Drymarchon couperi (Eastern Indigo Snake), a large, federally-protected species in North America that was recently divided into two species based on gene sequence data from three loci and heuristic morphological assessment. Here, we re-evaluate the two-species hypothesis for D. couperi by evaluating both population genetic and gene sequence data. Our analyses of 14 microsatellite markers revealed 6–8 genetic population clusters with significant admixture, particularly across the contact zone between the two hypothesized species. Phylogenetic analyses of gene sequence data with maximum-likelihood methods suggested discordance between mitochondrial and nuclear markers and provided phylogenetic support for one species rather than two. For these reasons, we place Drymarchon kolpobasileus into synonymy with D. couperi. We suggest inconsistent patterns between mitochondrial and nuclear DNA are driven by high dispersal of males relative to females. We advocate for species delimitation exercises that evaluate admixture and gene flow in addition to phylogenetic analyses, particularly when the latter reveal monophyletic lineages. This is particularly important for taxa, such as squamates, that exhibit strong sex-biased dispersal. Problems associated with over-delimitation of species richness can become particularly acute for threatened and endangered species, because of high costs to conservation when taxonomy demands protection of more individual species than are supported by accumulating data.Accurate species delimitation and description are necessary to guide effective conservation of imperiled species, and this synergy is maximized when multiple data sources are used to delimit species. We illustrate this point by examining  The mtDNA was sequenced for 72 specimens, while the nDNA was sequenced for a subset of 23 specimens. The authors estimated phylogenetic relationships among populations by analyzing a concatenated dataset including both mitochondrial and nuclear loci using maximum likelihood (ML) and Bayesian inference (BI) analyses. Because both analyses generated similar phylogenetic hypotheses, the authors described results from only the concatenated Bayesian analysis. However, a customary practice in phylogenetic studies is to use both mitochondrial and nuclear loci and to describe phylogenetic patterns inferred from these two components of the genome separately . –71. D. c [et al.  combinedet al. [b, ND4 and non-coding tRNAs Histidine and Serine) included 72 individuals of D. couperi  and the nuclear data included 23 individuals ; both datasets had five outgroup samples . We fit different models of nucleotide substitution, ranked them using Bayesian Information Criterion (BIC), and inferred phylogenetic trees using IQTREE v. 1.6.4 . Gene sequence data are available on GenBank with accession numbers from The use of live snakes for research was approved by Auburn University IACUC protocols  and a federal research permit from the United States Fish and Wildlife Service (TE32397A-O). All DNA samples used to generate the microsatellite data are accessioned in the Auburn University Museum of Natural History or the Florida Museum of Natural History; our microsatellite data and accession numbers for the DNA samples are available on the digital data repository ‘figshare’ . Therefore, we eliminated these loci from further analysis and retained the remaining 14 loci. We estimated an error rate of 0.05 while re-running samples. Values of hreshold ; becausenference . There wnference  and Genenference , especianference . SamplesK = 6 (SI Geneland identified clear support for K = 6 SI  across aK = 6 SI .r ≥ 0.93). The global test was significant  while the local test was not . The first PC axis explained the most variation followed by the second PC axis , the Gulf lineage , and among all samples . When comparing genetic distance within and between mitochondrial lineages, values within lineages overlapped greatly with values observed among all samples between lineages ; the nuclear locus was best fit by the Kimura [D. couperi, and the ML phylogeny from mtDNA recovered a Gulf lineage that rendered the Atlantic lineage paraphyletic  three Georgia population clusters and five peninsular Florida population clusters (Structure), and (2) three Georgia population clusters, two south Florida clusters, and one wide-spread, peninsular-Florida cluster (Geneland). Both Structure and Geneland produced fairly consistent results, particularly with respect to samples from Georgia, but neither analysis supported the Gulf-Atlantic dichotomy predicted by the two-species hypothesis. Second, and most importantly for the two-species hypothesis, both Bayesian clustering analyses and the sPCA analyses documented widespread contemporary admixture of alleles among populations along the Florida peninsula. Admixture occurred across the entire range of D. couperi and cannot be characterized as a narrow hybrid zone. In fact, the greatest degree of admixture occurred across the contact zone between the two putative species with no evidence of genetic differentiation. While our MLPE analyses found support for isolation by distance both across all samples and within each mtDNA lineage, those analyses also found a high degree of overlap in the genetic distances within and between the mitochondrial lineages used to generate the two-species hypothesis. Further, our sPCA results, which also accounted for spatial autocorrelation among samples, were consistent with isolation by distance along a north-south gradient and are not consistent with the nearly-discrete distributions and low dispersal ascribed to the Gulf and Atlantic lineages by previous authors [Our analyses found evidence of genetic structure within populations of  authors . These rD. couperi [D. couperi; however, our separate analyses of mitochondrial and nuclear gene sequence data failed to support this hypothesis. Our phylogenetic analysis of the nuclear locus NT3 recovered no support for the two-species hypothesis because this marker was identical for all individuals; instead, all phylogenetic structure of D. couperi inferred from sequence data was associated with the mitochondrial genome. Phylogenetic structure inferred from mtDNA recovered a monophyletic Gulf lineage that was nested within a paraphyletic Atlantic lineage, a result inconsistent with previously reported phylogenetic structure for D. couperi [Drymarchon kolpobasileus into synonomy with D. couperi.The two-species hypothesis for  couperi ,26 was f couperi ,26. BecaD. couperi. First, because D. couperi is federally-protected as Threatened under the Endangered Species Act [D. couperi is a single species. For example, D. couperi is now being repatriated to Conecuh National Forest, Alabama, and Apalachicola Bluffs and Ravines Preserve, Florida. Genetic stock from southeastern Georgia is primarily being used for these efforts; founder females used to generate captive stock for repatriation were sampled from Georgia and represent individuals from the three Georgia populations revealed by both Geneland and Structure. Our evaluation of population genetic structure indicates that no error of releasing the wrong historical entity  to repatriation sites is being made, as was argued by Krysko et al. [D. couperi in northern populations are thought to be dependent upon Gopher Tortoise (Gopherus polyphemus) burrows for winter refugia, likely because of milder winter temperatures experienced by populations at those latitudes [D. couperi, Georgia source populations were chosen for repatriation stock to account for potential genetic components associated with refuge-seeking behavior and cold-climate physiology.Our study has broad implications for the conservation management of cies Act ,29, effoo et al. . Rather,atitudes –40,79,80D. couperi life history and spatial ecology allows us to generate hypotheses for the discordant patterns of phylogenetic and population genetic structure observed here. Drymarchon couperi, particularly males, have extremely large home ranges and great vagility [D. couperi gene flow is consistent with other reptile systems for which life-history strategies generate contrasting patterns of gene flow among populations. First, female philopatry of Loggerhead Sea Turtles (Caretta caretta) causes structuring of mtDNA in the Atlantic Ocean, but high male dispersal drives significant nuclear gene flow among populations [D. couperi is similar to other reptile species in that life history provides explanations for why phylogeographic patterns from mtDNA are predicted to be inconsistent with historical and/or contemporary patterns of nuclear DNA.Our knowledge of vagility ,39. Malevagility . Given tvagility . This liulations . Second,ulations . Last, aulations –87; therulations , a patteulations ,25. ThusD. couperi adds to a growing number of examples of southeastern North American organisms that appear, based on modeling of one or a few genetic loci, to represent species that are distinct from other mainland counterparts, but for which microsatellite or similar data demonstrate substantial contemporary gene flow. Burbrink and Guiher [Agkistrodon piscivorus) in peninsular Florida and the mainland that speciation must have occurred between those two regions, a hypothesis immediately contested by data from Strickland et al. [Macrochelys temminckii) from the Apalachicola River and adjacent rivers to be a distinct species, despite microsatellite data from Echelle et al. [A. piscivorus, the wetland habitats occupied by this species are abundant and extensive throughout the range of the species, which limits opportunity for population isolation within the core of the geographic range. Agkistrodon piscivorus are known to move among wetlands [M. temminckii [D. couperi. We point to papers that carefully meld phylogenetic and population genetic analyses [The North American Coastal Plain is a global biodiversity hotspot , and pend Guiher  estimated et al.  who deted et al.  describee et al.  that aree et al. . While awetlands , providiwetlands ,92. Simimminckii  indicatemminckii . Thus, lanalyses –100 as eanalyses . When seanalyses ), the utS1 FigDrymarchon couperi estimated through the Bayesian clustering algorithm Structure with (A) K = 6, (B) K = 7, (C) K = 9, and (D) K = 10. The y-axis is the proportion of individual ancestry for each cluster; in the x-axis, number represents county where sample was collected. County names for each number are shown in K = 8.Bar plots of population genetic clustering of (TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 FigI (right) plotted against the variance for each axis.The eigenvalues (left) for each axis where positive values indicate global structure and negative values indicate local structure, and the Moran’s (TIFF)Click here for additional data file.S4 FigDrymarchon couperi using a Delaunay triangulation connection network demonstrating the spatially lagged scores from the first (A) and second (B) axes. Atlantic lineage samples are displayed using circles while Gulf lineage samples are displayed using squares. Samples with more extreme values/colors are more genetically differentiated.Additional results of spatial principle components analysis for population genetic data of (TIFF)Click here for additional data file.S5 FigI plotted against the variance for each axis.The left figure shows the eigenvalues for each axis where positive values indicate global structure and negative values indicate local structure and the right figure shows the Moran’s (TIFF)Click here for additional data file.S1 TableSee  for an e(DOCX)Click here for additional data file.S2 TableThe names of loci are as in .(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableEach metric is estimated at four different thresholds of cluster membership.(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


This study illustrates the difficulties in assessing the impacts on Ne in populations that have suffered demographic crashes but have recovered rapidly and received gene flow, particularly in species with long generation times in which genetic time lags are longer. This work provides a starting point and baseline information for genetic monitoring of the GKNP elephants.Savannah elephant populations have been severely reduced and fragmented throughout its remaining range. In general, however, there is limited information regarding their genetic status, which is essential knowledge for conservation. We investigated patterns of genetic variation in savannah elephants from the Greater Kruger Biosphere, with a focus on those in previously unstudied nature reserves adjacent to Kruger National Park, using dung samples from 294 individuals and 18 microsatellites. The results of genetic structure analyses using several different methods of ordination and Bayesian clustering strongly suggest that elephants throughout the Greater Kruger National Park (GKNP) constitute a single population. No evidence of a recent genetic bottleneck was detected using three moment-based approaches and two coalescent likelihood methods. The apparent absence of a recent genetic bottleneck associated with the known early 1900s demographic bottleneck may result from a combination of rapid post-bottleneck population growth, immigration and long generation time. Point estimates of contemporary effective population size (N Conservation threats, such as illegal hunting, habitat fragmentation and confinement to small isolated areas, contribute to the decline of animal populations and can lead to their extinction ,2,3. TheLoxodonta africana) is a charismatic flagship species that continues to decline in numbers due to poaching and habitat destruction  and Ne (T = 3000) 90% CR = ) (In the ) = 951) . When di overlap . However11,697]) .Bottleneck under the SMM or TPM, except those using the latter model with 78% single-step mutations (which were not significant at α = 0.05), revealed significant heterozygosity deficiency over all loci in all the three data sets analysed (APNR and KNP separately and pooled together). In contrast, assuming the IAM, most of the tests indicated significant heterozygosity excess over all loci , APNR (0.905) and the two combined (0.904) were higher than the respective critical values cM for the four different combinations of Θ and mutational processes tested, except in the case of the KNP assuming Θ = 0.1, 90% single-step mutations and Δg = 3.5 (cM = 0.871)  . This laIn this study, we conducted a conservation genetic assessment of savannah elephants in the Greater Kruger Biosphere, with a focus on those in previously unstudied nature reserves (APNR) adjacent to KNP. Our aims were: i) to characterize the genetic status of the APNR elephant population; ii) to examine genetic structure across the Greater Kruger; iii) to provide estimates of local effective population size; and iv) to investigate demographic history and in particular to test for genetic bottlenecks, which might be expected given the known history of South African elephants.Bottleneck and M ratio analyses in this and other studies [M ratio method in particular might have been expected to be able to detect a possible recent Ne reduction in the Kruger elephants, as it can be powerful for detecting bottlenecks when they were severe and lasted several generations, the population had made a demographic recovery and the pre-bottleneck population size was large [Bottleneck and M ratio tests clearly did not support the hypothesis of a genetic bottleneck may be due to the rapid post-bottleneck growth of the Kruger elephant population [e [M ratio method has less power [Bottleneck and M ratio tests [Bottleneck and M ratio tests [e when they occurred ≤ 10 generations ago [e between samples from the Kruger area from the 1920s–1930s (after the demographic contraction but before the population rebound) and the mid-19th century [The sample sets from the APNR and from across the KNP showed similar and moderate mean observed microsatellite heterozygosity . These values are also comparable to those obtained in a previous study  for the  studies ,30. The as large ,152. Thepulation ,30 that ation [e ,153, a sss power ,152; in ss power  but see ss power . Concomiss power  and possio tests . Also, gio tests . Simulations ago ,120,126.ions ago . Lastly,ions ago ,139. A cst and related measures between APNR and KNP were significant based on permutation, their values were very small (0.004–0.011) and the 95% bootstrap CIs included zero. Moreover, Mantel tests of IBD, using different inter-individual genetic distances, were either not significant or yielded very low correlation coefficients (XYr ≤ 0.02). Overall, these different results indicate a lack of genetic structure across the GKNP. It has been shown that exact tests using microsatellite data can be very powerful in detecting even very small departures from panmixia [The exact G-test indicated significant genetic differentiation between APNR and KNP but the results of genetic structure analyses using several different methods of ordination and Bayesian clustering strongly suggest that elephants throughout the GKNP constitute a single population. Concordantly, although estimates of Fpanmixia ,157 but panmixia , which cst and related statistics) between APNR and KNP were significant for females but not for males. The weaker fine-scale genetic structure observed in the SAC analyses using only the APNR adult samples from the second (dry) season may potentially be due to the contraction of elephant home ranges in the dry season [The SAC analyses including both sexes indicated positive and statistically significant fine-scale genetic structure up to 2–3 km. Fine-scale structure can exist even when dispersal is restricted in only one sex ,159, whiy season ,160. They season . In any y season ,57. For y season . Fine-scy season ,17,57 anMigraine and VarEff also did not support the hypothesis of a significant Ne reduction in the recent past. The Migraine analysis using the ‘OnePopVarSize’ model yielded a non-significant signal of a strong ancient decrease in Ne. In turn, VarEff inferred a monotonic decline in Ne during the last ~50,000 years, with two steeper drops 12750–8500 years ago and 4250–3400 years ago, resulting overall in noticeably lower point estimates of contemporary Ne; however, the 90% CR of the Ne estimates overlapped substantially through time, even between today and 50000 years ago. Thus, neither Migraine nor VarEff analyses could find unambiguous and statistically significant evidence for past demographic changes. The non-significant signal of a strong ancient population contraction inferred by the ‘OnePopVarSize’ model of Migraine may reflect the fact that the Kruger elephant population was part of a historical metapopulation [Migraine may have moderate false contraction detection rates if the Ne of demes is neither small nor large (a few hundred) and migration rates between demes are low (≤1%), so that the genealogy of a sample taken from a single deme is closer to that expected in a Wright-Fisher population [e, sampling a single deme resulted in low contraction detection rates; these increased when levels of gene flow were higher [VarEff is also sensitive to gene flow and the demographic trend inferred by VarEff might reflect recurrent immigration at low to moderate rates  than for the APNR alone (200–400) but the respective 95% CIs overlapped substantially. To our knowledge, these are the first available estimates of Ne for Kruger’s elephants. Given our relatively limited sample size for the KNP, a better sampling of this area should be obtained for Ne estimation. The LD estimates of Ne may be underestimated due to several factors, all of which may have had an impact. Firstly, the underlying model of the LD method assumes discrete generations, with the estimated Ne referring to the parental generation of the sample [b) that produced the cohort(s) from which the sample was taken or of a quantity intermediate between Nb and Ne [e from LD when generations overlap [e, which in several species studied was about 20–30% when the ratio of adult life span (=maximum age – age at maturity + 1) to generation length is in the range 2–3 [e based on a demographic model assuming a population of constant size [e estimates to be affected by Ne in generations prior to the one sampled [e because the signal from current (reduced) Ne is strong relative to the signal from larger Ne in previous generations but if the population has recently increased, as is the case of the Kruger elephants, LD estimates of Ne can be biased downwards for a few generations, with duration and magnitude of bias proportional to the severity of the previous population reduction and relative increase in Ne [IS, potentially substantially underestimating Ne even for low values of FIS , that i02–0.05; ,171 that0.05; [e . It has 5; [e [e . Neverthstimates ,172.e estimates to those obtained by the LD and EPA methods were inferred from analyses in Migraine (~700), VarEff (~400) and FΘ (~600), in all three cases assuming an average mutation rate of 10−3. The analyses in VarEff suggest that this rate fits the data better than a rate of 10−4 and it agrees well with reported overall average mutation rates for mammalian tetranucleotides [−3 can be considered as in the upper bound estimate of the average microsatellite mutation rate in mammals and the typical average rate, in particular for dinucleotide repeats, is probably somewhat lower [−4, a figure often used in the literature , VarEff (~800) and FΘ (~1200). On the other hand, since the microsatellites used in this study were selected for their polymorphism, their mutation rates may actually on average be higher than the mean mutation rates observed in microsatellite panels unfiltered for polymorphism [e estimates from these different methods are comparable, given their differences in model assumptions and estimation algorithms. For instance, mutation-drift equilibrium is assumed by FΘ but not by VarEff. However, all three methods assume an isolated population. It is well known that even low levels of immigration may strongly influence genetic diversity in local populations and this can lead to biased estimates of local Ne by Θ-based Ne estimators [e that even a small amount of immigration can cause has been illustrated for the island model [e estimates could be biased upwards. Nevertheless, the magnitude of this bias may be limited. The genetic structure analyses did not consistently indicate the presence of genetically divergent immigrants that could have strongly affected the Ne estimates. Moreover, the effect of immigration on estimates of local Ne may be modest if allele frequencies are similar between the focal and source demes, so that immigrants have less effect on allele frequency changes [e using methods that account for migration , would morphism . In any timators ,134,166.nd model . Howevernd model  and thennd model . This ge changes ,176. A fferences ,178).e for the GKNP were 500–700. These estimates are close to values of Ne that have been proposed as absolute minimum for the long-term maintenance of quantitative genetic variation and evolutionary potential of a population (Ne = 500–1000) [e to population size is important because if this ratio is very small (<0.1), then genetic drift can be strong although the population is large [e in the population of breeders that produced the sample; therefore, to estimate Ne/N, we used as denominator the mean of the estimated number of adult elephants (Na) [e/N using the mean census size (Nc) over the same period in the denominator. The ratio Ne/Nc is directly proportional to the total reproductive value of a population [e, the values obtained for Ne/Na and Ne/Nc were 0.27–0.37 and 0.10–0.14, respectively. These ratios, however, may be overestimated because, when temporal trends in N are evident , it may be more appropriate to use the more recent values of N to estimate the denominator of Ne/N [a and Nc for the period 1960–1990, the Ne/Na and Ne/Nc ratios were 0.19–0.26 and 0.07–0.10. Either way, the Ne/Nc ratios are similar to the mean (0.11) of 92 comprehensive empirical estimates of Ne/N reviewed by Frankham [e/N of 0.23 based on 31 correctly linked Ne/N ratios but these only included ectotherms. The main reasons for the low Ne/N ratio in the Kruger elephants should be a fluctuating population size, variance in reproductive success and a high ratio of adult life span to age at maturity [e/Nc ratio is not unusually low [e did not change much in the meantime and given a present census size of 21,000 individuals, of which 8,000 are estimated to be adults [e/Nc and Ne/Na ratios could be about 0.02–0.03 and 0.06–0.09, respectively. The long generation time of elephants may slow the rate of genetic drift but there may only be a delay in the reduction of inbreeding Ne and the restoration of genetic variation by mutation will be very slow [e [e are needed to better understand the effects on it of this complex and dynamic history [The LD and EPA point estimates of contemporary N00–1000) ,182,183.00–1000) , can giv00–1000) . Consideis large . The LD nts (Na) ,186 in tnts (Na) , over thnts (Na) . We alsopulation  and givepulation . Based o of Ne/N . Using tFrankham  reportedmaturity ,191. Theally low  but it iery slow ,192. Foc slow [e , can be  slow [e . Given t history ,136,137.e of recent dramatic changes in population size can only be understood through follow-up analyses in subsequent generations of the target population.We consider that conservation genetic assessments should be as thorough as possible, covering genetic diversity, fine- and population-scale genetic structure, genetic bottleneck testing, demographic history inferences and estimates of effective population size. Only then is it possible to obtain an accurate and detailed picture of the genetic status of populations, clues to causes of this status and a solid basis for further investigations and conservation and management plans. This study also illustrates the difficulties in determining the conservation genetic status of populations that have suffered catastrophic demographic collapse but have recovered rapidly and received gene flow, so that the genetic impacts of this complex history in a short time interval are difficult to ascertain. This is further complicated in species with long reproductive lifespan and generation time, since in these the effects on the genetic dynamics of a population of the different demographic events in the last generations may actually reflect history over the last century or more and time lags are expected to be long. In such cases, we argue that the full extent of impacts on genetic variation and Ne or deviation from mutation-drift equilibrium [e are at the lower end of the range of values that have been suggested as the critical minimum for the long-term maintenance of genetic variation and adaptive potential [e/N may be quite low (<0.1). Clearly, in the future there is a need for genetic monitoring [e and Ne/N, ideally also using genomic data [For reasons discussed above, such as a rapid population recovery following the demographic bottleneck during the 19th–20th century transition, immigration from neighbouring populations and a long generation time, the Kruger elephant population does not appear to have significantly lower levels of genetic diversity than other Southern African elephant populations and different methods of demographic inference also did not detect consistent signs of a recent reduction in Nilibrium ,30. Howeotential ,181,183 nitoring  and analmic data . This ismic data , and submic data ,197 and mic data .


Extended parental care is a complex reproductive strategy in which progenitors actively look after their offspring up to – or beyond – the first juvenile stage in order to maximize their fitness. Although the euarthropod fossil record has produced several examples of brood-care, the appearance of extended parental care within this phylum remains poorly constrained given the scarcity of developmental data for Palaeozoic stem-group representatives that would link juvenile and adult forms in an ontogenetic sequence.Fuxianhuia protensa from the early Cambrian Chengjiang Lagerstätte in South China. Our data demonstrate anamorphic post-embryonic development for F. protensa, in which new tergites were sequentially added from a posterior growth zone, the number of tergites varies from eight to 30. The growth of F. protensa is typified by the alternation between segment addition, followed by the depletion of the anteriormost abdominal segment into the thoracic region. The transformation of abdominal into thoracic tergite is demarcated by the development of laterally tergopleurae, and biramous walking legs. The new ontogeny data leads to the recognition of the rare Chengjiang euarthropod Pisinnocaris subconigera as a junior synonym of Fuxianhuia. Comparisons between different species of Fuxianhuia and with other genera within Fuxianhuiida suggest that heterochrony played a prominent role in the morphological diversification of fuxianhuiids. Functional analogy with the flexible trunk ontogeny of Cambrian and Silurian olenimorphic trilobites suggests an adaptation to sporadic low oxygen conditions in Chengjiang deposits for F. protensa. Finally, understanding the growth of F. protensa allows for the interpretation of an exceptional life assemblage consisting of a sexually mature adult alongside four ontogenetically coeval juveniles, which constitutes the oldest occurrence of extended parental care by prolonged cohabitation in the panarthropod fossil record.Here, we describe the post-embryonic growth of F. protensa, offers insights on the macroevolutionary processes involved in the diversification of this clade, and contributes towards an improved understanding of complex post-embryonic reproductive ecology in Cambrian euarthropods.Our findings constitute the most detailed characterization of the post-embryonic development in a soft-bodied upper stem-group euarthropod available to date. The new ontogeny data illuminates the systematics, trunk segmentation and palaeoecology of The online version of this article (10.1186/s12862-018-1262-6) contains supplementary material, which is available to authorized users. Leanchoilia illecebrosa .  Figs. ,-d, or 13F. protensa indicate that overall body size  is positively correlated with the number of trunk tergites, and that there is a substantial degree of body size variation among later ontogenetic stages  the primary effects responsible for controlling body size in F. protensa will only become possible through the additional input of completely articulated specimens corresponding to early juvenile stages.Morphometric data obtained from previously published and our newly documented specimens of ges Fig. . This isges Fig. . This caF. protensa allows the interpretation of a complex fossil specimen that consists of one stage 29 individual preserved in close association with four stage 8 juveniles  [F. protensa juveniles and adult specimens reflect ontogenetic change, as demonstrated by the gradual broadening of the thoracic tegites during stages 24 and 25  , 30, and 25 Fig. .F. protensa indicates that this taxon underwent anamorphic post-embryonic development  salopiensis [S. salopiensis alternates between three or four segments through part of its meraspid stage [F. protensa in which the abdomen count varies between 12 and 13 tergites.The ontogeny of nt Figs. , 12, 13,ilobites , 31, 32 ilobites , 33, 34.ilobites . Althougilobites , the bipopiensis , 36; botid stage , 36, andF. protensa as recorded by the morphometric data , but becomes much more variable during late ontogeny resulting in mature adults (holaspids) with a polymorphic trunk segment count [A. konincki favours a developmental model where the total number of thoracic segments was determined during early ontogeny, and once this maximum was reached, growth continued epimorphically in the holaspid stage. The small sample size of measurable juveniles available for F. protensa  compared to the meraspids, namely those that have between 18 to 22 thoracic tergites . Ho in ref. ; thoracithropods , 37, 38.thropoda , 21, 24,thropoda , 12, 29.nsa Fig. , the samF. protensa evokes comparison with that of Cephalocarida, a relatively poorly understood but highly ecologically specialized clade of meiofaunal pancrustaceans [F. protensa  Hongjinshao Formation at Chenggong. This specimen is highly significant, as it unequivocally demonstrates the presence of reduced anterior tergites underneath the head shield, and traces of multiple limbs associated with the thoracic tergites. Although the posterior half of the Chenggong euarthropod is not preserved, its morphology and size fit comfortably within those of stage 8 or 9 juveniles of F. protensa . In this context, the overall exoskeletal dorsal morphology and size of Pisinnocaris sp. most closely resemble those of the aglaspidid euarthropod Tremaglaspis [Pisinnocaris sp. within the Chengjiang, we refrain from formalizing this interpretation until more specimens of this problematic taxon become available for study and comparison with the type material of Tremaglaspis.Xu  describeure 3 in ). Pisinnes Figs.  and 10. aglaspis –48, poteF. protensa is characterized by the addition of tergites from a posterior growth-zone, as well as the corresponding increase in size  Yu’anshan Member of the Chiungchussu Formation of the Kunming region in South China . The matAdditional file 1:Table S1. Morphometric and locality data of articulated individuals of Fuxianhuia. (XLSX 15 kb)


Ciona robusta, during recent range expansion to the harsh environment in the Red Sea.Adaptive evolution is one of the crucial mechanisms for organisms to survive and thrive in new environments. Recent studies suggest that adaptive evolution could rapidly occur in species to respond to novel environments or environmental challenges during range expansion. However, for environmental adaptation, many studies successfully detected phenotypic features associated with local environments, but did not provide ample genetic evidence on microevolutionary dynamics. It is therefore crucial to thoroughly investigate the genetic basis of rapid microevolution in response to environmental changes, in particular on what genes and associated variation are responsible for environmental challenges. Here, we genotyped genome-wide gene-associated microsatellites to detect genetic signatures of rapid microevolution of a marine tunicate invader, The Red Sea population was significantly differentiated from the other global populations. The genome-wide scan, as well as multiple analytical methods, successfully identified a set of adaptive genes. Interestingly, the allele frequency largely varied at several adaptive loci in the Red Sea population, and we found significant correlations between allele frequency and local environmental factors at these adaptive loci. Furthermore, a set of genes were annotated to get involved in local temperature and salinity adaptation, and the identified adaptive genes may largely contribute to the invasion success to harsh environments.Ciona robusta within a few generations. Such a rapid microevolutionary process is largely responsible for the harsh environmental adaptation and therefore contributes to invasion success in different aquatic ecosystems with largely varied environmental factors.All the evidence obtained in this study clearly showed that environment-driven selection had left detectable signatures in the genome of The online version of this article (10.1186/s12862-018-1311-1) contains supplementary material, which is available to authorized users. Dissecting evolutionary dynamics of environmental adaptation in organisms is one of the fundamental research themes in evolutionary biology and ecology, particularly in the context of rapid global climate change and distributional shift of organisms in the past five decades –3. SpeciRapid microevolution is usually associated with changes of genetic variation in a population, such as changes of allele frequency and generation of new alleles through mutations at functional genes , 13, 14.Ciona robusta, is a notoriously fouling organism with wide salinity tolerance ranging from 12‰ to 40‰ and temperature varying from 3 °C to 30 °C [C. robusta is usually considered to be native to Northwest Pacific and has invaded the coasts of Mediterranean Sea, Atlantic, Pacific and Oceania oceans [C. robusta, such as the growth rate, life span and spawning time, vary depending on local environmental factors, particularly the water temperature and salinity [Biological invasions are promising ‘natural’ experiments that provide opportunities to study rapid microevolutionary processes , 26, 27.to 30 °C . C. robua oceans –33. Mosta oceans , 35. In salinity ; howeverC. robusta has been reported in the Red Sea, a representative tropical region which is well known for its high temperature and salinity [C. robusta was detected in the Eilat marina in 2015 [C. robusta reported so far. Genetic analyses based on mitochondrial DNA suggested that the Red Sea population might be introduced from the Mediterranean Sea through the Suez Canal [C. robusta populations, the optimal temperature range was 14–23.4 °C [A recent introduction of salinity . The fir in 2015 . Many enez Canal . Since tez Canal , 38. Forez Canal –39. Howeez Canal , mainly ez Canal , 42. For–23.4 °C ; howeverC. robusta in the Red Sea and genotyped genome-wide gene-associated microsatellites to reveal the genetic signatures of rapid microevolution in the harsh environment. In order to cover various temperature and salinity gradients globally, we included five other populations  from Lin et al. [In this study, we sampled the recently established population of n et al. . At the p < 0.01), suggesting significantly different environmental conditions in the Red Sea. The Red Sea population had significantly higher temperature and salinity than the other populations  was significantly lower than that in the other five global populations , especially two Pacific populations . In comparison, the highest genetic differentiation was found between the Red Sea population and one Pacific population collected from New Zealand . Individuals from the Red Sea population were assigned into the same cluster formed by three other populations , whereas two Pacific populations  in ARLEQUIN, including 17 loci (11.6%) for directional selection and 15 loci (10.3%) for balancing selection , indicating strong effects of local environmental factors on genetic variation. A total of 41 loci were associated with temperature, including 22, 6 and 33 loci for annual average water temperature (AveT), the highest monthly average water temperature (MaxT) and the lowest monthly average water temperature (MinT), respectively. A total of 57 microsatellite loci were detected with the strong correlation with salinity, including 40, 30 and 56 loci for annual average water salinity (AveS), the highest monthly average water salinity (MaxS) and the lowest monthly average water salinity (MinS), respectively. Altogether, 28 loci showed significant associations with both temperature and salinity. Furthermore, a total of 19 loci detected by ARLEQUIN or BAYESCAN approach were significantly associated with environmental factors , MaxS  and MinS  was performed to test the correlation between allelic frequency and environmental factors. Interestingly, a total of 93 alleles 9.5%) at 70 microsatellite loci 47.9%) showed significant association with at least one of the six environmental parameters  with the functions of DNA damage response and Hsp90 protein binding. Indeed, many other genes play crucial roles in various important biological processes related to responses to environmental stresses, such as cell apoptosis, fatty acid metabolic process, and ion transport .Deep insights into the rapid adaptive changes during range expansion will facilitate our understanding on species’ distribution ranges and responses to future environmental changes , 18, 45.ns Table ; Fig. 1,C. robusta samples from the same sampling site in 2015 suggested that the Red Sea population might be introduced from the Mediterranean Sea, where C. robusta were detected much earlier in the end of nineteenth century [C. robusta in a short timescale. Similar strong and rapid adaptation was also detected in other marine species. For example, significant reproductive isolation was detected within 13 generations among introduced salmon populations in novel environments [For newly established populations, gene flow and some events such as genetic bottleneck, genotype sorting and drift are likely to influence population genetic structure –48. Give century , 44. In  century . Althoug century , 52. Herronments . Bernardronments  reportedC. robusta could be as narrow as 9.8 kb, suggesting the soft selective sweep with standing genetic variation. However, recent studies documented that novel mutations could provide opportunities, especially for small founding populations, to rapidly adapt to the new environments [C. robusta exhibits a high level of mutation rate, accompanied with relatively high fecundity, rapid growth rate and short life history, which may provide new mutations for natural selection [Rapid local adaptation during biological invasions primarily depends on two sources of genetic variation: standing genetic variation and new beneficial mutations , 14, 54.ronments . C. robuelection , 57. ThuC. intestinalis in high temperature treatment. In addition, this gene can cooperate with Cullin-type ubiquitin ligase to get involved in spermatogenesis and fertilization in C. intestinalis [trans-Golgi network and the plasma membrane was required for salinity stress adaptation [C. intestinalis includes at least three pathways: the canonical pathway, the planar cell polarity (PCP) pathway and the Wnt/Ca2+ pathway based on the KEGG database . Multiple studies have revealed that the Wnt signaling pathway played a crucial role in many biological processes, such as cell proliferation [C. robusta under high temperature and salinity in laboratory conditions can contribute to our understanding which adaptive genes and related biological processes and pathways play a key role in rapid microevolution.Understanding the function of adaptive genes can provide insights into the genetic basis of rapid microevolution in changing environments , 44. In stinalis , 62. Anoaptation , 64. Thiaptation , 64. Twoferation , embryogferation , and bioferation . A recenferation . Thus, fC. robusta is generally considered as a temperate-water species, it has currently successfully established in a Red Sea marina since 2015. Rapid local adaptation is a crucial mechanism for invasive species to cope with novel environments and/or environmental stresses during range expansion. Although it is well-known that phenotypic plasticity is a crucial mechanism for temporal adjustment during range expansion, while microevolution is more important for successful population establishment in a long-term perspective. Interestingly, the results obtained in our study suggest that rapid microevolution could occur within a few generations. We detected a large number of adaptive loci and candidate genes responsible for temperature and salinity adaptation in C. robusta, and these genes are candidate loci for further studying adaptation dynamics based on single genes or gene networks using C. robusta as a model. Our study confirms the rapid microevolution hypothesis that rapid local adaptation is largely responsible for the harsh environmental adaptation and therefore contributes to invasion success in different aquatic ecosystems with largely varied environmental factors.Although C. robusta samples were collected from the Eilat marina  on March 2018 (experiencing ~ 10 generations after introduction). All collected individuals were immediately preserved in absolute ethanol at 4 °C for further genetic analyses. Total genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen). To confirm the species identification based on morphology, one mtDNA fragment cytochrome c oxidase subunit 3–NADH dehydrogenase subunit 1 (COX3-ND1) [OX3-ND1) , was useHere, we used a total of 146 genome-wide gene-associated microsatellite makers , 70 to ghttp://www.nodc.noaa.gov/OC5/SELECT/woaselect/woaselect.html), including three metrics of SST: annual average water temperature (AveT), the highest monthly average water temperature (MaxT), the lowest monthly average water temperature (MinT) and three metrics of salinity: annual average water salinity (AveS), the highest monthly average water salinity (MaxS), the lowest monthly average water salinity (MinS). We used a non-parametric Kruskal-Wallis test to assess differences in temperature and salinity between the Red Sea population and the others in SPSS v.18. In addition, a principal component analysis (PCA) with the ‘prcomp’ package in program R was performed to illustrate the high salinity and temperature in Red Sea population.Water temperature and salinity are among the most crucial environmental variables in marine ecosystems, as these factors largely influence survival, development and many physiological processes of marine species. The Red Sea was characterized with a high level of temperature and salinity . TherefoAR), observed heterozygosity (HO), expected heterozygosity (HE) and inbreeding coefficient (FIS) using FSTAT v. 2.9.3.2 [AR) between the Red Sea population and the others. Recent bottleneck effect was tested using program Bottleneck v.1.2.02 [Population genetic diversity was measured by allelic richness , with 100,000 Markov chain Monte Carlo iterations preceded by 100,000 burn-in, and we performed ten independent runs for each K value. In addition, we further conducted Bayesian analyses to assess population assignment between the Red Sea population and Mediterranean-Atlantic population group. The optimal K value was identified with STRUCTURE HARVESTER [We assessed population genetic differentiation with pairwise RE v.2.3 . For theARVESTER , 76. TheARVESTER  was usedq-value < 0.05).In this study, two different population differentiation approaches were used to search for microsatellite loci putatively under selection. Firstly, we screened all microsatellite loci with the fdist2 method under a hierarchal island model in ARLEQUIN . This anIn order to identify loci that have strong association with particular environmental variables and further confirm that the outlier loci were the outcome of selection resulting from local environment factors, the environmental association analysis (EAA) was conducted in MatSAM v.2 . MatSAM C. robusta KH assembly of Satou et al. [www.ensembl.org). In addition, we chose 20 kb up- and down-stream of each adaptive outlier loci as a selective sweep window against the C. robusta KH assembly as suggested by Lin et al. [To annotate gene functions of the putatively adaptive outlier loci, we firstly located these loci on the chromosomes based on the u et al.  at Ensemn et al. , and then et al. , 83 weren et al. .Additional file 1:Figure S1. (a) Temperature and (b) salinity data in six populations. Figure S2. PCA plot on environmental factors for six Ciona robusta populations. Figure S3. Outlier detection in 146 microsatellite loci in ARLEQUIN. Purple line represents 99% confidence intervals; red and green lines represent 95 and 5% confidence intervals, respectively. Figure S4. Manhattan plot showing the distribution of FST-based outliers detected by BAYSECAN across different chromosomes of Ciona robusta KH assembly. The q-value of given locus is the minimum false discovery rate (FDR) at which this locus may become significant. Table S1. Protein MB21D2 (MB21D2 gene). (ZIP 1091 kb)


Triple negative breast cancer (TNBC), which is typically lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), represents the most aggressive and mortal subtype of breast cancer. Currently, only a few treatment options are available for TNBC due to the absence of molecular targets, which underscores the need for developing novel therapeutic and preventive approaches for this disease. Recent evidence from clinical trials and preclinical studies has demonstrated a pivotal role of signal transducer and activator of transcription 3 (STAT3) in the initiation, progression, metastasis, and immune evasion of TNBC. STAT3 is overexpressed and constitutively activated in TNBC cells and contributes to cell survival, proliferation, cell cycle progression, anti-apoptosis, migration, invasion, angiogenesis, chemoresistance, immunosuppression, and stem cells self-renewal and differentiation by regulating the expression of its downstream target genes. STAT3 small molecule inhibitors have been developed and shown excellent anticancer activities in in vitro and in vivo models of TNBC. This review discusses the recent advances in the understanding of STAT3, with a focus on STAT3’s oncogenic role in TNBC. The current targeting strategies and representative small molecule inhibitors of STAT3 are highlighted. We also propose potential strategies that can be further examined for developing more specific and effective inhibitors for TNBC prevention and therapy. Due to y needed –7. More y needed , 9. The 2), a coiled-coil domain (CCD) for binding with interactive proteins, a DNA binding domain (DBD), a linker domain, a SRC homology 2 (SH2) domain for phosphorylation and dimerization, and a C-terminal transactivation domain (TAD) [The STAT family of transcription factors is comprised of seven members with high structural and functional similarity, including STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6 , 11. Allin (TAD) . STAT3 win (TAD) , 14. Ovein (TAD) , 16. TheConsidering that there is an unmet medical need for TNBC treatment and innovative therapeutic agents are urgently required, an in-depth understanding of the roles of STAT3 in TNBC will facilitate the development of STAT3-targeted therapeutics and pave the way for a novel TNBC treatment approach. In this review, we focus on the recent findings related to STAT3’s role in TNBC as well as STAT3 inhibitors and current targeting strategies. We also discuss other potential strategies for developing new STAT3 inhibitors for TNBC treatment., Src and Abl [The classical STAT3 signaling pathway that is activated through the binding of cytokines or growth factors to their corresponding cell surface receptors has been extensively reviewed –18. Here and Abl . The pho and Abl , 22. Wit and Abl , 18.Fig.STAT3 mRNA, thus down-regulating STAT3 expression in TNBC cells [STAT3 is also highly expressed in some normal tissues and organs, including the bone marrow, peripheral nervous system, and digestive tract and plays a physiological role –25. In tBC cells . Due to BC cells . Herein,The oncogenic potential of STAT3 has been widely recognized through its involvement in regulating the expression of genes related to cancer cell proliferation, anti-apoptosis, migration, invasion, angiogenesis, chemoresistance, immune suppression, stem cell self-renewal and maintenance, and autophagy  has been found to inhibit breast cancer cell proliferation and induce apoptosis, independent of ER expression [Several studies have demonstrated that STAT3 promotes cell proliferation and inhibits apoptosis in TNBC by increasing the expression of target genes, including survivin, c-Myc, cyclin D1, B-cell lymphoma-2 (Bcl-2), and B-cell lymphoma-extra large (Bcl-xL) . In TNBCcription , 44, whicription . In addicription . Converscription . Wwox alpression . A furthpression .STAT3 also promotes TNBC cell proliferation and inhibits apoptosis through the crosstalk with SET and MYND domain 2 (SMYD2) and nuclear factor-kappa B (NF-κB) . SMYD2 iThe role of STAT3 in promoting cell migration and invasion has been linked to the upregulated expression of matrix metalloproteinase 2 (MMP2), MMP9, TWIST, and Vimentin . As discRecent studies reported that several upstream regulators of STAT3 signaling are involved in TNBC metastasis. Wwox blocks JAK2-STAT3 interaction and inhibits STAT3 phosphorylation, therefore repressing STAT3-driven TNBC metastasis . G proteThe pro-angiogenic role of STAT3 has been partially attributed to the upregulation of vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), hepatocyte growth factor (HGF), and basic fibroblast growth factor (bFGF) via STAT3 transactivation . A recenIt has frequently been observed that blocking STAT3 signaling enhances the anticancer activity of chemotherapies in TNBC cells in vitro and in vivo, which endorses a critical role of STAT3 in chemosensitivity of TNBC –61. SeveMIR181A1 promoter but also recruits MSK1 (mitogen- and stress-activated protein kinase-1) and stabilizes its binding to MIR181A1 promoter, facilitating the transactivation [MIR181A1 transactivation for sensitizing cells to chemotherapy and preventing metastasis has also been validated in a TNBC orthotopic model.STAT3-mediated microRNA (miRNA) expression is emerging as a mechanism for regulating chemoresistance in TNBC. Niu et al. found that miR-181a expression is increased in TNBC due to doxorubicin treatment and contributes to acquired resistance and metastasis of this disease through repressing the expression of its target gene Bax (Bcl-2-associated x protein) . Furthertivation . The effSTAT3 is also involved in hypoxia-induced chemoresistance in TNBC . Under hPD-L1 promoter and activate its transcription [Recent findings have established STAT3 as a powerful regulator of tumor-mediated immune suppression , 71. STAcription . Anothercription . Moreovecription , 74.Early studies on STAT3 signaling disclosed an important role in stem cells self-renewal and differentiation . The inc, Leptin are also involved in the constitutive activation of the STAT3 signaling pathway. Leptin and its long form of leptin receptor (LEPRb) are enriched in breast cancer tissues and promote cell proliferation, migration, and angiogenesis [Except for cytokines and growth factors, adipokines, e.g.ogenesis . Recentlogenesis . Moreoveogenesis . HN1L isogenesis .Autophagy is capable of regulating STAT3 phosphorylation status in TNBC cells . MaycottAbundant evidence has suggested that STAT3 may be a promising molecular target for TNBC therapy . Various, carfilzomib [Ganoderma lucidum extract [, deguelin [The majority of STAT3 inhibitors have been identified to target the upstream regulators of STAT3 signaling. STAT3 activation is often initiated through the binding of cytokines and growth factors to their corresponding cell surface receptors. Therefore, small molecules and natural products that are able to inhibit IL-6 secretion and production, e.g.filzomib , manuka filzomib , bazedoxfilzomib , 90, and extract  or supprdeguelin , picrasideguelin , canthardeguelin , and sildeguelin  have shodeguelin . FurtherAs discussed earlier, several protein tyrosine kinases, such as JAK2 contribute to STAT3 phosphorylation and activation in both receptor-dependent and/or receptor-independent manners. JAK2 inhibitors, including silibinin  and ganoAs one of the major client proteins of heat shock protein 90 (HSP90), STAT3 can be degraded through inhibiting HSP90. Two deguelin-derived HSP90 inhibitors, termed compound 57 and L80 have been observed to inhibit STAT3 expression and phosphorylation by interacting with the C-terminal ATP-binding pocket of HSP90 and blocking its function , 105. BoDue to advances in the understanding of the structural biology of STAT3, small molecule inhibitors have been developed to directly bind to STAT3 and inhibit its activity. Currently, many small molecule inhibitors have been designed to target the SH2 domain and block its phosphorylation, dimerization, and nuclear translocation. Several STAT3-binding small molecule inhibitors that are under preclinical and clinical investigations have shown excellent efficacy in TNBC cells in vitro and in vivo.Recently, a dual-luciferase assay-based screening of 1563 compounds for STAT3 inhibitors was performed, leading to the identification of Bt354 . FurtherInula helenium L. have been shown to suppress tumor growth in vitro and in vivo by inhibiting STAT3 phosphorylation and decreasing the expression of the downstream target genes, including cyclin D1, c-Myc, and Bcl-2 [Rhus coriaria was also discovered to inhibit angiogenesis, tumor growth and metastasis in TNBC models in vitro and in vivo by repressing STAT3 phosphorylation and STAT3-mediated VEGF expression [Except for the STAT3-binding small molecule inhibitors that we discussed above, a number of natural products and their derivatives were found to inhibit STAT3 phosphorylation and/or nuclear translocation without affecting the upstream regulators. Sesquiterpene lactones, which are enriched in the hexane fraction from nd Bcl-2 . Anotherpression . Moreovepression , eupalinpression , galiellpression , and urspression  have shoOf note, several approved drugs have shown potent inhibitory effects on pSTAT3 and may be repositioned as anticancer drugs. Niclosamide, an FDA-approved anthelmintic drug was identified as a potent STAT3 inhibitor. A recent study demonstrated that niclosamide not only inhibits TNBC cell viability but also sensitizes TNBC cells to ionizing irradiation (IR) by blocking IR-induced STAT3 phosphorylation and activation . FlubendRecent studies have disclosed that targeting STAT3 acetylation may be a potential therapeutic approach for treating cancer. SH-I-14, a newly synthesized carbazole was shown to inhibit STAT3 phosphorylation through increasing SHP-1 expression . A folloVEGF promoter and STAT5 to IGF-1R (IGF-1 receptor) promoter and repressing the expression of VEGF and IGF-1R [Nanog and MMP2 promoters, respectively [STAT3 induces the expression of its downstream targets through binding to DNA and activating the transcription. Therefore, inhibition of STAT3-DNA binding has been considered as a promising strategy to develop targeted cancer therapies. Several STAT3-DNA binding inhibitors have been developed and shown potent anticancer efficacy in TNBC cells. Methylsulfonyl-methane (MSM), a dietary supplement was found to inhibit TNBC cell viability and induce apoptosis by blocking the DNA binding abilities of STAT3 to d IGF-1R . Consideectively , 129. Ho, NCOA/SRC1a, APE/Ref-1, and CBP/p300) and repressing its transcriptional activity, 2) modulating the binding of STAT3 with other interactive proteins  that regulate its activity and stability, and 3) developing STAT3-targeting PROTACs (proteolysis targeting chimeras) for promoting STAT3 ubiquitination and proteasomal degradation. Because most of the small molecule STAT3 inhibitors have been developed to inhibit its phosphorylation and activation but not affect the protein stability, long-term treatment of these inhibitors may result in the compensatory activation of other signaling pathways, finally causing drug resistance. Therefore, small molecules, such as PROTACs that can induce STAT3 protein degradation may be used more efficiently in combination with current inhibitors for cancer therapy.As discussed above, several strategies (as shown in Fig. TNBC is still a treatable but incurable disease with complex genetic heterogeneity. The STAT3 oncogene is overexpressed and constitutively activated in TNBC and is associated with the high metastatic risk and poor survival outcomes. Moreover, STAT3 not only acts as a transcription factor to activate the expression of its downstream target genes but also localizes to mitochondria and regulates its functions, then regulating the various aspects of TNBC cells. Many STAT3-targeted therapies have been successfully developed and shown efficacy in preclinical models of TNBC in vitro and in vivo; several STAT3 inhibitors even enter clinical trials and are currently under investigation in various human cancers, including TNBC. In addition to its role in cancer cells, STAT3 also plays a pivotal role in the immune system. Indeed, STAT3 inhibitors have been found to suppress tumor cells but also boost immune cell responses. Therefore, the STAT3 oncogene is a promising target for TNBC prevention and therapy.Of note, targeting STAT3 alone has shown excellent anti-TNBC activities in preclinical settings. However, TNBC has been reported to harbor multiple genetic alterations, including STAT3 overexpression and constitutive activation which contribute to the initiation, progression, metastasis, and drug resistance of this disease. Therefore, STAT3 inhibition combined with other targeted therapies may be more effective in treating TNBC. Considering that STAT3 plays a crucial role in chemoresistance, the combination of STAT3 inhibitors with other chemotherapies may exert synergistic effects in treating TNBC. Therefore, further studies are warranted to demonstrate the preventive and therapeutic efficacy of STAT3 inhibitors alone or in combination with chemotherapy and/or other targeted therapies in clinical studies. Moreover, new targeting strategies, i.e. inducing the degradation of STAT3 protein through PROTAC or inhibiting the binding of STAT3 to its co-activators and other interactive proteins can be examined, which may lead to more specific and effective inhibitors for TNBC prevention and therapy.


Lepista nuda is a popular wild edible mushroom that grows in China. In this study, we used ISSR and SRAP molecular markers to analyze the genetic diversity of 72 samples of L. nuda from eight populations in Northeast China. In total, six ISSR primers and five pairs of SRAP primers that produced clear and polymorphic banding profiles were selected for assessing L. nuda genetic diversity. The results revealed a high level of genetic variation among the 72 samples (94.4% polymorphism) but a low degree of gene flow among the populations. Among L. nuda populations, genetic distance was not correlated significantly with geographic distance. The antioxidant activity of the samples from each population was also tested and the result showed that all the selected samples had more than 60% DPPH scavenging activities. Nonetheless, the antioxidant activity diversity is not coincident with both the genetic diversity and the geographic distribution. The results indicate that ISSR and SRAP molecular markers are useful for studying the genetic diversity of L. nuda. The results also suggest that L. nuda populations in Northeast China require protection. Lepista nuda (Bull.) Cooke is a popular edible mushroom in China [Lepista nuda can be distinguished from other species by its lilac to purple-pink pileus, its white to pale-pink spore print, and its distinctive odor [L. nuda extracts can inhibit the in vitro formation of biofilms by multi-drug-resistant bacteria [in China , and natin China , 3. Lepiive odor . This fuive odor  and polyive odor . In addibacteria .L. nuda have been extensively collected for their commercial value, and the habitat of the species has been frequently destroyed [L. nuda. An analysis of ITS sequences of 66 samples of L. nuda revealed a low level of genetic diversity, but these samples were collected from only one site in China [In recent years in China, wild specimens of estroyed , suggestestroyed  and perhin China .Auricularia auricula-judae (Bull.) Quél. [Lentinula edodes (Berk.) Pegler [Pleurotus citrinopileatus Singer [Pleurotus eryngii (DC.) Quél. [Pleurotus pulmonarius (Fr.) Quél. [Tricholoma matsutake (S. Ito & S. Imai) Singer [L. nuda has obvious antioxidant activity [L. nuda in Northeast China. In addition, relationship between antioxidant capacity and genetic diversity of each population was studied.DNA fingerprinting methods have been widely used to study the genetic diversity of fungi. These methods, including the use of ISSR (inter-simple sequence repeats)  and SRAP.) Quél. , Lentinu) Pegler , Pleurots Singer , Pleurot.) Quél. , Pleurot.) Quél. , and Tri) Singer . Antioxi) Singer , 20. Preactivity , 22. In Lepista nuda is neither protected nor endangered in the sampled areas, and all samples were collected by researchers following current Chinese regulations. None of the sampled locations are privately owned or protected by law.Lepista nuda were drew by R Statistical Software [A total of 72 samples (basidiomata) were collected from eight sites in Northeast China from September 2012 to August 2015 . SamplinSoftware  and packSoftware . The samSoftware , 26 for Lepista nuda. Primers ITS5/ITS4 [Genomic DNA was extracted from the dried tissue blocks using a modified cetyltrimethyl ammonium bromide (CTAB) method . The extTS5/ITS4  were useTS5/ITS4 . The ampet al. [Clitocybe favrei (GU234009) and C. vibecina (GU234049) were used as the outgroup. The data matrice for ITS sequences analysis was produced. Bayesian analysis was conducted with MrBayes v.3.1.2 [Based on the results of Alvarado et al. , a totalet al.  and Cluset al. . Two spe v.3.1.2 . The besavrei GU2009 and C2, and 2.5 U of Taq DNA polymerase), 1 μL of primer, 1 μL of template DNA, and 10.5 μL of ddH2O. The amplification included an initial denaturation at 94°C for 2 min; followed by 37 cycles of 35 s at 94°C, 45 s at 42–60°C, and 90 s at 72°C; and a final extension of 10 min at 72°C. For SRAP analysis, the reaction mixtures contained 12.5 μL of 2×Power TapPCR Master Mix, 1 μL of each primer, 1 μL of template DNA, and 9.5 μL of ddH2O. The amplification included an initial denaturation at 94°C for 5 min; followed by 35 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C; and a final extension of 10 min at 72°C. All of the PCR products were separated by electrophoresis on a 1.5% agarose gel with 1 × TBE buffer at 80 V for 3 h. The gels were stained with ethidium bromide and photographed under ultraviolet light . The analyses were repeated at least twice, and molecular weights were estimated using a DNA marker .A total of 14 primers that produced clearly distinguishable and reproducible fragments were selected and used in this study for ISSR and SRAP analyses . All of Image Lab software  combined with visual assessment was used to score DNA bands on the gels as “1” for present or “0” for absent, which generated a binary matrix. To increase the number of genetic loci, the DNA bands produced by ISSR and SRAP markers were combined. Genetic diversity analysis was performed using POPGENE version 1.31 . Componeet al. [Control—ASample)/AControl, where A Control is the absorbance of the control reaction  and A Sample is the absorbance of the test compound.One represented sample was randomly selected from each location and the dried mushrooms were ground into a fine powder by a crushed mill, and then were through a 0.15-mm sieve for further analysis. Mushroom powders were extracted by using distilled water for 3 hours at 75°C. After centrifuging, the extracting solution was tested the antioxidant activity. The scavenging activity of the water extracts from each sample on DPPH radicals was measured according to the method of Cheung et al.  with somL. nuda formed a monophyletic clade with strong support (PP = 0.96). The result confirmed that the samples used in this study were the species of L. nuda , and the percentage of polymorphism was as high as94.4%. The results showed that the average polymorphic loci of each SRAP primer pair was much higher than some other species (22 loci for L. nuda vs. 12 loci for Lentinula edodes [Pleurotus citrinopileatus [L. nuda (15 loci) were similar to the other species, such as L. edodes (14 loci) [P. citrinopileatus (8 loci) [L. nuda.SRAP molecular marker were originally developed for gene tagging in e genome . SRAP ha strains , then it strains , 17, 18.pileatus ). The av[L. nuda  loci werL. nuda in Northeast China is weak. In comparison, the gene flow of Pleurotus eryngii var. tuoliensis was found to be 1.794 [L. nuda has at least two possible explanations. First, the species usually grows during the rainy season, and rain might limit the spread of spores. Second, habitat fragmentation in Northeast China is likely to reduce the exchange of spores between populations.Generally, gene flow was low when Nm value was less than 1.0 . In thisbe 1.794 . The weaL. nuda populations was not correlated with their geographical distance. For example, the geographic distance was greatest between the YC population and BX population (909 km), but the genetic distance was highest between the MDJ and the CBS populations (0.3731). The results also showed that all of the genetic distances between the CBS population and the other populations were very high. A similar phenomenon was reported for the plant Liparis japonica in Northeast China [L. nuda populations and might thereby promote the development of genetic differences unrelated to geographic distance.The genetic distance between eight st China . This last China , 45. A rL. nuda currently has a wide geographical distribution in Northeast China, the species may be endangered. On the one hand, the populations are small, and the habitat is increasingly fragmented. On the other hand, the results of this study indicate that gene flow among populations is low. We therefore suggest that measures are needed to protect the genetic resources of the species. We suggest that harvesting of L. nuda in Northeast China should be limited. It might also be useful to collect and store basidiomata as a gene bank for the species. The introduction of basidiomata from other places could increase the number of genotypes. Finally, domestication of L. nuda could reduce the harvesting of wild populations.Although In the present study, the samples of each population showed antioxidant activity to some extent, while the trend of antioxidant activity diversity is not consistent with either the genetic diversity or the geographic distribution of each population. Samir and Mathilde  obtainedS1 FigThe node support is indicated by Bayesian posterior probabilities on branch. Only support values greater than 0.60 in Bayesian are shown.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


Oenocarpus bataua var. bataua which are located within four biogeographic regions in northwestern South America: (a) Chocó rain forest, (b) Amotape‐Huancabamba Zone, (c) northwestern Amazonian rain forest, and (d) southwestern Amazonian rain forest. We hypothesize that these clusters developed following three genetic diversification events mainly promoted by Andean orogenic events. Additionally, the distinct current climate dynamics among northwestern and southwestern Amazonia may maintain the genetic diversification detected in the western Amazon basin. Genetic exchange was identified between the clusters, including across the Andes region, discarding the possibility of any cluster to diversify as a distinct intraspecific variety. We identified a hot spot of genetic diversity in the northern Peruvian Amazon around the locality of Iquitos. We also detected a decrease in diversity with distance from this area in westward and southward direction within the Amazon basin and the eastern Andean foothills. Additionally, we confirmed the existence and divergence of O. bataua var. bataua from var. oligocarpus in northern South America, possibly expanding the distributional range of the latter variety beyond eastern Venezuela, to the central and eastern Andean cordilleras of Colombia. Based on our results, we suggest that Andean orogenesis is the main driver of genetic structuring and diversification in O. bataua within northwestern South America.Andean orogenesis has driven the development of very high plant diversity in the Neotropics through its impact on landscape evolution and climate. The analysis of the intraspecific patterns of genetic structure in plants would permit inferring the effects of Andean uplift on the evolution and diversification of Neotropical flora. In this study, using microsatellite markers and Bayesian clustering analyses, we report the presence of four genetic clusters for the palm  Samples were obtained mostly from primary and secondary forests, although some sampling was done in pastures where deforestation had left few individuals standing. A particular case was the Ecuadorian Amazon locality of Chiriap (a Shuar indigenous community) where 2.3O. bataua or related (species within the tribe Euterpeae) were screened for amplification within our sampling  Genetrop facilities in Montpellier, France. DNA extraction was done using the DNeasy plant mini kit (Qiagen) and its concentration was analyzed with a Thermo Scientific NanoDrop™ ND‐100 spectrophotometer. A total of 35 microsatellite markers (simple sequence repeats) of SSR loci were amplified in two separate multiplex polymerase chain reactions (PCR): the first included four loci , while the second included three . Forward primers were fluorescently labeled using VIC, NED, 6‐FAM, and PET dyes. PCR amplifications were performed in a final volume of 10 μl as follows: 4 μl of DNA template (5 ng/μl), 5 μl of 2× Multiplex Mastermix, and 1 μl of 10× primer mix (containing 3 or 4 pairs of primers at 0.2 μM each primer). Thermal cycling conditions consisted of an initial denaturation step for 15 min at 96°C followed by 30 cycles of denaturation for 30 s at 94°C, annealing for 90 s at 56°C, and elongation for 90 s at 72°C. A final 10 min extension step at 72°C was added. For each PCR product, a final dilution factor of ~1/300 was obtained using 0.15 μl of size marker 500 LIZ (GeneScan™) and water. The final product of each PCR was then analyzed in a 3500 Genetic Analyzer sequencer (Applied Biosystems™), and the resulting chromatograms were examined using GeneMapper software v3.7 (Applied Biosystems™).2.4For all the sampled localities and individuals, we performed a linkage disequilibrium test for each pair of loci using the software Genepop v4.2 with default parameters , Structure was run under the default model of ancestry and population intercorrelation  without prior information about the samples’ geographic origin. Five independent Markov chain Monte Carlo (MCMC) runs were performed using 104 burn‐in generations followed by 105 sampling generations, K ranging = 1–10. From these data, the ΔK statistic, developed by Evanno, Regnaut, and Goudet (K).In order to determine a major population structure in d Goudet , was comO. bataua, a spatial Bayesian clustering analysis was performed using Geneland software  and differentiation values (FST) among pairwise clusters were also obtained from Geneland. Based on the Bayesian clustering analyses, we obtained a hierarchical AMOVA in order to understand how the genetic variation is partitioned between varieties, between populations within varieties, and within populations, using the software Arlequin v3.5.2.2  in order to infer the population structure within this variety. Next, we used the mean F value computed by Structure to explore the dispersal history among genetic clusters of var. bataua. Under population admixture and correlated allele frequencies, the program returns an F value  that describes the degree of genetic differentiation of a certain cluster from a hypothetical ancestral population . Additionally, phylogenetic relationships between clusters were depicted to determine the sequence of divergence between the clusters. A neighbor‐joining (NJ) tree was constructed in MEGA7  previously identified as var. bataua by the first Bayesian clustering analyses, and each locality was treated as a population. In order to determine how genetic diversity was spatially distributed, allelic richness (A) was calculated using the rarefaction procedure implemented in FSTAT v2.9.3  to check whether genetic diversity decreased as we moved away from a center of diversity. Expected heterozygosity (He), observed heterozygosity (Ho), and the inbreeding coefficient (FIS) for each population were estimated with Arlequin.At this level, we worked with 18 localities  and after (0.175) correction for null alleles.Seven of the 21 pairs of loci were detected in linkage disequilibrium  and western  populations. An exception to this pattern was the relatedness of the San Francisco population (Colombia), with the individuals identified as var. oligocarpus by the Bayesian analyses  with posterior probabilities of membership of 1. Despite the clear divergence between the two varieties, the FST value obtained was moderate (Table p < 0.01**).Based on the Δs Figure , even thte Table . Geneticbataua, the Structure analysis identified a peak at K = 4 , Amotape‐Huancabamba Zone (AMO), northwestern Amazonian rain forest plus northwestern Bolivia (NWA), and southwestern Amazonian rain forest (SWA)  the SWA cluster was formed by all samples from southwestern Amazonia along the Ucayali and Madre de Dios River basins in Peru. The average F values obtained for the clusters in Structure suggest that the NWA and SWA clusters experienced the lowest genetic drift after the ancestral population subdivided, whereas CHO experienced the highest. The highest genetic differentiation  between clusters was found between CHO compared to SWA and CHO compared to AMO, whereas the lowest was between NWA and SWA  Figure . Based os Figure  and the s Figure  showed. WA Table , showing3.2NWA cluster, had the highest genetic diversity  in southwestern Ecuador. The population of San Buenaventura in Bolivia, located at the southern limit of our sampling, showed low diversity in terms of allelic richness. No significant regression was found between A and altitude (p = 0.062) or between A and the distance from each population to the most diverse population ; however, their p values were in the threshold of statistical significance. As all trans‐Andean (west of the Andes) populations  showed values of A under the regression line  was detected between A and the distance from each population to Intuto, whereas no significant correlation was found between A and altitude (p = 0.054).The lowland Amazonian populations of Intuto and Jenaro Herrera, located near Iquitos in Peru within the ty Table . Other AFIS value was observed in Villaseca. High and significant FIS values were also found in the Andean populations Shuaro and Rioja in Peru and for the Amazonian population of San Buenaventura in Bolivia, where deforestation processes have occurred recently and were observed during sampling. An unexpected moderate inbreeding coefficient was observed in Yasuní in western Ecuador, located within a protected national park. A particular case with excess of heterozygosity came from Chiriap in Ecuador.The inbreeding coefficient tended to be low with few exceptions Table . The hig44.1bataua  and SWA correlated with the historical division of the northern and southern foreland basins. After the basins overfilled, no geographical barriers have been present in the western Amazon basin  proposed by Haffer  to the O. bataua that have experienced strong genetic divergence. The geographic distribution of var. oligocarpus coincided with the location of the Guiana Shield, which is made up of exposed Precambrian rock that formed about 2 Ga. The geology of this area allowed for the development of a forest with a different floristic composition compared to the forests of western and central Amazonia  in 1864 as a distinctive species from O. bataua , which spatially coincides with the high palm and tree species diversity found in these forests  harbors the highest Oenocarpus species diversity, with six described species  was lower when compared with other lowland palms such as Euterpe edulis  due to population structuring , located in an isolated and heavily deforested area. This population also represents the edge of the species’ ecological range, growing in semideciduous forest; therefore, its conservation would secure this ecological adaptation of 4.4O. bataua var. bataua that promoted the structuring of four genetic clusters in northwestern South America. In order to determine the effective role of the Andean orogenesis as a driver of intraspecific genetic diversification in the region, we encourage the realization of dated phylogenies or coalescence analyses in other Neotropical plant species with similar distribution to O. bataua in a similar or bigger geographical extent as this study. Based on our results, we can suggest that Andean uplift may be the main driver of genetic diversification for this palm in northwestern South America, showing the influence of this orogenic event on the evolution and diversification of Neotropical flora. As gene flow is present among the identified genetic clusters of var. bataua, it seems improbable that one of these clusters would diversify as a different intraspecific entity (variety) unless gene flow becomes restricted. Addressing the role of dispersal corridors in the genetic structure of widely distributed plants would provide more accurate information about the influence of the Andes in the diversification of Neotropical flora. Additional studies are necessary to understand whether populations with high inbreeding coefficients are under the Wahlund effect. Furthermore, we confirmed the existence of two varieties with strong divergence within the wide distribution of O. bataua whose divergence is not related to Andean orogenesis. We strongly recommend a more detailed exploring of genetic diversification in the northern Andes in Colombia and Venezuela in order to have a better understanding of the influence of the Andes within this region. Furthermore, we suggest the study of the flowering phenology of the populations within the NWA and SWA clusters to determine whether differences in climate are altering flowering times and gene flow patterns in these populations.We detected three events of genetic diversification within R. M., J.‐C. P., and H. B. developed the study; S. E., R. M., and J.‐C. P. made the sampling; J.‐C. P., R. B., M. M. R., and B. M. provided logistics for sampling and obtained collection permits; J.‐C. P. provided logistics for laboratory work; S. E. performed laboratory work and did the statistical analyses. S. E. and R. M. wrote the manuscript; R. M., H. B., R. B., M. M. R., B. M., and J.‐C. P. corrected the manuscript.https://doi.org/10.5061/dryad.1r4p8.Microsatellite and geographic data are available at  Click here for additional data file.


Microtus longicaudus), northwestern deermice (Peromyscus keeni), and dusky shrews (Sorex monticola), and then tested hypotheses derived from Species Distribution Models, reconstructions of paleoshorelines, and island area and isolation. In all three species, we identified paleoendemic clades that likely originated in coastal refugia, a finding consistent with other paleoendemic lineages identified in the region such as ermine. Although there is spatial concordance at the regional level for endemism, finer scale spatial and temporal patterns are less clearly defined. Demographic expansion across the region for these distinctive clades is also evident and highlights the dynamic history of Late Quaternary contraction and expansion that characterizes high‐latitude species.Although islands are of long‐standing interest to biologists, only a handful of studies have investigated the role of climatic history in shaping evolutionary diversification in high‐latitude archipelagos. In this study of the Alexander Archipelago (AA) of Southeast Alaska, we address the impact of glacial cycles on geographic genetic structure for three mammals co‐distributed along the North Pacific Coast. We examined variation in mitochondrial and nuclear loci for long‐tailed voles ( LimitedDuring the Last Glacial Maximum , ice covered much of North America Figure , restricAs glaciers receded and sea levels rose, the islands of the AA became increasingly fragmented, although the sequence of fragmentation is complex due to isostatic rebound  tends to prefer more open herbaceous habitats, P. keeni (an omnivore) typically occurs in forest and scrub habitats, and S. monticola (an insectivore) usually occupies forested and non‐forested habitats with dense ground cover . Sampling covered 44 localities across Southeast Alaska and Haida Gwaii. All recognized subspecies  were also sequenced. Additionally, we used GenBank sequences representing 41 M. longicaudus, and 18 P. maniculatus  and three nuclear loci per genus, chosen based on variability observed in previous work and preliminary assessment within this study (Microtus: Protein C‐est‐2 (ETS2), β‐fibrinogen (FGB), and Recombination Activating Protein 1 (Rag1); Peromyscus: β‐fibrinogen (FGB), interphotoreceptor retinoid‐binding protein (IRBP), and zona pellucida 3 (ZP3); Sorex: Alcohol Dehydrogenase 2 (ADH2), Apolipoprotein B (ApoB), and β‐fibrinogen (FGB); Supporting Information Appendix We extracted total genomic DNA to a final concentration of 50 ng/µl using either Omega Bio‐Tek  E.Z.N.A. or standard salt extraction , aligned in mega v5.2 . Climate suitability was limited by the low median threshold values over all replicates , LGM (~21 kya) , 14, and 10 kya , while all rates for phased nuclear loci were estimated and assigned strict clocks. Each run consisted of random start trees with a Species Tree: Yule process prior and piecewise linear and constant root population size model with MCMC chain lengths of two billion iterations, sampling every two million. tracer,awty,logcombiner, and treeannotator were used as in the beast analysis , and selection and expansion statistics Tajima's  within cyt 33.1M. longicaudus, 0.777 ± 0.080 for P. keeni, and 0.754 ± 0.082 for S. monticola across replicate runs, and all performed significantly better than random. No model clamping was detected. Suitable climate conditions for all three species in Southeast Alaska were present across all four temporal periods , followed by P. keeni (97.8%), and S. monticola (76.4%). Nuclear haplotype diversity for M. longicaudus ranged from 12.4% to 18.2%, P. keeni 6.5%–39.1%, and S. monticola 4.1%–33.8%. Selection was not detected through HKA tests.All loci across all species had varying levels of polymorphism and genetic diversity Table , with mtM. longicaudus reveal a single supported clade containing both Island and Northern cyt b clades. The species tree for Peromyscus lacked support for the Island clade (P. keeni). Species tree for S. monticola supports the Island clade and the Southern clade, and also indicates that S. monticola is monophyletic.The multilocus species trees Figure  for M. lMicrotus longicaudus populations on Forrester and Chichagof Island each have unique haplotypes for ETS2  from a small ancestral population. In contrast to sudden growth in M. longicaudus, P. keeni and S. monticola show a steady increase in Ne. Additionally, growth occurs post‐LGM within S. monticola and concurrent with the LGM for both M. longicaudus and P. keeni. Relative to mainland clades, high genetic diversity values for the Island clades of M. longicaudus, P. keeni, and S. monticola likely are the cumulative result of independent genetic drift within each island, with each of these fragmented island populations having relatively small Ne.Overall, measures of genetic diversity for the Island clades were low for all three species  between contemporary populations representing the Island and Northern cyt b clades, rather than post‐glacial expansion of mainland populations into the AA , consistent with deglaciation of these areas Figure . All thrM. longicaudus, P. keeni, and S. monticola, but the idiosyncratic influence of mutation rates, selection and drift, combined with independent population‐level responses to historical climate and variable pathways are potentially reflected in incongruent aspects of the phylogeographic patterns. Nuclear loci for all three species lack consistent signatures of geographic structure across the region  indicate that M. longicaudus and S. monticola likely persisted in coastal refugia along the southern extent of the AA. In contrast, P. keeni, likely persisted in a combination of northern and southern refugia within the AA, as suggested by the distinct cyt b lineage for the northern islands and differentiation across the southern islands. Unique haplotypes for P. keeni individuals from the islands of Haida Gwaii also present the option of coastal refugia near Haida Gwaii  extend beyond SE Alaska, but divergence dates, net genetic distance, genetic diversity, and expansion statistics, as well as models of refugial migration for P. keeni  in the region  and black bear (Ursus americanus), where the distribution of the island lineages now extend far beyond the boundaries SE AK (Dawson et al., Diversification of fauna in coastal refugia and then subsequently on the Alexander Archipelago also raises the possibility that continental diversity in northwestern North America has been influenced through recolonization of the mainland from islands (e.g., Filardi & Moyle, n Figure . The dis5Historical climate and coastal refugia shaped genetic structure of species of the high‐latitude Alexander Archipelago. Multiple lines of evidence suggest all three small mammals have paleoendemic lineages in the region, a finding consistent with other recent work on endemics in the region such as the Prince of Wales/Haida Gwaii ermine (Colella et al., Assessments of genetic structure across an array of species in complex landscapes, such as this coastal archipelago which experienced dynamic sea level fluctuations (e.g., Dolby et al., None declared.This research was part of the doctoral dissertation of Y.E.S. She is interested in the effects of historical climate on contemporary small mammals, particularly in the high latitude islands of Southeast Alaska. Y.E.S and J.A.C. developed the ideas; S.O.M. and J.A.C collected the specimens; Y.E.S. collected the genetic data in the laboratory of J.A.C; Y.E.S. analyzed the data; and Y.E.S., S.O.M., E.P.L. and J.A.C contributed to the writing. Click here for additional data file.