a modified CTAB method was used to extract genomic DNA  and expected heterozygosity (HE), number of alleles (A), number of private alleles (PA) and inbreeding coefficient (FIS) were calculated in GenAlEx 6.5 [Matching allele profiles between different individuals were detected using GenAlEx 6.5 . DescripAlEx 6.5 .ST values between all populations with more than five samples with Arlequin v3.5.2.2 [ST for all A. cirratum populations was also calculated with Arlequin. For both global and pairwise FST values, statistical significance was tested by 10 000 permutations whereby for each permutation individuals were randomly exchanged between populations and a new FST calculated.Loci were tested for deviation from Hardy–Weinberg equilibrium (HWE) with GenAlEx 6.5. Fisher's exact tests of linkage disequilibrium between loci were calculated in Genepop v.4.2 . The Bonv3.5.2.2 . Global ST and geographic distance, we performed Mantel tests between the pairwise FST values and spatial distance among the nine natural populations with more than five samples in Arlequin v3.5.2.2. Both straight-line distances and minimum coastline distances were tested and significance tested with 1000 permutations. Straight-line geographic distances between populations were calculated using the Geographic Distance Matrix Generator [To test for correlations between Fenerator  and coasK) was obtained by calculating the ΔK statistic [Population structure was examined with STRUCTURE v2.3.4 ,19, withtatistic  in STRUCtatistic . CLUMPP tatistic  was usedtatistic .In some populations, including natural populations demonstrating variation at other loci, all of the individuals at two of the 12 loci, ArtCir18 and ArtCir38, had fixed heterozygotes. These two loci were excluded from further analyses. Microchecker found no evidence of large allele dropout or stuttering. Possible null alleles were inferred for seven loci, each in a single population. Three of these loci were in the Matapouri Bay population (population 11), which has within-population sub-structuring , likely explaining this result. For the remaining four loci the null allele frequencies was estimated to be low (<0.2) and, because previous research has shown that low frequency null alleles have little influence on the detection of genetic differentiation , we retaA. cirratum the 10 loci displayed a total of 87 alleles, with 4 to 20 alleles per locus (mean ± SD: 8.7 ± 4.6). The six A. bifurcatum samples had 23 alleles, with 1 to 4 alleles per locus (mean ± SD: 2.3 ± 1.1). Arthropodium bifurcatum had three private alleles. Within A. cirratum, 12 populations had private alleles with one to three private alleles in each .IS for Arthropodium cirratum was high and positive (FIS = 0.72±0.09), indicating inbreeding. At the population level, the FIS values varied from -0.23 to 0.57  had very low genetic diversity compared to the natural populations. Across the 10 loci only 21 of the 87 Genotype matching revealed that all individuals sampled from the cultivated Tora and Wainuiomata populations (47 and 48) shared an identical genotype but this genotype was not detected from any individuals from natural populations. All the alleles found in the Tora and Wainuiomata populations were also found in the natural populations at Haparapara and Motu (populations 38 and 39), except for the fixed allele at locus ArtCir43.All individuals from the cultivated Miramar and Paekakariki populations (populations 49 and 50) plus all of the South Island individuals (populations 51–55) had an identical genotype and this matched that found in all individuals from the natural populations at Tohora Pirau (42) and Otiki (45) and four individuals from the Hick’s Bay population (43).The only cultivated population that was not monomorphic across the 10 loci was Kairakau (population 46), whose three individuals showed variation at six loci. None of the three individuals from Kairakau matched any other individuals sampled across all loci.A. cirratum was very high (FST = 0.84). Pairwise FST values for A. cirratum, calculated for populations with >5 individuals, were generally high and significant  and the comparisons between the three cultivated South Island populations . Pairwise FST values between A. bifurcatum and the A. cirratum populations were also high and ranged from 0.14 to 0.93 , including the Maungaraho population, which exhibited the A. bifurcatum chloroplast haplotype. The other A. cirratum population shown to have an A. bifurcatum chloroplast haplotype, Papanui Point (population 34), showed affinities to both the cluster containing A. bifurcatum and also to a second cluster that included populations in close geographic proximity (populations 29–33).The Structure Harvester analysis of ΔK indicated that the optimal K was 5 , S1 Fig,A. bifurcatum and a number of other A. cirratum populations (see above). The Tora and Wainuiomata populations (47 and 48) clustered with populations from the eastern Bay of Plenty (36–41). The Miramar (49), Paekakariki (50) and South Island populations (51–55) formed a cluster with samples from East Cape (42–45).At K = 5 the cultivated populations were assigned to three clusters. The population at Kairakau (46) clustered with A. cirratum was not present. The genetic split detected by STRUCTURE corresponds to this geographic separation.At higher values of K additional population structuring was evident , with maArthropodium bifurcatum did not form a separate cluster until K = 16. At this value of K the Papanui Point A. cirratum population (34), which shares a chloroplast haplotype with A. bifurcatum, was split between the A. bifurcatum cluster and populations 29–33. Individuals from the second population of A. cirratum with an A. bifurcatum chloroplast haplotype, the Maungaraho population (17), were assigned with high probability to the A. bifurcatum cluster until K ≥ 16, when they formed their own cluster.At K = 14 and K = 21 the cultivated populations 49–55 clustered with those from East Cape (42–45). At K = 14 the Kairakau population (46) grouped with Lake Rotoehu (35) but it formed an independent cluster at K = 21. At K = 14 the Tora (47) and Wainuiomata populations (48) were split between clustering with the Lake Okataina population (36) and a second cluster that included samples from Motu (38), Tauwhare Pa (37) and some individuals from Haparapara (39). At K = 21 the Tora and Wainuiomata individuals were assigned with high probability to a distinct cluster, with individuals from Lake Okataina (36) also showing some affinity to this cluster.A Mantel test indicated that there was a moderate significant correlation between straight-line geographic and genetic distance  for the natural populations with more than 5 individuals. This relationship weakened slightly when coastal distances were used Arthropodium cirratum exhibited very strong genetic differentiation between populations within its natural distribution, and some of this structure can be explained by isolation-by-distance. Population structuring was even detected within the Matapouri Bay site, where two headlands 200m apart had different subpopulations. The nuclear microsatellite results confirm that the high level of genetic structuring previously reported [A. cirratum seeds are dispersed by gravity [IS values among populations; i.e., some populations have sufficient pollinators for outcrossing, while others do not. It is possible that clonal propagation has contributed to the population structuring and low diversity observed within some A. cirratum populations. However, since this species tends to form distinct clumps in different crevices on rock bluffs, the observed patterns cannot solely be explained by clonal propagation and our sampling targeted distinct clumps to avoid collecting clones.reported  is not r gravity  and/or w gravity . The spe gravity . This poA. cirratum populations reinforces our previous recommendation [The high structuring at nuclear microsatellites and presence of private alleles in many endation  that numA. cirratum populations indicated that these populations had been sourced from the Bay of Plenty/East Cape region. However, it was unclear whether there had been multiple introductions from different sites or a single introduction from a single unsampled variable population [A. cirratum into cultivation.The two chloroplast haplotypes previously detected from the cultivated pulation . The micThe cultivated samples with chloroplast haplotype C (populations 49–55) had identical microsatellite profiles. This genotype was also found at four locations in the natural populations: Onepoto, Otiki, Tohora Pirau and Hick’s Bay (populations 42–45). The Tohora Pirau sample has a different chloroplast haplotype, AC , thus exA. cirratum was more widespread in this region in the past. Alternatively, the Kairakau population may derive from multiple introductions from different source populations from this region.Cultivated samples with chloroplast haplotype B were more variable at the microsatellite loci than those with haplotype C. The population at Kairakau (46), showed variation at six of the ten microsatellite loci, despite only three individuals sampled. This was considerably more variation than that detected from Tauwhare Pa and Motu (populations 37 and 38), the only natural populations with haplotype B. Those populations only showed variation at two and one of the loci, respectively. The variation found at Kairakau may result from an introduction from an unsampled, possibly extinct, genetically variable population in the vicinity of populations 37 and 38. There is little natural coastal vegetation remaining in the Bay of Plenty  and it iThe other cultivated populations with haplotype B, Tora (47) and Wainuiomata (48), had identical microsatellite profiles, suggesting a common origin. However, this genotype did not match any of the individuals we sampled from the natural population. STRUCTURE analyses indicated that these cultivated populations are most similar to natural populations 36–41. However, populations 36 and 39–41 do not have haplotype B, excluding them as the source, and populations 37 and 38 are fixed for a different allele at locus ArtCir43 to the Tora and Wainuiomata samples. This suggests the source of Tora and Wainuiomata was an unsampled, possibly extinct, population from the Bay of Plenty, in the vicinity of populations 37 and 38. This source population is unlikely to be the same as the Kairakau population because the Kairakau samples were fixed for different alleles than those from Tora and Wainuiomata at two loci (ArtCir32 and Art43). This also suggests that the Tora and Wainuiomata populations have an independent origin to the plants from Kairakau and are unlikely to derive via stepping-stone dispersal from Kairakau. Overall our results indicate at least three independent translocation events out of the Bay of Plenty/East Coast region and suggest that both chloroplast and nuclear data can provide complementary sources of information.A. cirratum populations, except for Kairakau, were fixed across all microsatellite loci, indicating the loss of diversity through a ‘domestication bottleneck’. Overall only 41.6% of the nuclear diversity detected in the natural A. cirratum populations was maintained through the domestication bottleneck, as measure by HE. This value is low compared with other perennial crops [A. cirratum domestication bottleneck including the high level of population structuring within the natural distribution of A. cirratum, the narrow area from which cultivated plants were sourced and the physical isolation of the translocated populations from the natural populations, which has prevented gene flow from introducing additional wild diversity into the cultivated populations.All of the cultivated al crops , where aal crops ). As poial crops  a numberA. cirratum was cultivated vegetatively through the division of plants. Alternatively the observed low diversity in these populations may be a consequence of low diversity in the source populations, selfing or loss of diversity through the domestication bottleneck (see below) and/or population bottlenecks following the cessation of cultivation. Next-generation sequencing methods that allow screening of many more DNA markers may be able to discriminate between these hypotheses.The lack of variation within most of the cultivated populations (populations 47–55) may indicate that A. cirratum had previously been shown to share a chloroplast haplotype with A. bifurcatum. The nuclear microsatellites also indicate a mixed ancestry for these populations. In the STRUCTURE analyses the Papanui Point population (34) was assigned to two genetic clusters: one containing A. bifurcatum and the other comprising geographically adjacent A. cirratum populations. The Maungaraho population (17) clustered with A. bifurcatum in the STRUCTURE analyses until it formed its own cluster at higher K. Pairwise FST values also indicated a closer relationship between A. cirratum from Maungaraho and A. bifurcatum than any other comparisons (including within A. cirratum).Two populations of A. cirratum favours a hybridization hypothesis (shared polymorphism is more likely in large effective population sizes [A. cirratum cluster (as well as to the A. bifurcatum cluster) in STRUCTURE also points to an origin involving hybridization rather than shared ancestral polymorphism. For the observed pattern to have arisen through shared ancestral variation, A. bifurcatum must have diverged subsequent to the development of the genetic structuring within A. cirratum. However, the divergent chloroplast haplotype in A. bifurcatum [A. bifurcatum to Papanui Point is over 200 km away could infer a chance long distance dispersal event in A. bifurcatum or indicate that this species had a wider distribution in the past. Heenan et al. [A. bifurcatum. Since that publication our own fieldwork has extended the mainland range of this species, discovering highly fragmented A. bifurcatum populations–sometimes comprising single plants still occur scattered as far south as Whangamata and Ngatutura Point. Otherwise A. bifurcatum is a common plant on predator-free offshore islands of northern North Island [There are two possible explanations for the patterns observed: shared ancestral variation and interspecific hybridization, and they are often difficult to distinguish because they produce similar patterns of allele sharing . The smaon sizes ). The asfurcatum  suggestsn et al.  had noteh Island . In thosh Island –34. As tS1 FigAccording to the ΔK, K = 5 represents the optimal structure partition in our dataset, with secondary optima at K = 14 and K = 21.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Glossina brevipalpis Newstead and Glossina austeni Newstead present in South Africa. These populations were also compared with flies from adjacent areas in Mozambique and Eswatini. For the molecular markers, deoxyribonucleic acid was extracted, a r16S2 Polymerase chain reaction (PCR) was performed and the PCR product sequenced. Nine landmarks were used for the morphometrics study as defined by vein intersections in the right wings of female flies. Generalised Procrustes analyses and regression on centroid size were used to determine the Cartesian coordinates for comparison between populations. Both methods indicated an absence of significant barriers to gene flow between the G. brevipalpis and G. austeni populations of South Africa and southern Mozambique. Sustainable control can only be achieved if implemented following an area-wide management approach against the entire G. brevipalpis and G. austeni populations of South Africa and southern Mozambique. Limited gene flow detected between the G. austeni population from Eswatini and that of South Africa or Mozambique may imply that these two populations are in the proses of becoming isolated.The effective control of tsetse flies , the biological vectors of trypanosome parasites that cause human African trypanosomosis and African animal trypanosomosis throughout sub-Saharan Africa, is crucial for the development of productive livestock systems. The degree of genetic isolation of the targeted populations, which indicate reinvasion potential from uncontrolled areas, will be critical to establish a control strategy. Molecular and morphometrics markers were used to assess the degree of genetic isolation between seemingly fragmented populations of  African animal trypanosomosis is endemic in South Africa with an estimated 350 000 cattle at risk in the rural north east of the KwaZulu-Natal province  of Mozambique, represent the southern most distribution of tsetse flies in Africa  approach, that is, a strategy that targets the entire pest population  markers were used to determine the level of genetic isolation between the southern and northern tsetse fly pockets in South Africa. In addition, the South African populations were compared with flies collected in the adjacent areas in southern Mozambique and Eswatini. The level of genetic isolation between the various populations within South Africa and between these three countries will provide an approximation of the invasion potential of flies from neighbouring areas , Pretoria, South Africa, were included for the genetic analyses as out-groups. The colonies of G. austeni and G. brevipalpis at the ARC-OVR originate from laboratory colonies at the Vector and Vector-Borne Diseases Research Institute, Tanga, Tanzania, and the FAO/IAEA Insect Pest Control Laboratory in Seibersdorf, Austria, respectively . The wings and legs of the flies were removed, and the body crushed in fresh PBS using a micro-pestle in a 1.5 mL polypropylene tube. After centrifugation, 200 µL of the supernatant suspension was used for DNA extraction with a DNeasy Blood & tissue kit . The purified DNA was suspended in 100 µL of the supplied elution buffer and either processed immediately or stored at −20 °C.Deoxyribonucleic acid (DNA) was extracted from ethanol preserved µL of the extracted DNA as described previously  Ltd, Johannesburg) using the Big Dye Terminator V 3.1 Cycle Sequencing Kit  Ltd).The N1-J-12585 [5′GGT CCC TTA CGA ATT TGA ATA TAT CCT 3′] and LR-N-12866 [5′ACA TGA TCT GAG TTC AAA CCG G3′] primer pairs , Kosi Bay (n = 31) and False Bay Park (n = 37) for G. brevipalpis and Mozambique (n = 14) and the Hluhluwe-iMfolozi Park (n = 13) for G. austeni. The right wings of 345 G. brevipalpis and 346 G. austeni females were removed, dry mounted between two microscope slides  of wings of flies collected from the various sites. The data were normally distributed, standard (parametric) methods were used and the Tukey’s test was applied. Multivariate regression of partial warps on size was used to estimate the residual allometry and the statistical significance was estimated by the 10 000 runs permutation tests  has been granted for tsetse fly collection (Ref 12/11/1/1/9).Materials used in the study posed no health risk to researchers and no vertebrate animals were involved. The study was conducted as part of a project funded by the Department of Science and Technology on National Assets (P10000035) at the ARC-OVR  in collaboration with the Food and Agriculture Organization (FAO) or International Atomic Energy Agency (IAEA) Division of Nuclear Techniques in Food and Agriculture and the Department of Technical Cooperation of the IAEA under project RAF 5069. Permission to carry out research in terms of Section 20 of the G. austeni and 116 G. brevipalpis specimens were used in multiple alignments, which included sequences from four G. austeni and two G. brevipalpis from the tsetse colonies as out-groups. The phylogenetic trees  sequences from 79 ic trees , construG. brevipalpis from South Africa and southern Mozambique did not show any significant branching or grouping of specimens, with the exception of the colony flies (p-distances < 0.01). The only exceptions were the colony flies and G. austeni from Eswatini which formed a distinct phylogenetic grouping  had a unique haplotype , whereas the level of genetic differentiation was moderate between the populations of southern Mozambique and Eswatini (0.15).The Fst values indicate a great level of genetic differentiation between the G. brevipalpis and G. austeni was analysed to assess the degree of genetic isolation between the populations within South Africa as well as between the South African and the southern Mozambique populations. The analysis for the G. austeni populations was extended to include the population in Eswatini. The allometric effect was removed so that shape could be analysed independently.The shape of the right wings of female G. brevipalpis and G. austeni, respectively. These discriminant factors indicated that there was no clear wing shape separation between the G. brevipalpis populations collected from the sites in South Africa and southern Mozambique (p = 0.30), indicating that there was no residual allometry.The two species were evaluated separately. The principal component analyses indicated that the first two discriminant factors (shape components) accounted for 71% and 69% of the variance for zambique . The mulG. austeni, as the multivariate regression was significant (p = 0.01), might indicate an environmental effect. There was no separation within the first discriminant factors between the G. austeni collected from the different sites in South Africa as well as those from southern Mozambique , Ndumu (p < 0.05), Kosi Bay (p < 0.05) and St Lucia (p < 0.01) , Tembe versus St Lucia (p < 0.01) and Boomerang versus St Lucia (p < 0.01) (The wing size variation as indicated by the isometric estimator known as the average wing centroid size was determined for both species. For St Lucia  and 5a.  < 0.01) . Signifi < 0.01) .p < 0.01) between G. austeni from Phinda (916 ± 26) and St Lucia (941 ± 52) (G. austeni population from Eswatini (892 ± 37) was significantly different (p < 0.01) from that of the flies from southern Mozambique and South Africa  . Similarh Africa .This indicates a longitudinal (coast to interior) trend in wing size for both species. Flies collected at the coastal sites were, on average, bigger than flies collected in the interior . A trendG. brevipalpis and G. austeni extend into South Africa and Eswatini (G. austeni)  indicate limited gene flow and potential incomplete isolation  may not be sustainable because of reinvasion from uncontrolled neighbouring areas. Should the data be confirmed by the ongoing microsatellite work, this would entail the need for an AW-IPM approach against the entire tsetse fly belt of South Africa and southern Mozambique. The entire area should be controlled simultaneously or in a sequential way using temporary barriers of impregnated traps and/or targets between tsetse free areas following the ‘rolling-carpet’ principle (Vreysen et al. Within the known limitations of the used markers, the data of this study seem to provide some evidence of the absence of significant barriers to gene flow between the populations in South Africa and southern Mozambique and justify the need for further investigation. This seems to imply that the


STF. Based on a qualitative survey, numerical analyses, simulations, and empirical data, I here argue that STF does not express the relationship to allele frequency differentiation between populations generally considered interpretable and desirable by researchers. In particular, STF (1) has low sensitivity when population differentiation is weak, (2) is contingent on the minor allele frequency across the populations, (3) can be strongly affected by asymmetry in sample sizes, and (4) can differ greatly among the available estimators. Together, these features can complicate pattern recognition and interpretation in population genetic and genomic analysis, as illustrated by empirical examples, and overall compromise the comparability of population differentiation among markers and study systems. I argue that a simple differentiation metric displaying intuitive properties, the absolute allele frequency difference AFD, provides a valuable alternative to STF. I provide a general definition of AFD applicable to both bi- and multi-allelic markers and conclude by making recommendations on the sample sizes needed to achieve robust differentiation estimates using AFD.Measuring the magnitude of differentiation between populations based on genetic markers is commonplace in ecology, evolution, and conservation biology. The predominant differentiation metric used for this purpose is  STF, interpreted broadly as a measure of the proportion of the total genetic variation at a genetic locus attributable to differentiation in allele frequencies between populations [STF was conceptualized in the middle of the last century as a descriptor of genetic structure among populations [STF to be calculated with empirical genetic data, based on different assumptions about the sampled study populations and/or the mutation process of the genetic markers [STF with multi-allelic genetic markers such as microsatellites [STF estimators is firmly established in population genetics and genomics; STF is currently among the most widely used statistics in these fields. In this note, I will argue that despite its popularity, STF has shortcomings that complicate the analysis of population differentiation, and that a powerful alternative differentiation metric is available.Biological studies measuring the magnitude of genetic differentiation between populations, for example to explore levels of gene flow between populations, to discover genome regions influenced by natural selection, or to inform decisions in conservation biology, are published on a daily basis. A differentiation metric used frequently in such work is ulations . FST wasulations ,3,4. Ove markers ,12,13,14tellites ,19,20,21STF, we will start from the very beginning and ask what properties a metric of genetic differentiation should exhibit. First, the scale of the metric should range from zero (no genetic differentiation among populations) to one . This familiar scale greatly facilitates interpretation and allows for convenient comparisons of differentiation among genetic markers and study systems. STF estimators satisfy this scale criterion; they are generally designed to range from zero to one.To approach the problems inherent in Y-axis) an ideal metric of differentiation should exhibit if such a metric was to be invented from scratch. Specifically, the respondents were presented in The second, perhaps even more crucial requirement of an appropriate differentiation metric is that it should show an intuitive and traceable relationship to the magnitude of genetic differentiation between populations, so a researcher can understand and interpret what they are measuring. But what should this relationship look like? The answer to this question cannot be derived from theory but depends on the needs and expectations of the researchers measuring differentiation among their study populations. To develop a sense for these expectations, I performed a qualitative survey involving a total of 15 haphazardly chosen colleague researchers (advanced postdocs and faculties) having years of experience in population genetics and/or evolutionary genomics, including both empiricists and theoreticians. I confronted these researchers with a graphic displaying a continuum of symmetrically increasing genetic differentiation between samples of nucleotides (n = 40) drawn from two hypothetical populations at a single-nucleotide polymorphism (SNP) . I then asked them to specify the corresponding magnitude of population differentiation  More generally, the calculation of AFD between two populations at a genetic polymorphism can be formalized asn represents the total number of different alleles observed at the polymorphism, and the if-terms specify the frequency of allele i in the two populations . This fferences ,25,26,27AFD particularly intuitive and interpretable. Note that throughout this paper, (non-)linearity refers only to the immediate relationship of a given differentiation metric to population allele frequencies, and hence does not imply any specific relationship of the estimator to biological factors influencing allele frequencies, such as gene flow, mutation, selection, population size, or divergence time. Now let us consider how STF behaves along the continuum of differentiation in allele frequencies. For this, we will initially focus on the two most popular STF estimators, STG [Theta (θ) [STF, but they are clearly under-appreciated by empiricists.As suggested above, a substantial proportion of researchers appear to find the linear relationship to continuous genetic differentiation exhibited by ors, GST  and Thetheta (θ)  (given iheta (θ) ) and conSTG is zero, as one would expect , is maximal . It is instructive to also explore the behavior of our focal differentiation metrics when the MAF is minimal . Under this condition, the relationship between genetic differentiation and AFD remains straightforward to interpret. For instance, an allele frequency differentiation exactly intermediate between the absence of differentiation  and the complete fixation for alternative alleles between the populations still yields an intuitive AFD value of 0.5  allele, as determined based on the e of 0.5 b. ReduciAFD and STG rely exclusively on allele frequencies and thus ignore the sample sizes used to estimate these frequencies. Therefore, the expected (parametric) value of these differentiation metrics is not dependent on sample size. . By contrast, the expected value of Theta at a given marker does depend on sample size. As long as sample sizes are similar between the two focal populations, the absolute size of these samples has a relatively minor influence on Theta, at least for typical  sample sizes used by empiricists (details not presented). However, imbalance in the size of the samples from the populations can have a dramatic influence on Theta. To appreciate this point, we assume that we sample nucleotides at a genetic marker from two populations exhibiting intermediate differentiation in allele frequencies (AFD = 0.5). Sample size for the first population is always constant . By contrast, choosing allele frequencies in the two populations such that the MAF is maximal, we find that the influence on Theta of sample size imbalance between the populations is reversed in direction, and weaker in magnitude . STF estimators, like Theta, are sensitive to the balance between populations in the number of nucleotides sampled at a given SNP. This property must be considered a serious nuisance to empiricists, especially those working with high-throughput sequencing data from population pools [STF among markers and among studies when using Theta as estimator. Moreover, that the expected value of differentiation at a marker should shift when the samples drawn from the focal populations differ in size—even when the allele frequency estimates remain exactly the same—and that this influence of sample size imbalance on STF is itself contingent on the MAF, can hardly be considered intuitive.The third conclusion is that at least some on pools ,33. EvenSTF were based on the commonly used estimators STG and Theta. A number of less widely applied alternative STF estimators have been introduced, however, hence it is valuable to examine if these estimators share with the former the weaknesses identified above. For this, I repeated the analysis of the magnitude of differentiation along the two continua of differentiation in allele frequencies visualized in STF [STPhi (Φ) [STG. Similarly, repeating the calculations with Hudson’s STF [Theta. Clearly, the problems identified for STG and Theta extend to the whole family of STF estimators. Moreover, ESTD (Formula (13) in Reference [STF, exhibited differentiation values qualitatively similar to the STF estimators  and PhihiST (Φ) . For theon’s FST  (Formulaon’s FST ) produceeference ), proposSTF  makes clear that STF was not designed as a simple descriptor of allele frequency differentiation. Instead, STF estimators aim at quantifying progress in population differentiation, or at partitioning genetic variation among hierarchical levels, in the light of specific models of mutation, gene flow, and drift [STF, however, will rarely be aware of the underlying assumptions, and even for those who are, real-world situations will generally not allow evaluating what—if any—evolutionary model is meaningful for any given population pair, genome region, and marker analyzed . It is  decades ,40,41,42AFD, I further considered a metric derived from information theory called Shannon differentiation (hereafter ShannonD) that has recently been claimed to exhibit a ‘straightforward relationship to allele frequency differences’ [ShannonD exhibits even less sensitivity than STF in the domain of weak to modest allele frequency differentiation between populations . I eulations a. Conseqp-values derived from statistical tests of differentiation between populations at genetic markers  quantitatively into progress toward complete genetic differentiation (the effect size itself). In addition, p-values are a direct function of sample size, further reducing the comparability among markers and studies.The latter conclusion also applies to ad hoc differentiation metrics based on ferences ). The diSTF (and related metrics) were exposed based on simple numerical analyses. Given the ubiquity of STF in empirical research, I next illustrate implications of quantifying population differentiation by STF in real-world genetic analyses based on two examples from threespine stickleback fish (Gasterosteus aculeatus L.).In the previous section, the drawbacks of Sbf1 enzyme restriction) in 28 female and 26 male stickleback from a single population inhabiting Misty Lake, Vancouver Island, Canada  than for AFD (upper mode near 0.5)  X- and Y-limited alleles, one needs to be aware of the specific function linking allele frequency differences to STG .The first example re-uses SNP data generated through individual-level RAD sequencing . ear 0.5) a. The ans to GST a,b. NoteSTG and AFD across genome-wide SNPs. For this, the original raw marker data set .STG and AFD as differentiation metrics reveals an important difference: the distribution of SNP-specific STG values has a striking mode near zero, tapering off steeply into a thin tail of higher differentiation values  across the pool of the two samples, noting that with increasing differentiation, the range of possible MAFs decreases  and exhibiting a maximal MAF of 0.5 , as in the left bar plot of p = 0.125) to draw two identical alleles from one population and two opposite alleles from the other, and hence to observe complete differentiation (AFD = 1). Such overestimation, however, is not possible when the populations are undifferentiated but the MAF is minimal  per population, AFD (and STG; AFD value became relatively stable with sample sizes of around 20–30 nucleotides per population (AFD; STF shows a very similar sensitivity to sample size and to the associated precision in population allele frequency estimation, as presented in To examine this recommendation with empirical data, I again used the SNP data set from the lake-stream stickleback population pair described above. I here assessed how genome-wide mean and GST;  changes STF statistics also permit estimating the overall genetic structure across a larger collection of populations. With AFD as a differentiation metric, this option is not available. A straightforward ad hoc solution, however, is to simply average multiple AFD values for SNPs or genome windows across the multiple population contrasts of interest [As a final methodological remark, I highlight that this article has so far considered only the situation in which the number of populations to be compared is exactly two. Although such pairwise population comparisons arguably represent the most common analytical situation, it should be noted that interest .STF statistics and related metrics—do not necessarily measure the quantities most meaningful in genetic and genomic research. As a point in favor of STF, one may argue that its long tradition would promote the comparability of differentiation among studies [STF is highly contingent on the specific estimator, is sensitive to the MAF spectrum of the markers, and sometimes to imbalances in sample size. Combined with the general insensitivity across the differentiation range most relevant in many analytical situations—weak population differentiation—STF falls short of being a reliable standard for measuring genetic differentiation. I argue that in many analytical contexts, the simple absolute allele frequency difference AFD will provide a sufficient, meaningful, and robust differentiation metric, thus promoting the discovery of patterns in differentiation, and their interpretation.The purpose of this note was to show that metrics of population differentiation used routinely in the analysis of genetic data— studies . This vi


Pinus cembra L.), a long-lived conifer species that inhabits the subalpine treeline ecotone in the western Alps to investigate how the genetic processes contribute to the modern-day spatial distribution. Moreover, we corroborate our findings with paleoecological records, micro and macro-remains, to infer the species’ possible glacial refugia and expansion scenarios.Genetic processes shape the modern-day distribution of genetic variation within and between populations and can provide important insights into the underlying mechanisms of evolution. The resulting genetic variation is often unequally partitioned within species’ distribution range and especially large differences can manifest at the range limit, where population fragmentation and isolation play a crucial role in species survival. Despite several molecular studies investigating the genetic diversity and differentiation of European Alpine mountain forests, the climatic and demographic constrains which influence the genetic processes are often unknown. Here, we apply non-coding microsatellite markers to evaluate the sporadic peripheral and continuous populations of cembra pine (FST based approaches, across the range of the species, situated in the northern, inner and south-western Alps. We found that genetic differentiation is substantially higher in marginal populations than at the center of the range, and marginal stands are characterized by geographic and genetic isolation due to spatial segregation and restricted gene flow. Moreover, multiple matrix regression approaches revealed effects of climatic heterogeneity in species’ spatial genetic pattern. Also, population stability tests indicated that all populations had experienced a severe historical bottleneck, no heterozygosity excess was detected, suggesting that more recently population sizes have remained relatively stable.Four genetically distinct groups were identified, with Bayesian and Our study demonstrated that cembra pine might have survived in multiple glacial refugia and subsequently recolonized the Alps by different routes. Modern-day marginal populations, at the edge of the species’ range, could maintain stable sizes over long periods without inbreeding depression and preserve high amounts of genetic variation. Moreover, our analyses indicate that climatic variability has played a major role in shaping differentiation, in addition to past historical events such as migration and demographic changes. Amounts and distributions of genetic variation among populations and across species’ ranges are results of complex interplay of gene flow, genetic drift and natural selection . GeograpFragmentation segregates a continuously distributed population into smaller, spatially isolated habitats, leading to differences in the genetic architecture within and between the resulting populations . NumerouIsolation, together with reduction in habitat quality and small population size, is predicted to result in the decline, and ultimately extirpation, of a population , 9. UsuaFragmented and isolated populations, especially those at the margin of a species’ range, often inhabit suboptimal, heterogeneous environments , 14. FluPinus cembra L.) provides an interesting model to measure genetic variation within its distribution area and investigate the drivers that contribute to genetic differentiation within and among populations. This tree is a keystone species which grows up to the treeline in high mountains from the western Alps in western Europe to the Carpathians in eastern Europe. Historically, the range of this species also encompassed the Euro-Asian boreal forests from eastern Siberia to the western Urals [P. cembra dominates forests in central valleys and massifs of the Alps, where the continental-type climate is optimal for its growth, the climate of the peripheral mountain massifs varies from oceanic in the north to Mediterranean in the south. Populations in these peripheral areas become increasingly sporadic and sparse and, ultimately, absent. Pinus cembra is a long-lived species (up to 500–1000 years) with long generation intervals, which should intuitively limit its capacity for genetic differentiation during a short period like the interglacial Holocene. However, other properties may promote genetic differentiation. For example, P. cembra has heavy seeds and strict zoochory, which prevents long-distance seed dispersal and hinders its colonization potential, but this also enhances the founder effect of initial colonizers. In practice this may have caused a bottleneck effect in isolated peripheral mountains when they were first colonized during the postglacial period , ultimately resulting in the reduced genetic diversity that is believed to exist in the present day peripheral populations  may have preserved boreal-type habitats, and thus provided previously unsuspected glacial refugia for P. cembra, in the southwestern Alps [The intraspecific genetic variability and structure of cembra pine populations in the central Alps and the Carpathians have been previously investigated, but not the genetic structure within the western Alps, which encompasses naturally fragmented forests Fig. c. We hypern Alps . TherefoPinus cembra) trees (727), from 22 populations, were genotyped with seven nuclear microsatellite markers (nSSR) from western range of the species’ in the French and Italian Alps . Generally, all populations conformed to Hardy-Weinberg equilibrium (HWE) with no significant deviations and although a few loci showed some deviation, this does not appear to be associated with null alleles or non-neutral behavior. Additionally, no loci appear to be consistently linked across all populations (data not shown).Ho) was 0.627 and expected heterozygosity (He) 0.581. Both of these indices were slightly lower for the marginal group  than the central group . The estimated inbreeding coefficient (FIS) ranged from − 0.186 for population CLP to 0.115 for population MDE. Mean values were − 0.077 for the central group and − 0.031 for the marginal group. However, significant inbreeding was only detected in the marginal populations MDE, MGI and MMO , based on 95 detected alleles at seven nSSR loci, was estimated at an overall mean of 5.162; however, the mean was higher for populations in the central group (5.350) than for populations in the marginal group (4.973). The overall mean observed heterozygosity  = 1.693, p = 0.106; Hot (20) = 1.503, p = 0.148; Het (20) = 1.445, p = 0.164) and inbreeding (FISt (20) = − 1.194, p = 0.246) between the central and marginal groups (N = 11 populations in each group). In addition, Levene’s F test indicated that the assumption of homogeneity of variances between the central and marginal groups was met (AR F (20) = 3.555, p = 0.074; HoF (20) = 2.801, p = 0.110; HeF (20) = 0.322, p = 0.576; FISF (20) = 0.515, p = 0.481).Independent sample M-ratio test indicated that all populations had experienced a historical reduction in size. The population-specific M-ratios ranged from 0.209 (MBR) to 0.356 (MRO), and were all below the threshold of M < 0.680 indicating a pronounced historical bottleneck , indicating that differentiation among populations accounts for > 6% of the total genetic variance. However, the regional comparison indicated that differentiation between central and marginal populations accounts for just 1% of the total variation , indicating the presence of population structure in P. cembra. In addition, the pairwise FST values were substantially higher for marginal populations (mean FST 0.096 ± 0.115) than for central populations (mean FST 0.027 ± 0.027) . In addition, scatterplots showing local densities of distances . Similarly, partial-Mantel tests using FST as the constant variable indicated a significant correlation with geographic distance when controlling for climatic distance , and with climatic distance when controlling for geographic distance . However, the regression coefficient was larger for climatic distance than for geographical distance . MMRR analysis resulted in a highly significant correlation between FST and the climatic distance matrix , while the contribution of geographic distance to genetic differentiation was non-significant, only if evaluated with altitudinal distance  was slightly lower in marginal populations than in central populations, these differences were non-significant. For example, mean expected heterozygosity (i.e. gene diversity) was 0.567 in marginal and 0.594 in central populations, which is not significantly different (p < 0.05). Furthermore, the overall mean He for the 22 populations was 0.581, which is slightly higher than values previously obtained for P. cembra populations using the same set of nSSR markers: 0.505 in the eastern Alps, and 0.547 [P. cembra populations appear to have preserved genetic variation and avoided large scale genetic erosion.Generally, genetic diversity is expected to decrease towards margins of species’ ranges due to factors including intensified fragmentation, isolation, restricted gene flow and genetic drift , 37. Simnd 0.547  and 0.56nd 0.547  in the CSeveral factors could promote high genetic diversity, including several common features of many conifers, such as longevity, an outcrossing mating system, wind-dispersed pollen and high fecundity . In addiFIS) values were slightly negative or around zero, indicating no heterozygote deficiency and thus no inbreeding. Additionally, mean FIS values did not differ significantly between the C and M regions. Interestingly, inbreeding depression was previously reported in Carpathian populations [We did not detect any substantial deviations from Hardy-Weinberg equilibrium (HWE) and intrapopulation inbreeding , applied to detect historical reductions (if any) in population size. First, bottleneck analysis using both SMM and TPM models detected no evidence of a significant excess of heterozygosity in any population. Moreover, the mode-shift indicator detected a normal L-shaped allele frequency distribution, as expected for populations that are near to mutation-drift equilibrium. Based on this allele frequency distribution, we can assume that present day populations are randomly mating. However, the Garza-Williamson indices  suggested that all present-day populations are remnants of a substantially larger population [M-ratios and lack of heterozygosity excess are regarded as indicative of severe, old bottlenecks, while significant heterozygosity excess is expected following weak, relatively recent (within a few millennia) bottlenecks [Larix-type forests [M-ratio, which is expected to have a long recovery time, heterozygosity excess and allele frequency distributions will recover relatively quickly when conditions allow the addition of new and rare alleles [We obtained conflicting results from the two population stability tests  between the marginal populations, especially at the northwestern and southern limits of the range. This pattern was corroborated by higher values of the gene flow parameter Nm for the marginal group than for the central group.Both the structure analysis and pairwise Our findings are consistent with the ‘central-marginal’ theory, which predicts a general increase in genetic differentiation towards margins of species’ ranges . High leP. cembra most likely survived the Last Glacial Maximum (LGM) near or within the western Alps [P. cembra populations , and one from the east though the Italian plains or foothills  that was dominant in one population of the southern Alps [nunataks emerged [Paleoecological evidence suggests that ern Alps , 35, 60,ern Alps . This scern Alps  and paleern Alps , 60. Ourcenario, . The foucenario, . Interesern Alps  at a sit emerged , 63, whi emerged  and subsP. cembra . Micro- and macro-remains indicate that the species’ abundance in the subalpine belt peaked between 9000 and 6000 BP, and in the mid-Holocene P. cembra became a common tree at high elevations in both the marginal and central western Alps, as shown (for example), by [P. cembra started to decline between 6000 and 3000 cal yr BP, depending on the favorability of its mountain habitats for human societies, in terms (for example) in distance from existing settlements, slopes, exposure or rock cover. These human-driven processes are likely to have promoted further fragmentation, segregation and decline in population sizes, especially at the margins of the species’ range. Indeed, the first postglacial fragmentation processes began towards the end of the Lateglacial period and the early Holocene , when a warming climate drove migration of cembra pine from peripheral plains and central valleys to higher altitudes, thus disconnecting marginal and central populations. Peripheral fragmented populations are often prone to severe stress because of the temporal and spatial variation of the environment, demographic stochasticity and edge effects, such as genetic isolation and drift [P. cembra populations could have separately evolved genetic variation, ultimately resulting in the observed genetic differentiation.Regardless of the postglacial migration route, it is therefore likely that present-day marginal populations were connected to central populations during the postglacial expansion of ple), by , 64, resnd drift , 14. ThuAbies alba, Pinus sylvestris and Pinus uncinata) and boreal regions  located at margins of their respective ranges are particularly vulnerable to climatic changes  theory states that if dispersal (i.e. gene flow) between regions or populations is geographically restricted, genetic differences can accumulate locally [P. cembra populations. In addition, while marginal populations in the western Alps are spaced out along the edge of the distribution, central populations are close to each other, thereby promoting higher gene flow between the stands, which further supports IBD. Although previous studies found only weak or non-significant IDB [Mantel and partial-Mantel tests detected significant correlation between  locally , 77. Ourcant IDB , 28, 29,cant IDB .FST is more strongly correlated with climatic distance than with geographical distance . Furthermore, MMRR analysis indicated a significant linear relationship between climatic distances. Together our partial-Mantel and MMRR results suggest that IBE explains most of the genetic differentiation, although IBD also contributed significantly. Alternatively, meaning that genetic divergence resides among far spaced populations inhabiting different environments with contrasting ecological conditions [However, partial-Mantel tests and MMRR analysis suggest that climatic constraints have an even stronger influence on genetic differentiation than geographical distance, in accordance with ‘isolation by environment’ (IBE) theory, i.e., that genetic differentiation increases with environmental differences –80. Our nditions , 21, 81.P. cembra populations inhabiting the western Alps, especially the marginal regions, are highly affected by regional topographical features of the Alps (such as isolated mountain massifs) that control gene flow, and microclimatic conditions that influence population genetic processes such as selection that can contribute to population divergence. Previously, the climate-topography relationship has been widely studied in the Alps and the results have highlighted the substantial effect of climate variability [P. cembra, and the results indicate that precipitation and temperature predominantly influence genetic differentiation and structure [P. cembra in the western Alps.Our results suggest that iability , 83. Mortructure –86. TogeP. cembra. While central populations are homogenous with high admixture, the fragmented marginal stands are structured, heterogeneous and geographically-genetically isolated. The highest degree of genetic differentiation is present in populations at the margins of the species’ range, partly due to their geographical distance from central populations, but mainly due to climatic heterogeneity. In addition to the demographic history, environmental differences between the central and marginal regions, and between populations, may also have contributed to current levels of genetic diversity and divergence in P. cembra. Finally, our results support a published hypothesis that P. cembra survived in multiple refugia within the western Alps.Our study suggests that both central populations, situated along the main axis of the western Alps, and marginal populations at the edge of the species’ range, can maintain stable sizes over long periods and preserve high amounts of genetic variation. Surprisingly, there was no significant partitioning of genetic variation within the investigated area and isolated populations did not develop substantial inbreeding, indicating that there is recurrent gene flow between modern-day populations through seed and/or pollen distribution. However, there is clear regional-scale genetic differentiation and structuring of Twenty-two natural cembra pine populations, consisting 11 central (C) and 11 marginal (M) populations, were selected from French and Italian Alps and classified based on their geographical position, isolation, size and habitat characteristics  following the manufacturer’s instructions. Seven polymorphic microsatellite (nSSR) loci  developed by  were useNa) and number of effective alleles (Ne), were calculated for each population using GenAlEx v.6.5 software [Np) and allelic richness (AR) were computed in R [He), observed heterozygosity (Ho), and inbreeding coefficient (FIS) were calculated using the “poppr” package [FIS values was calculated separately with FSTAT software [FIS statistics [Ho, He) and inbreeding (FIS) differed significantly between the central and marginal groups of populations we applied independent sample two-tailed t-tests in SPSS v.22 . To test whether distributions of variables met normality requirements, we applied one-sample Kolmogorov-Smirnov tests, and where necessary variables were log-transformed. Throughout the analysis, 95% confidence intervals (CI) and bootstrapping with 1000 replicates were applied. The homogeneity of variances assumption was tested with Levene’s F test for all variables.Genetic diversity indices, including the number of different alleles  and observed heterozygosity (Ho) at mutation-drift equilibrium. For this, as well as the most conservative Stepwise Mutation Model (SMM), we also applied the Two-Phase Model (TPM) allowing multiple-step mutations, which is recommended for microsatellite data [M or M-ratio), which is the number of alleles (k), divided by the allelic range (r), using Arlequin v.3.5 [M < 0.680 is regarded as indicative of a pronounced historical bottleneck [We used two approaches to investigate population stability. First, we tested for evidence of population bottlenecks by calculating the heterozygote excess relative to the number of alleles using BOTTLENECK 1.2.02 , which cite data , 98. Theite data . In a sein v.3.5 . The calin v.3.5 , and genttleneck , 101.FST matrix [FST values, between all populations , and between populations in the central and marginal groups using GenAlEx software.We used hierarchical analysis of molecular variance (AMOVA), implemented in Arlequin software, to determine the partitioning of the genetic variation among genetic groups, within populations, and among populations. We also compared central and marginal population groups using regional-level AMOVA. The significance of differences was evaluated using a permutation approach with 999 replications. In addition, a pairwise T matrix  and a PrT matrix  and “FacT matrix  packagesK value (the estimated number of genetic groups) to 1–10 with a burn-in period of 105 iterations followed by 106 Markov Chain Monte Carlo (MCMC) steps, with 20 repetitions for each run. Subsequently we used the Evanno method [K value that best explained the data. Finally, the average matrices of individual membership proportions for each population were estimated using CLUMP v.1.1.2 [To investigate the spatial genetic structure, and identify groups or subpopulations within the nSSR dataset, we implemented a Bayesian clustering approach using STRUCTURE 2.3.4 . The anao method  to detec v.1.1.2  and the DA distance matrices  in Micdistance  by bootsP. cembra. First, since marginal populations positioned separately, considerably far apart from the central population group we tested the correlation between geographical distances (kilometers) and genetic distances (Nei’s) between population pairs, IBD [FST values using the “MASS” package [P-value was calculated using a Monte Carlo simulation with 999 permutations. Second, to evaluate the relationships between pairwise FST and geographic/environmental distances we conducted a partial-Mantel test while controlling for environmental/geographic distances using the “vegan” package in R [http://www.worldclim.org/). Finally, we applied Multiple Matrix Regression with Randomization analysis  and present environmental conditions on the pattern of genetic differentiation  of irs, IBD . We geneirs, IBD  implemenirs, IBD . In addi package . The kerage in R . The Eucis (MMRR ; the R sis (MMRR . To perfis (MMRR , 116. PoAdditional file 1: Supplemental materials.


Notopterygium herb species in the QTP and adjacent regions. We sequenced 10 nuclear loci, 2 mitochondrial DNA regions, and 4 chloroplast DNA regions in a total of 72 natural populations from the 4 species, and tested the hypothesis that the population history of these alpine herbs was markedly affected by the Miocene–Pliocene QTP uplifts and Quaternary climatic oscillations. We found that the four Notopterygium species had generally low levels of nucleotide variability within populations. Molecular dating and isolation-with-migration analyses suggested that Notopterygium species diverged ~1.74–7.82 million years ago and their differentiation was significantly associated with recent uplifts of the eastern margin of the QTP. In addition, ecological niche modeling and population history analysis showed that N. incisum and N. franchetii underwent considerable demographic expansions during the last glacial period of the Pleistocene, whereas a demographic contraction and a expansion occurred for N. forrestii and N. oviforme during the antepenultimate interglacial period and penultimate glacial period, respectively. These findings highlight the importance of geological and climatic changes during the Miocene–Pliocene and Pleistocene as causes of species divergence and changes in population structure within cold-tolerant herbs in the QTP biodiversity hotspot.Historical geological and climatic events are the most important drivers of population expansions/contractions, range shifts, and interspecific divergence in plants. However, the species divergence and spatiotemporal population dynamics of alpine cold-tolerant herbal plants in the high-altitude Qinghai-Tibetan Plateau (QTP) and adjacent areas remain poorly understood. In this study, we investigated population evolutionary history of four endangered  Historical and ecological factors such as geological and/or climatic processes are strongly linked with the origin and evolution of biodiversity Coyne . The int2, stretching from the southern edge of the Himalayan Range to the northern edge of Kunlun Mountains, and from the eastern edge of Pamir and Karakoram Mountains to the eastern edge of Hengduan Mountains  sensu lato is a massive plateau with an area of about 2.5 million kmCautleya Royle and Roscoea Smith. Phylogenetic and biogeographic analyses of Spiraea L. also indicated the close relationship between species diversification and the first two QTP uplift events  D. Y. Hong  have caused important population dynamics shifts and lineage divergence of alpine plant species that are distributed in the QTP itself and adjacent areas  is an interesting model for studying species divergence and the spatiotemporal population dynamics of cold-tolerant herbal species in the high-altitude QTP and adjacent areas. In general, Notopterygium plants are insect-pollinated perennial herbs; their fruit type, a cremocarp with wings, favors dispersal by wind and water streams , whereas N. franchetii is found at much lower ones (1700–3500 m) on moist river banks and in mountain valleys. N. oviforme is distributed mainly in the Qingling mountain ranges in central and western China at elevations of 1800–2700 m. N. forrestii has a much smaller range, being only distributed in the southwestern part of Sichuan at altitudes of 2000–3000 m in forest margins and grasslands. The other two species, N. tenuifolium and N. pinnatiinvolucellatum, have very restricted distribution areas, with the former growing only in the valley meadows of Litang County in western Sichuan (at 4300 m) and the latter being distributed only in Xiaojin County in western Sichuan at about 3400 m  based on three chloroplast DNA (cpDNA) fragments and internal transcribed spacer (ITS) sequence variation. Phylogenetic analysis based on the ITS region showed that the four Notopterygium species  constituted four independently evolved genetic lineages that corresponded to four taxonomically recognized species. In contrast, the phylogenetic analysis based on cpDNA was not able to separate N. oviforme and N. franchetii  across their entire geographic distributions in the high-altitude QTP and adjacent areas. We employed maternally inherited cpDNA sequences, mitochondrial DNA (mtDNA) sequences, and biparentally inherited nuclear genes, to determine the population genetic variability, evolutionary history, and species divergence in these alpine medicinal herbal plants. We mainly addressed the following questions: (1) how did the effects of the QTP uplift contribute to genetic variation and species divergence among Notopterygium populations? (2) how did Quaternary climatic changes  affect the population demography and the distribution range of these four Notopterygium species? and (3) how did the interspecific gene flow proceed (and how was this related to the speciation processes) in the cold-tolerant Notopterygium herb species?In this study, we investigated the same four N. incisum, 27 populations of N. franchetii, 15 populations of N. oviforme, and 4 populations of N. forrestii; the details of the sampled populations are shown in Table Notopterygium species, N. incisum and N. franchetii  as follows: 5 min at 94 °C, followed by 32 cycles at 94 °C for 40 s, 40 s at the specific annealing temperature (Tm) for each marker, and 90 s at 72 °C, with a final extension at 72 °C for 10 min. The PCR products were then purified and sequenced using an ABI 3730xl genetic analyzer . We sequenced the PCR products directly on both strands for the cpDNA and mtDNA fragments. The PCR products were cloned into pGEM T-easy vectors (Promega) for the nuclear genes where heterozygous individuals existed. We randomly selected five clones for Sanger sequencing. The newly obtained sequences were submitted to GenBank under accession numbers MK312210–MK312239, MK305312–MK305813, and MK258173–MK258185. Meanwhile, we have also combined the three previously published  with the currently obtained cpDNA OG28079 (ndhF) for further population genetic analysis of Notopterygium species.Initially, 340 individuals were collected from 72 wild populations (26 populations of S), Watterson’s parameter  and diversity (Hd) based on the number of segregating sites  Arcang. , Heracleum moellendorffii Hance (LOC108225228), Pleurospermum franchetianum Hemsl. (LOC108995448), and Pleurospermum prattii H. Wolff (LOC108224998) were used as outgroups  using various statistics in DnaSP, including Tajima’s c Tajima , Fu and Notopterygium species was investigated to assess the correspondence between genotypic clustering and taxonomic delimitation based on variations in nuclear loci using STRUCTURE v2.3  in a sample of individuals. K-values were explored from 1 to 10 using 20 independent runs per K, employing an admixture model with a burn-in of 100,000 iterations and a run length of 200,000 iterations. The best K-value was estimated using the ΔK method ] reached a plateau  frequencies as variance component ratios using the locus-by-locus analysis of molecular variance  was estimated using DnaSP.In order to quantify the extent of differentiation among species, we first estimated Wright’s fixation index T Wright  and the FST as the distance measure with 10,000 permutations, to investigate the genetic variation among and within populations for each species (Excoffier and Lischer HS), total gene diversity (HT), and the values of genetic differentiation over all populations (GST), as well as the values of differentiation by considering genetic distance (NST) using PERMUT  using a Bayesian inference method. The best-fit model of nucleotide substitution was determined by jModelTest v2.1.5 based on Akaike’s information criterion and the MrBayes v3.2 program  instead of calibration points to calculate the divergence time  model (Hey and Nielsen μ), including the contemporary and ancestral effective population sizes , migration rates , and divergence times  among populations. We analyzed the species in a pairwise manner using a basic two-population model and, because of the absence of fossils or any published mutation rate of Umbelliferae, we used an evolutionary rate of μ = 6.1 × 10−9 per site per generation, to calculate the divergence times . Scenario 1 assumed ancient population growth (t2), followed by a larger and stable population size (N1). Scenario 2 assumed an ancient population growth (t2) followed by a larger but stable population size (N2), and finally a recent expansion . Scenario 3 assumed an ancient population growth (t2) and a subsequent larger but stable population size (N3), followed by a recent bottleneck . Scenario 4 assumed an ancient bottleneck event (t2) with a subsequent smaller stable population size (N4) and a recent expansion . Finally, scenario 5 assumed an ancient bottleneck event (t2) and a smaller but stable population size (N5)  in DIYABC v1.0 . For the LIG period, we used the dataset of Otto-Bliesner et al.  and our sampling location information . We did not model the distribution of N. forrestii, because the number of localities (four) was not sufficient to make reliable predictions. To avoid multicollinearity, we assessed Pearson’s correlation coefficients between all of the layers within the study area; the selection of variables from pairs or groups of highly correlated ones (r ≥ |0.9|) was done on the basis of their relative contribution to the model  and the shape of their response curves, making sure that the top most influential variables for each species were selected. Variables with neither significant contribution to the models  nor clear response  were not considered. The final selected variables that were used to detect changes in the distribution ranges of N. incisum, N. franchetii, and N. oviforme were bio1, bio2, bio3, bio4, bio5, bio12, bio14, bio15, and bio18.The sites of occurrence for We used the default parameters for MaxEnt and employed the “subsample” method (setting the number of replicates to 20), with 75% of the species records for training and 25% for testing the model. The overall model performance was assessed using the area under the curve (AUC) of the receiver operating characteristic. AUC scores range between 0.5 (randomness) and 1 (exact match), and a value above 0.9 is considered a good performance of the model Swets . As threNotopterygium species. The analysis was based on environmental space (E-space) under the PCA-env approach developed by Broennimann et al.  with 100 randomly simulated overlap values (Dsim) under a null distribution. When Dobs is greater or smaller than Dsim (with a P < 0.05), niche conservatism or divergence could be proposed, respectively. Alternatively, when Dobs is within 95% of Dsim (P > 0.05), the hypothesis of conservatism or divergence can be neither accepted nor rejected with confidence. Finally, we quantified the three niche dynamic metrics of niche unfilling, stability, and expansion.As input data, we used the same as for the ENM assessment: the nine relatively uncorrelated climatic variables (see above) and the geographical coordinates for the three a et al. . The PCASchoener . In addiNotopterygium species. The concatenated fragments of cpDNA and mtDNA comprised 1804 bp and 755 bp, respectively. The sequenced nuclear loci ranged from 214 to 842 bp, with a total concatenated length of 4580 bp after excluding gaps and missing data. In general, the levels of nucleotide polymorphism (θwt and πt) over all nuclear loci were higher in N. incisum , followed by N. franchetii (0.0048 and 0.0027) and N. oviforme (0.0043 and 0.0035), and finally those of N. forrestii (0.0021 and 0.0023) (Table πs) was ~3 times greater than that in nonsynonymous sites (πn), where each gene had a πn⁄πs ratio of < 1 in all four species (Table θwt and πt) were similar over the cpDNA and mtDNA loci  and total cpDNA diversity (HT) in N. incisum, N. oviforme, and N. franchetii were 0.227 and 0.801, 0.205 and 0.910, and 0.177 and 0.371, respectively. In terms of within-population and total mtDNA diversity, N. incisum (HS = 0.142) and N. oviforme (HT = 0.529) had the highest values among the taxa studied  and high-frequency-derived values (negative H). In addition, the average MFDM test values were not significant for all nuclear loci, which suggested no likelihood of natural selection acting on individual loci at the species level.The values of Tajima’s Notopterygium species , N. franchetii (H23–H38), N. oviforme (H27 and H39–H54), and N. forrestii (H55–H57) had 22, 15, 16, and 3 private haplotypes, respectively, whereas one haplotype (H27) was shared between N. franchetii and N. oviforme. In total, five haplotypes were identified based on the mtDNA . Moreover, AMOVA based on the cpDNA data showed that the genetic variation occurred mainly among species (63.8%), whereas most of the variation in terms of mtDNA was due to differences among populations within species   contained N. franchetii, N. oviforme, and N. forrestii individuals. A second species (N. forrestii) could be roughly assigned to a single cluster only at K = 4, whereas N. franchetii and N. oviforme individuals apparently exhibited a pattern of genetic admixture mainly between the green and yellow clusters , as N. incisum and N. forrestii formed their own groups, whereas a pattern of strong genetic admixture was found between N. franchetii and N. oviforme. The maximum-likelihood phylogenetic analysis based on the concatenated nuclear genes showed a similar topology , 3.12–15.93 Ma] based on the cpDNA variation , whereas N. forrestii had the lowest one . The effective sizes of the descendant populations of these four species were ~12–13 times larger than those of the ancestral populations  between N. incisum and N. oviforme, 6.32 Ma (90% CI: 3.35–11.7 Ma) between N. incisum and N. forrestii, 1.74 Ma (90% CI: 0.997–3.08 Ma) between N. franchetii and N. oviforme, 4.45 Ma (90% CI: 2.80–6.47 Ma) between N. franchetii and N. forrestii, and 4.39 Ma (90% CI: 2.66–6.38 Ma) between N. oviforme and N. forrestii  but also out of current Chinese borders including Myanmar and NE India  but it underwent a slight contraction. In the future, the six assayed scenarios showed similar distributions among them, with the potential species range remaining superficially unchanged compared with the present , coupled with the appearance of an area of suitability in the border region of Xinjiang with several central Asian countries , current, LGM, and LIG distributions for dia Fig. . In the tii Fig.  and S7, els Fig.  and S7. N. oviforme  of the total climatic variation occupied by the species and background areas Fig. . As the ii Table . In addies Table , althouges Table . In simice Table . The higum Table .Notopterygium species, we randomly selected some orthologous nuclear genes by comparative transcriptome analysis of N. franchetii and N. incisum  at ten nuclear loci for N. oviforme (0.0055) is higher than those for N. franchetii (0.0050), N. incisum (0.0036), and N. forrestii (0.0029). The average total nucleotide diversity is also slightly higher in N. oviforme (πt = 0.0035) than in N. incisum (πt = 0.0031), N. franchetii (πt = 0.0027), and N. forrestii (πt = 0.0023). These values are comparable to the levels of diversity in Umbelliferae herbs and higher than those in other alpine herbaceous plants, e.g., Cardamine nipponica Franch. and Sav. (Brassicaceae): πt = 0.0011–0.0015  (Umbelliferae): πt = 0.0033–0.0037  Hill (Polygonaceae): πt = 0.0007–0.0032  S. H. Fu (Crassulaceae): πt = 0–0.0016 : πt = 0–0.0002  and their high genetic diversity is consistent with the role of these mountains as Pleistocene refugia  and N. franchetii (HT = 0.371). These diversity values are also similar with the previously reported results  based on the three cpDNA fragments  , whereas the genetic variation in mtDNA is due mainly to differences among populations within species. These differences might be attributable to the limited number of informative sites and the slow evolutionary rate in the mtDNA markers  were even higher than the GST values reported for other QTP endemic alpine herbs, including Pomatosace filicula Maxim. (GST = 0.518)  . The existence of a significant geographic genetic structure for most of Notopterygium species may be associated with the heterogeneous environments inhabited by their natural populations in the QTP and the associated mountains in its eastern margin.The population genetic structure may be affected mostly by dynamics of climate and landscape across the evolutionary history of populations  started to uplift later, probably after 10 Ma . The other divergence events (with the exception of the split between N. franchetii and N. oviforme) occurred during the following two million years (Table Notopterygium was between N. oviforme and N. franchetii at 1.74 Ma (95% HPD: 0.997–3.08 Ma), which closely corresponds with the largest glaciations of the early–middle Pleistocene in this region (0.5–1.2 Ma) and the deformed uplift of the QTP at ca. 1.6–3.6 Ma . Similarly, the gene flow from N. forrestii to N. franchetii and N. oviforme was much higher than that from N. franchetii and N. oviforme to N. forrestii . In addition, the incongruent phylogenetic topologies of cpDNA vs. nuclear genes should also be noted, which could be signals of hybridization and/or backcrosses between these species  S. H. Fu  do not change significantly compared to the present time; even, a small increase can be inferred for the first two species (with a somewhat westwards expansion). Such pattern, contrary to what one might expect, has been widely reported for a series of subalpine and alpine plants of the Hengduan Mountains and the same reasons provided by the authors of such study can be advocated here: that the large adjacent mountain ranges of the QTP (toward the west) might have had an important role as an escape region  under the following Accession IDs MK312210–MK312239, MK305312–MK305813, and MK258173–MK258185.Nucleotide data have been deposited at NCBI (Supplemental material infoSupplementary material