Herbivory on plants with high δ15N values cannot be ruled out based on this method, however. Here we apply an approach using the δ15N values of individual amino acids from collagen that offsets the baseline δ15N variation among environments. The analysis yielded strong signals of reliance on plants for Romanian cave bears based on the δ15N values of glutamate and phenylalanine. These results could suggest that the high variability in bulk collagen δ15N values observed among cave bears in Romania reflects niche partitioning but in a general trophic context of herbivory.Heavy reliance on plants is rare in Carnivora and mostly limited to relatively small species in subtropical settings. The feeding behaviors of extinct cave bears living during Pleistocene cold periods at middle latitudes have been intensely studied using various approaches including isotopic analyses of fossil collagen. In contrast to cave bears from all other regions in Europe, some individuals from Romania show exceptionally high  The paradox of the cave bear is that their diet has been said to be herbivorous despite their large body sizes while extant herbivorous Carnivora species are smaller7. After their divergence from the brown bear lineage 1.2–1.6 million years ago, cave bear populations showed substantial morphological and genetic variability and multiple forms have been recognized8, although their taxonomic status and the relationships among them continue to be debated9. The possible causes of the extinction of these bears are also intensively debated, involving climate change, human impacts, and (lack of) flexibility in feeding behavior15. Understanding cave bear feeding behavior is therefore important as it might give insights into the extinction of this species, and also it could be relevant for the conservation of extant, herbivorous carnivoran species that are under threat of extinction .Bears represent the largest terrestrial members within the Carnivora alive today and the vast majority of them have carnivorous or omnivorous feeding habits. Until around 25,000 years ago, the coldest period in the Pleistocene, additional, now extinct bear species were living23. This is also the case for stable carbon and nitrogen isotope analyses (δ13C and δ15N) on collagen extracted from bone/teeth31. Relatively low δ15N values of most of these bears so far indicate their highly-plant-dependent feeding habits with possible exceptions for some groups in today’s Romania that exhibited relatively high δ15N values33. The δ15N value of bulk bone/teeth collagen is known to increase from prey to its consumer in a food web, thus being an important indicator to evaluate the trophic position of organisms35. Other factors also influence δ15N values of collagen, such as variability in the consumed plants and environmental factors including climate38, leading to possible spatiotemporal shifts of the isotopic baseline due to heterogeneities among local environments42. Hence, some herbivores may exhibit similar δ15N values as predatory species in the same context45.More recent studies have shown mixed results based on different lines of evidence including anatomical properties like craniodental morphologies, tooth wear analyses, mortality patterns , etc. wherein the conclusions were highly context dependent and differed by sample-setsδ15N values of individual amino acids (AA) can be classified into several categories, “trophic” and “source” AAs that exhibit more 15N enrichment and less/little 15N enrichment, respectively, in each trophic step of food webs53. It is worth mentioning that the comparison of the “source” AAs like phenylalanine (Phe) with the “trophic” AAs like glutamate (Glx) enables the evaluation of animal trophic position regardless of baseline δ15N fluctuations in ecosystems54. We aim at testing the two hypotheses that high collagen δ15N values observed for cave bears in the Romanian region were attributed to (i) omnivorous/carnivorous feeding behavior associated with a trophic level effect as a corollary or (ii) consumption of plants with high bulk δ15N values. To this end, δ15N values of individual AAs in collagen were measured for adult cave bears from several sites in this region , due to the wide confidence interval depicted based on molecular tip dating, given that USR67 is the only representative within its clade. In each cave and related timespan, both high (above 8‰) and lower δ15N values of bulk collagen have been recorded (see more details in Table Judging from the C content 26.0–46.1%), N content (9.1–16.2%) and C/N atomic ratio (3.3–3.5), all the cave bear as well as the cave lion and horse bone collagen are well preserved . Other AA such as hydroxyproline  also showed strong correlations with bulk collagen δ15N values. The scatterplot presenting δ15NPhevs. δ15NGlx values shows that some cave bear individuals from the Romanian caves exhibited isotope values similar to those analyzed in previous studies for cave bears from Late Pleistocene Belgian sites, though three individuals with bulk collagen δ15N values above +8‰ analyzed in our study exhibited higher δ15NPhe values than the others without any overlaps. Most relevant for this study are the canonical trophic position  estimates based on eqn. 1 (see Methods section) indicating that all cave bears, including those with high δ15N values for their bulk collagen, had a TP around 2 (TP = 1.8–2.2), similar to published TP-values for cave bears from other sites  significant contribution of aquatic resources such as fish to their diets; (ii) carnivory as the main feeding behaviour54. The TP estimates for all individuals based on δ15N of glutamate and phenylalanine were around 2, which rather indicates a highly plant-dependent feeding behavior for all the analyzed cave bears, including those with a high δ15N value of their bulk collagen.The bulk collagen δ15N value of bulk bone collagen and the δ15N value of phenylalanine that represents δ15N of the nitrogen source in a food web. This indicates that the bulk collagen δ15N values of the Romanian cave bears reported here can be explained by the following two possibilities, that are not mutually exclusive: (i) a δ15N shift of the baseline in this local ecosystem, and/or (ii) the consumption of some specific plants with high δ15N values by these cave bear individuals. This means that the reported high δ15N values in the Romanian cave bears’ bulk collagen can be explained by the exclusive consumption of plants rather than by the trophic level effect caused by consumption of animal meat. As shown in Fig. δ15NGlx and δ15NPhe values for some individuals, especially the ones with high bulk δ15N values, overlap with those of strictly herbivorous species, such as late Pleistocene mammoths from other regions, suggesting that a purely herbivorous species could exhibit such carbon and nitrogen isotopic values of bulk collagen in this palaeoenvironmental context58.The strongest correlation was observed between the 59. Considering this error together with a general offset of 0.1–0.2 (accuracy) between calculated TP based on eqn.1 and known TP that were estimated based on well-controlled field observations or laboratory feeding experiments for extant animals, our estimates for all individuals are well within the range of acceptable values for herbivorous mammals. In addition to predatory carnivores, fossils that so far yielded TP-values above 2.3 originate from brown bears, wild boars, foxes and badgers60, in contrast to the cave bears with TPs less than 2.2 analyzed in the present study.It was shown that there is currently a potential error of ~0.3 units for the TP estimation calculated by the propagation of errors for each parameter in eqn.1δ15N values. Mammoth and some Pleistocene fallow deer have been found to exhibit higher δ15N value and lower δ13C values relative to the other coeval herbivorous species44. Some horses from the Late Pleistocene of western Germany also exhibited similar high δ15N and low δ13C values and are interpreted as having the same ecological niche as mammoths62. It is conceivable that the cave bears with high bone collagen δ15N values fed on plants, since the high values were also found in species with indisputable herbivorous dietary behavior. Possible candidates of such plants with high bulk δ15N value consumed by cave bears were those consumed by some fallow deer in the Pleistocene44, while those consumed by mammoth such as dry, mature grasses and sedges were less likely based on the lower resistance of cave bear teeth against abrasion. Mushrooms, another plant food with high δ15N values, can be also excluded as an explanation for the high δ15N values of some cave bears for the two following reasons: (1) mushrooms typically have high δ15N and high δ13C values , (2) mushrooms are consumers, like animals, and they display a trophic position value higher than 252, therefore their consumption would lead to a TP significantly higher than 2.The question remains which kind of plants could have been consumed by the cave bears with bulk collagen high Homo sapiens, bears hibernate. Indeed, it has been demonstrated that a longer hibernation period caused by climate cooling could result in higher δ15N of cave bear bulk collagen64. Although we did not see any statistically significant chronological trend in δ15N values of AAs, only a limited number of individuals have been analyzed so far with this approach. Further investigations on the possible influence of this factor on the δ15N values of individual AAs of Ursids should be performed on more extinct cave bears as well as extant bears in order to clarify how nitrogen metabolism such as urea recycling during hibernation possibly affects the δ15N of AAs in bone collagen66. Yet, our data currently does not support a major physiological control of the high bulk collagen δ15N value in bone that remodels intensively during the warm season30.One additional factor specific to bears that has to be considered is hibernation, since unlike the other mammalian taxa discussed above, including 67. It is possible that, in contrast to other regions of Europe, the absence of mammoths played a role in allowing cave bears to have access to a more diverse feeding niche and that competition was stronger among cave bears in Late Pleistocene Romania than in other regions, although an exact contemporaneity between the populations with diverse bulk collagen δ15N values must be examined. If the observed variability in δ15N values of bulk collagen among the Romanian cave bears was the result of high competition among cave bears in a context of a feeding niche restricted to herbivory, it is possible that some individuals specialized on other plants rather than turned to omnivory including animal food resources. Such a dietary strategy is a very unusual case among bears. Assuming that the herbivory for individual cave bears observed in our study was a general trait across all Late Pleistocene cave bear taxa, the most parsimonious interpretation would be the loss of omnivorous feeding behavior after the divergence of cave bears from the brown/polar bear lineage and before the basal divergence of cave bear lineages . However, it still remains an open question why this extinct animal that could live over a wide geographical range reaching Transbaikalia in Eastern Siberia with diverse ecological conditions within the mammoth steppe ecosystem finally became extinct70. It is possible that a dietary adaptation similar to that seen in extant giant panda that consumes large quantities of bamboos but has a macronutrient profile, namely the percent of energy derived from protein, similar to that of carnivores, might have facilitated the full shift for the cave bear lineage towards highly plant-dependent feeding behavior while keeping some ecological plasticity in the mammoth steppe71.Despite a predominantly herbivorous diet, Romanian cave bears exhibit a very large variation of their isotopic values that probably reflects ecological differences. Such isotopic differences in bone collagen reflect long-term dietary differences, since the turnover of collagen is slow in bone, averaging diet isotopic composition over many years72. Cave bear populations were likely adapted to their local habitats, with different altitudes being associated with different features, such as body size, tooth morphology, and tooth wear patterns . Although the tooth microwear and the isotopic composition of bone collagen reflect the diet of the analyzed individuals at different pre-mortem time periods , the observed variation in the bulk collagen δ15N values for cave bears in the Romanian region possibly reflected a difference in foraging strategy on plants associated with different body sizes24. Extant Carnivora species with herbivorous feeding behavior  have smaller body size and live only in temperate and tropical/subtropical Asia, highlighting the paradox of cave bears that had much larger body sizes and lived in a colder and drier environment. Besides, these three extant species are all on very long evolutionary branches, namely they diverged more than 10 million years ago from their closest relatives. Cave bears, in contrast, share a much more recent ancestor within an omnivorous/carnivorous clade, which is another ecological paradox of this extinct taxon. It is possible that the modern herbivorous carnivorans are relicts of a dietary trend that was much more widespread in the past, and that their rarity is rather due to the recent extinction of the large mammalian species rather than to the impossibility for Carnivora to have a diet restricted to plant food. Our results emphasize the need to investigate more in detail the diversity in food sources, especially plants, consumed by cave bears, as a unique case among Carnivora to further understand the benefits, costs and limitations of herbivory.Our findings have interesting implications regarding the evolution of herbivory among carnivorans. The foraging strategy of an animal is largely determined by body mass via energetic needsδ15NGlx and δ15NPhe values. The high δ15N of bulk bone collagen of some cave bears can be explained by a higher δ15N baseline of the exploited food chain that is reflected in the δ15NPhe values, namely plants with high δ15N values. Therefore, the high δ15N value of bulk collagen of some cave bears in the Romanian region, a unique feature compared to cave bears from the other regions of Europe analyzed so far, can be explained by the consumption of plants. Further research is needed to clarify which plant species were eaten by those cave bears by using other approaches such as δ13C analysis of individual AAs to identify protein sources76. It is also of importance to see if this trend towards herbivory was common for other cave bear populations from broader regions including Asia.A highly plant-dependent feeding behavior for cave bears was demonstrated by the TP estimates based on 77. Well-preserved bone specimens of cave bears from these three sites allowed us to examine the feeding behavior of the individuals from the Romanian region. Măgura  is a 1,500m-long cave located in the eastern part of the Sighiștel valley part of the Apuseni Natural Park; Cioclovina Uscată  is a 2,002m-long cave formed in the Early Cretaceous reef limestones of the Luncani karst platform and situated on the foot of the Southern Carpathians, in the Grădiștea-Muncelului Cioclovina Natural Park; Răsuflătoare cave  is located in Caraş-Severin County, Banat Mountains and is a part of the Semenic-Cheile Carașului National Park.The Romanian cave sites of Măgura, Cioclovina and Răsuflătoarei, among others, yielded skeletal remains of cave bearsδ13C and δ15N analysis as well as AA-specific δ15N analysis. One cave lion collagen sample from the North Sea, an area that was terrestrial during the Late Pleistocene and one horse sample from the site of Lommersum (Germany) from the late Pleistocene, have been analyzed together with the cave bear collagen using the same analytical protocol.Bone collagen from 6 adult cave bears from the three caves was subjected to 78 with a minor modification as described in ref. 79. In summary, this acid-base-acid method includes a first step of demineralization with 1 M HCl, a second step in 0.125 M NaOH to remove humic acids and lipids, and a final step of gelatinization at pH 2 during 17 h at 100 °C. Isotopic measurements were done using an NC2500 elemental analyzer connected to a Thermo Quest Delta þ XL mass spectrometer. Using the ‘δ’ (delta) value, the isotopic compositions are expressed as follows: δ (‰) ≡ 103 [Rsample/Rstandard − 1], where the R denotes the 13C/12C ratio for carbon and the 15N/14N ratio for nitrogen, with the international reference (standards) being V-PDB for δ13C values and atmospheric nitrogen (AIR) for δ15N values. Isotopic measurements were normalized to δ13C values of USGS24 (δ13C = −16.00‰) and to δ15N values of IAEA 305 A (δ15N = +39.80‰). The reproducibility was ±0.1‰ for δ13C measurements and ±0.2‰ for δ15N measurements, based on one standard-deviation of the mean of multiple analyses of purified collagen from modern bones and international standards.The samples were washed in an ultrasonic bath in acetone, rinsed several times with demineralized water, dried at 35 °C for 72 h and crushed to powder of 0.7 mm grain size. Collagen extraction was performed after ref. δ15N values of the individual AA derivatives were measured at University of Tübingen using the following systems; A gas chromatography/IRMS (GC/IRMS) using an Agilent Technology 7890B GC coupled to an Elementar Isoprime100 IRMS  via combustion and reduction furnaces. AA derivatives were injected into the GC column  in splitless mode at 275 °C. The GC oven temperature was programmed as follows: isothermal hold at 40 °C for 3 min; temperature ramp to 110 °C at 15 °C min−1; ramp to 150 °C at 3 °C min−1; ramp to 240 °C at 2.5 °C min−1; ramp to 270 °C at 6 °C min−1; and subsequent holding isothermally at 270 °C for 4 min. Carrier gas (He) flow rate through the GC column was 1.4 ml min−1. The CO2 generated in the combustion furnace was eliminated by a liquid nitrogen trap. Standard mixtures of ten AAs with known δ15N values were injected into the GC/IRMS every two to five runs to confirm reproducibility of the isotope measurements. The precision of the reference mixtures was 0.65–0.90‰. Nitrogen isotopic composition of maximally the following eight AAs was determined as their N2 gas peaks from derivatives showed fine resolution on the GC/IRMS chromatogram: valine , leucine (Leu), isoleucine (Ile), proline (Pro), serine (Ser), glutamate (Glx), phenylalanine (Phe) and hydroxyproline (Hyp). All reported δ15N values for glutamate included the contributions from the α-amino group of glutamic acid and glutamine, as glutamine is converted to glutamic acid during acid hydrolysis. Data for alanine and glycine were not obtained for all samples due to relatively large peak sizes in this study.Bone collagen extracted from the bone samples was hydrolysed with 12 N HCl at 110 °C for 12 h, and derivatized with thionyl chloride/2-propanol  at 110 °C for 2 h and subsequently pivaloyl chloride/dichloromethane  at 110 °C for 2 h. The δ15N of glutamate and phenylalanine, we estimated the trophic position of cave bears in terrestrial C3-plant-based ecosystem: TP = [(δ15NGlx − δ15NPhe − β)/TDF] + 1 (eqn. 1), where δ15NGlx, δ15NPhe, β and TDF indicate δ15N of glutamate, δ15N of phenylalanine and the isotopic offset between glutamate and phenylalanine  in primary producers, the trophic discrimination factor, respectively60. The values in the equation are based on the fact that some “source” AAs show little isotopic change through each trophic step  whereas other “trophic” AAs fractionate with trophic steps (e.g. 8.0 ± 1.1‰ for glutamate)49. We adopted 8.4‰ for the β value for terrestrial C3 vascular plants80, and 7.2‰ for the TDF value81, in this study.Using the equation below based on 83. All dates were calibrated to BP dates with 2σ (95.4%) probability.Cave bear collagen was dated directly using AMS radiocarbon dating. Radiocarbon dating was performed by the Labor für Ionenstrahlphysik at Eidgenössische Technische Hochschule—ETH in Zurich (Switzerland). The obtained radiocarbon ages were calibrated using the OxCal v4.2.4 software using the IntCal13 atmospheric curve84, with reduced centrifugation speeds as described in ref. 85. Prior to library building, each DNA extract has been quantified with the Qubit 2.0 fluorometer with high sensitivity reagents (Thermo Fisher Scientific) by using one microlitre out of 25 μL extract. To support the validity of the measurements, positive and negative controls were also measured. Illumina single-stranded libraries were then prepared following the protocol of ref. 86, after the removal of uracil residues and abasic sites. The optimal number of library amplification PCR cycles has been assessed with qPCR as described in ref. 85. A volume of 80 μL including 20 μL template library, Accuprime Pfx DNA polymerase and eight base-pair tailed-primers was used for Indexing PCR to generate dual-indexed library molecules. Amplified libraries were purified using commercial silica spin-columns (Qiagen MinElute). Prior to single-end sequencing on Illumina platforms, library concentration and length distribution were quantified using Qubit 2.0 and 2200 TapeStation (Agilent Technologies), respectively.For two samples lacking radiocarbon dates (USR10 and USR67), molecular tip-dating was performed. DNA was extracted from 50 mg of cave bear petrous bones following the protocol of ref. 87. Raw reads were trimmed with minimum overlap of one nucleotide and reads shorter than 30 bp were discarded using cutadapt v1.1288. The processed reads were mapped to the reference mitogenome sequence of Ursus spelaeus  with default parameters using the bwa v0.7.15 “aln” algorithm90. The alignment was then filtered for mapping quality (Q > 30), sorted by read position and potential PCR duplicates were removed using SAMtools v0.1.1990.Shotgun sequencing was performed on an Illumina NextSeq 500 sequencing platform. We obtained 75 bp single-end reads using the NextSeq 500/550 High Output Kit v2 (75 cycles) sequencing kit, following the procedures described in ref. 91. Using the option -doFasta 3 that takes into account bases with highest effective depth92, the mitochondrial consensus has been constructed only considering read mapping and Phred base quality scores >30. The two mitogenomes were aligned to a published alignment of cave bear mitochondrial sequences15. The repetitive section of the d-loop was removed from the alignment because it could not be aligned unambiguously. The final alignment comprised 19 sequences of 16,468 nucleotides, publicly available (Dryad database and GenBank). The GenBank accession numbers of the mitogenomes generated in this study are: MN311249 for USR10 and MN311250 for USR67.ANGSDv0.920 was used to generate consensus sequences for both individuals93, a method shown to provide accurate age estimates for Late Pleistocene European cave bears15. PartitionFinder v.2.1.1 has been used for data partitioning and substitution model selection94. All substitution models available in BEAST v.1.8.2 were considered and the best partitioning scheme has been computed under the Bayesian Information Criterion using linked branch lengths and the greedy search algorithm (Supplementary Table 95.Ages of the two samples were estimated using Bayesian phylogenetic tip dating14C dates as calibration from the dataset in ref. 15 (Supplementary Tables 96. The Maximum Clade Credibility Tree was selected from the posterior sample using TreeAnnotator and visualised in FigTree (Supplementary Fig. Tip-dating was performed separately for each sample by analyzing its mitochondrial sequence in BEAST v1.8.2, using median Supplementary information.


Toggling of antiferromagnetic states is demonstrated in prototype device structures at CMOS compatible temperatures between 300 and 400 K. The boundary magnetization associated with the Néel vector orientation serves as state variable which is read via magnetoresistive detection in a Pt Hall bar adjacent to the B:Cr2O3 film. Switching of the Hall voltage between zero and non-zero values implies Néel vector rotation by 90 degrees. Combined magnetometry, spin resolved inverse photoemission, electric transport and scanning probe microscopy measurements reveal B-dependent TN and resistivity enhancement, spin-canting, anisotropy reduction, dynamic polarization hysteresis and gate voltage dependent orientation of boundary magnetization. The combined effect enables H = 0, voltage controlled, nonvolatile Néel vector rotation at high-temperature. Theoretical modeling estimates switching speeds of about 100 ps making B:Cr2O3 a promising multifunctional single-phase material for energy efficient nonvolatile CMOS compatible memory applications.Multi-functional thin films of boron (B) doped Cr 2O3.Voltage control of magnetization is critical for the development of antiferromagnetic spintronics. Here, using magnetic force microscopy and Hall measurements, Mahmood et al. demonstrate controlled rotation of the Néel vector in a heterostructure composed of Pt and antiferromagnetic B:Cr In contrast to their multiferroic counterparts5, ME antiferromagnets have one spontaneous ferroic order parameter whose temperature (T) dependence determines the T-dependence of the ME response6. In pristine chromia, the linear ME response, αij, sets in below the Néel temperature TN = 307 K, where magnetization, M,  is induced by an electric E-field (magnetic H-field) according to 7. Voltage-controlled switching of the Néel vector between 180° domain states can be achieved when their degeneracy is lifted. In chromia this is accomplished by simultaneously applying E and H along the easy axis (c-axis) such that the free energy difference 8. This mechanism has been exploited in voltage-controlled exchange bias (EB) heterostructures fabricated from chromia and an exchange coupled perpendicular anisotropic magnetic thin film such as CoPd12. Uncompensated AFM surfaces with equilibrium roughness often have a small interface magnetization. Therefore EB tends to be small in heterostructures based on antiferromagnets with uncompensated surfaces13. However, in ME antiferromagnets, an equilibrium magnetic moment associated with the AFM order parameter is symmetry allowed16. Note that due to the rigorous symmetry argument leading to boundary magnetization in linear ME antiferromagnets, the boundary magnetization is strictly tied to the orientation of the Néel vector. This moment can be sizable even in the presence of roughness and enables effective coupling with the magnetization of an adjacent ferromagnet. In such heterostructures, voltage-controlled reversal of the Néel vector switches the EB field between negative and positive values and can give rise to reversal of the remnant magnetization of the adjacent ferromagnet17. Note that switching based on the ME effect requires the simultaneous presence of E and H fields which, combined, have to overcome a critical product (EH)c. The need for H-fields, even when provided via stray-fields or exchange fields18, is detrimental to the implementation of practical devices. Despite this limitation, voltage-controlled EB heterolayers can serve as building blocks for voltage-controlled spintronics where the orientation of the exchange coupled magnetization defines the state variable2. Voltage-control promises energy efficient performance due to the absence of dissipative currents often required in spintronic devices which switch the state variable via Oersted fields, spin polarized electric currents through spin transfer torque19, spin–orbit or Néel torque in metallic antiferromagnets20.Magnetoelectric (ME) antiferromagnets, notably the archetypical ME insulator CrH-field and stable device operation above room temperature. High-temperature operation is an essential prerequisite to embed a ME device in a CMOS environment where typical load temperatures of central processing units reach 350 K or more21.Chromia is an insulating antiferromagnet whose ME properties can be exploited for voltage-controlled AFM spintronic devices. However, in its pristine form, chromia falls short of two prime objectives. Those are voltage-controlled switching in zero H-field and CMOS compatible operation temperatures. B:Cr2O3 simultaneously acquires new tunable functionalities in addition to the linear magnetoelectricity known from Cr2O3. Those include TN and resistivity enhancement, reduced and voltage controllable anisotropy, spin-canting and transient electric polarizability not observed in pure chromia. Our data imply that B-doping breaks local symmetry allowing for the formation of polar nanoregions (PNRs). The PNRs orient in an applied electric field giving rise to transient polarization and piezoelectricity. Piezoelectrically induced strain alters the reduced, nearly cubic anisotropy22 of B:Cr2O3 giving rise to a π/2 rotation of the Néel vector into a new stable state. Coupling between the AFM order parameter and the boundary magnetization allows to read out the AFM state via magnetoresistive detection in an adjacent Pt Hall bar. This structure demonstrates the fundamental building block of an ultra-low power electrically controlled AFM spintronic device which operates above room temperature and in zero applied magnetic field.Our work demonstrates that B-doping of chromia creates a single-phase material, which enables voltage-controlled nonvolatile rotation of the Néel vector in zero T > TN rendering device operation near or above TN impossible. Chromia’s Néel temperature of 307 K is insufficient for CMOS compatible applications. To mitigate this shortcoming, B-doping has been established as a viable path to increase TN24 with its only potential alternative being epitaxial or chemical straining26. Although AFM order and the persistence of magnetoelectricity have been predicted and demonstrated up to T = 400 K in B-doped chromia27, utilizing the high-TN material in device structures which rely on EB, remains elusive. In device architectures based on voltage-controlled EB, B-doping does not translate into improved device performance. Although B-doping can increase the blocking temperature, exchange coupling is not accompanied by effective pinning and thus voltage-control of the ferromagnet /Pd(0.5 nm)Co(3 nm)Pd(0.5 nm), where the ratio of Co to Pd film thickness tunes the anisotropy of CoPd heterolayers to be in the plane  is the method of choice to detect Néel vector reversal via reversal of the AFM boundary magnetization36. In antiferromagnets such as CuMnAs and Mn2Au direct measurement of the reorientation of the Néel vector require x-ray magnetic linear dichrosim-PEEM38.When growing B:Cr2O3  and down (blue) spins. For simplicity, canting present in B-doped chromia as well as the fact that the magnetic unit cell contains four sublattices are not shown.Figure H = 0 and μ0H = −1 T indicates that ME switching, which is the well-established switching mechanism in pure chromia, can be ruled out. In both cases (H = 0 and μ0H = −1 T), deterministic and nonvolatile switching between two distinct AFM states is observed. The high degree of asymmetry in Vxy on switching between different nonvolatile AFM states associated with Vxy ≈ 0 and Vxy ≈ −15 mV implies 90° rotation of the Néel vector in sharp contrast to Hall-signals observed for 180° switching29. Rotation of the Néel vector by π/2 is consistent with the fact that time reversal symmetry is not broken by an electric field. Equilibrium reversal of the Néel vector by π requires a field combination such as Remarkably, the toggling between AFM domain states in Vxy versus VG measured at T = 300 K in H = 0 . Because the switching is triggered by the applied electric field alone we exclude ME switching and investigate an indirect coupling between induced polarization and AFM order41. Although the magnetic field has no effect on switching, it does affect the Hall voltage. The dissimilar field dependence of Vxy in the two AFM states provides strong support for the magnetic origin of the switching shown in Figs. The findings presented in Fig. 2O3 thin film used for the devices in Figs. To provide independent evidence that the switching effects shown in Figs. 2O3 and show in a control experiment , we provide experimental evidence for the induced dielectric polarization in B:Crase Fig.  indicati44 or reorientation of PNRs45. Figure 2O3/V2O3 heterostructure, is not stable but relaxes logarithmically with a characteristic relaxation time varying in the range from several hundreds of milliseconds to tens of seconds. This behavior suggests that voltage pulse application produces a metastable polarization state in B:Cr2O3. Similarity of the PFM relaxation dynamics to that observed in ferroelectric relaxors allows us to assume that the induced polarization in B:Cr2O3 could be due to the alignment of the naturally existing PNRs (see discussion below).Previously, it has been shown that robust PFM signals could be registered in the non-ferroelectric materials due to the electrically induced polarization, which in turn could result from various mechanisms, such as redistribution of oxygen vacanciesd33,eff piezoelectric coefficient of B:Cr2O3 can be estimated by comparing its electromechanical response with the PFM signal detected in another material with the well-known piezoelectric properties47 -oriented Cr2O3 film the c-axis is normal to the film plane. However, stable in-plane domain variants can be produced through lattice mismatch with a substrate53 or substitutional defects. The chromia film surface itself can be considered as a defect where temperature-driven surface reconstructions take place, which include spin-canting effects56. First-principles calculations show that the magnetic anisotropy in unstrained Cr2O3 is close to cubic and the preference to perpendicular anisotropy is strongly modified by strain as 22. Magnetometry and inverse photoemission data , the piezoelectrically induced strain is compressive or tensile enabling rotation of the Néel vector between in-plane and perpendicular orientations in accordance with the switching condition E-field. This switching criterion is in agreement with the experimentally observed coercive electric field of Ec ≈ 15 V/200 nm for a piezo-response coefficient of dzz = 13 pm/V. A dzz on the order of a few pm/V is in agreement with our estimate obtained from the PFM data  = 100 ps for 90° planar relaxation of the Néel order, where Ms = 1.9 × 105 A/m is the sublattice magnetization, α = 2 × 10−4 is the Gilbert damping, J = 9.5 × 107 J/m3 is the intersublattice exchange and γ is the electron gyromagnetic ratio. This underlines the advantage of AFM spintronics outperforming device concepts which rely on notoriously slow reversal of ferromagnetic constituents taking place on the ns time scale determined by the inverse Larmor precession frequency.Although the magnetoresistive switching data shown in Figs. 2O3 can be tuned into a multi-functional high-TN material through B-doping. Emerging functionality associated with B-doping include purely electric-controlled 90° nonvolatile rotation of the Néel vector up to T = 400 K. Indirect coupling between polar and AFM order explains the experimental findings and allows to estimate switching speeds on the order of 100 ps. An energy efficient memory which operates up to 400 K in zero magnetic field has been fabricated on the basis of B-doped chromia. Future investigations will focus on device stability, switching speed, and scaling.Energy-efficient isothermal and deterministic switching of remnant magnetic states above room temperature and in the absence of a magnetic field is fundamentally challenging and of highest interest for ultra-low power memory and logic device applications. Switching of AFM states is of particular interest due to inherent switching speed advantages over ferromagnets and robustness against external magnetic field perturbations. The work presented here demonstrates that the prototypical magnetoelectric antiferromagnet Cr2O3 (chromia) and ferromagnetic CoPd with tuned perpendicular and in-plane anisotropy for temperature-dependent EB. Magnetometry together with temperature-dependent spin-polarized inverse photoemission spectroscopy were utilized to investigate canting of the AFM interface magnetization relative to the film normal.Magneto-optical polar Kerr effect and vibrating sample magnetometry are utilized to investigate heterolayers of B-doped Cr58. As described elsewhere58, the spin electron gun was used in combination with an iodine-based Geiger–Müller isochromat photon detector with a SrF2 window. As is typical of such instruments, the electron gun has 28% spin polarization, and the data have been corrected for this incident gun polarization. The direction of electron polarization is in the plane of the sample. The magnetoelectric cooling was accomplished in an axial magnetic field of >40 mT and a voltage of 3 kV applied across the film thickness. The Fermi level was established from tantalum and gold foils in electrical contact with the sample.Our spin-polarized inverse photoemission experiments utilize a transversely polarized spin electron gun based upon the Ciccacci design2O3 and subsequently, in the presence of a decaborane background gas, B-doped chromia. The V2O3 film, which is grown on the c-plane of a sapphire single-crystalline substrate, serves as bottom electrode in a gated Hall bar structure. A 5 nm Pt film is deposited via DC magnetron sputtering on top of the B-doped chromia film and various Hall crosses are lithographically patterned. The Hall cross serves as readout device which detects the voltage-controlled AFM states of the chromia film.The AFM constituent films of the EB heterostructures are grown by PLD while the anisotropy tuned CoPd multilayers are deposited via molecular beam epitaxy. PLD in ultra-high vacuum is used to grow (0001)-oriented films of the sesquioxides V2 sized capacitive structures. In all PFM measurements, the top electrode was biased while the bottom electrode grounded.PFM spectroscopy loops were obtained in the resonance tracking mode using a commercial atomic force microscope system  by employing Pt-coated tips . The AC driving voltage was varied between 0.75 and 1 V at a frequency of ~330 kHz. For the PFM studies, capacitive devices were fabricated on the same samples as were discussed throughout this manuscript. Focused Ion Beam was used to mill 2 × 2 μmThe switching criterion has been derived analytically by variation of the anisotropy energy. The switching speed of a thermally excited state has been determined by the time it takes for the state to relax from the top of the energy barrier to the bottom. The relaxation process has been mapped to damping of an equivalent inverted pendulum.Supplementary InformationPeer Review File


Parazoanthus axinellae morphotypes differing in size, color and preferred substrate are found in sympatry. In this study, we used COI and ITS sequence polymorphism to assess (1) the genetic divergence between the two morphotypes, (2) their connectivity patterns and (3) their phylogenetic position within the Parazoanthidae. Specimens of P. axinellae were sampled in 11 locations along the northwestern Mediterranean; in 6 locations, samples of the two morphotypes were collected in sympatry. Small genetic diversity and structure were found within morphotypes, while marked and consistent differentiation was detected between them. Moreover, the less widespread morphotype appeared to be closer to Pacific species as P. juanfernandezii and P. elongatus. Our findings confirmed the limited knowledge on Parazoanthus species complex, and how this gap can have important implication for the conservation strategies of this widespread and valuable genus in the Mediterranean Sea.Coralligenous assemblages are among the most species-rich and vulnerable habitats of the Mediterranean Sea. Nevertheless, data on connectivity patterns on species inhabiting these habitats, crucial to define management and protection priorities, are largely lacking. Moreover, unreliable species-level taxonomy can confound ecological studies and mislead management strategies. In the northwestern Mediterranean two  They are among the most productive and diverse benthic ecosystems, providing habitat, feeding grounds, recruitment and nursery areas for a variety of invertebrate and vertebrate species2. These complex habitats are threatened by several human activities , which lead to their fragmentation and loss4.In the Mediterranean Sea, the main biogenic reefs are the coralligenous reefs. They are among the most important Mediterranean ecosystems due to the high diversity of species and ecological processes that they supportMonitoring spatio-temporal changes in species composition is crucial to quantify human-induced biodiversity loss and habitat fragmentation, but the absence of clear taxonomic identifiers to distinguish between species makes this a challenging task.5. Moreover, in these species, with high morphological plasticity6, some characters such as shape and coloration might not be reliable for species delimitation. It happened that morphospecies have turned out to be a single morphologically variable species9, and, conversely, what was thought to be multiple growth forms of a single species turned out to be a complex of species11.Many relevant coralligenous taxa  lack diagnostic morphological characteristics, and therefore these groups are recognized as taxonomically problematic, in particular at lower taxonomic levels12 may be erroneously inferred when cryptic species are ignored14, which may provide biased conclusions about the overall capacity of the studied populations to resist stress15. To disclose cryptic species before estimating gene flow and indeed evaluate connectivity among populations, we should (1) use more than one independent molecular marker, (2) sample individuals all along the distributional range of the investigated species, and (3) sample closely related taxonomic species to understand the spectrum of population to species divergence13.The presence of species complexes in the sea can confound ecological studies and mislead management strategies. For example, connectivity patternsParazoanthus exist along the Mediterranean and North-East Atlantic coasts: Parazoanthus axinellae , P. anguicomus  and another Parazoanthidae yet to be described in the Macaronesian waters16. In the Mediterranean Sea, Parazoanthus axinellae  species complex17 is one of the most common cnidarians inhabiting coralligenous assemblages. The simplicity of the zoanthid body plan makes morphology-based species identification quite challenging. Numerous morphological identification criteria have been used to identify phylogenetic signal ranging from colour, sphincter muscle anatomy18, tentacles number19, type and distribution of nematocysts20. In addition, differences in substrate preference, overall ecology17, biochemical profiles23 and sequence divergence using various gene markers were also proposed as zoanthid identification criteria26. Original species descriptions were thereby based on different traits depending on the authors27. Due to the aforementioned lack of morphological characters to identify zoanthids, a comprehensive taxonomic identification of the P. axinellae species complex is still missing (but see17 for a detailed description of P. axinellae species complex). Historically, there were four described subspecies of P. axinellae: P. axinellae mülleri Pax, 1957, common across the whole Mediterranean coastline; P. axinellae liguricus Pax, 1937, in the Ligurian Sea; P. axinellae brevitentacularis in the Gulf of Lion and P. axinellae adriaticus Pax, 1937 in the Adriatic Sea30. However, the morphological descriptions of these four subspecies are quite imprecise and overlapping, and clear diagnostic characters to discriminate them are still lacking. Recently, Ocana et al.17 stated that these four subspecies can be grouped in two morphotypes based on their chemical profiles31 and ecological requirement17. These two morphotypes live in sympatry in the northwestern Mediterranean29. One of them displays an elongated trunk, longer and thinner tentacles, and a light yellow color. This morphotype  and nuclear (ITS) sequences polymorphism to asses: (1) the genetic diversity and structure among populations within and between morphotypes and (2) phylogeny and differentiation in the Parazoanthidae Family.In the present study, we aim to describe the patterns of diversity and connectivity for populations of the two A total of 322 sequences of 402 bp of COI were obtained Table , and 5 pParazoanthus specimens  and “Stocky” and “Slender” morphotypes were differentiated by a minimum number of 68 mutation steps (between haplotypes II and VI) Fig. . The twosee Fig. .ST values) between populations ranged between 0 and 1 for both markers   between groups of samples according to morphotype and biogeographic areas showed that for the “Slender” morphotype most variability is explained by differences between all populations , which might indicate a decrease in population size and/or balancing selection acting in this marker. Mismatch distributions in both markers showed a bimodal distribution34, but the observed distributions were not statistically different from those expected under a sudden expansion model . Two genetic pools were observed; one is corresponding to a recent expansion and a second one with older demographic history. These two genetic pools are more similar according to COI, but very distant according to ITS  and 38 (ITS) haplotypes within Parazoanthidae family (sequences from our study + those retrieved from GenBank) (Supplementary Table Haplotype function from phangorn R package), based on minimum AICc values, identified HKY + I and HKY + G + I models as best nucleotide evolution models for COI and ITS markers, respectively. The Maximum Likelihood trees were reconstructed for each marker using F81 nucleotide substitution model as this was the most similar model to HKY + I and HKY + G + I available in phangorn R package.Likelihood ratio test . Moreover, the phylogenetic trees with MAFFT alignment  and with MAFFT alignment + GBlock gap removal also give similar topology. We only show results for P. axinellae), and V (ALI and “Stocky” P. axinellae), whereas for ITS marker this occurred only in haplotype VI (ELO and JUA). Overall “Stocky” and Slender” morphotypes cluster in two well-separated clades . In the “Stocky” clade all the “Stocky” morphotype haplotypes showed high similarity with P. elongatus (COI and ITS), and P. juanfernandezii (ITS), both species from the southeastern Pacific. The “Slender” morphotypes grouped with P. anguicomus (COI and ITS) from the eastern Atlantic, P. parasiticus (COI), P. swiftii (COI) both Caribbean species and P. aliceae (COI) from the Azores Archipelago  that the “Slender” morphotype is characterized by a lower genetic differentiation among populations compared to the “Stocky” morphotype.In the present study we evidenced for the first time that (1) the two Mediterranean morphotypes of 31 and in morphological features17 between the two morphotypes are consistent with our results, suggesting that the Parazoanthus axinellae species complex includes two different taxa. Both COI and ITS haplotype networks showed the presence of two highly abundant haplotypes surrounded by low frequency haplotypes that clearly discriminate the two morphotypes. This low variability can be explained by the geographical distribution of the morphotypes. In fact, the “Slender” morphotype is widespread all along the Mediterranean Sea while the “Stocky” morphotype17 has a more restricted distribution. Although we cannot discard the existence of more populations of the “Stocky” morphotype in other unsampled areas of the Atlantic Ocean, information on the geographic distribution of this species in the Eastern Atlantic Ocean is scarce and refers only to the “Slender” morphotype . The widespread distribution of the “Slender” morphotype can also explain the low genetic structuring observed among populations.COI and ITS evidenced clear genetic isolation between the two morphotypes. Differences in chemical profilesCorallium rubrum36; Paramuricea clavata37; Patella caerulea38) related to the presence of a large‐scale separation of currents that occurs in the region39. Biological and reproductive characteristics of the species can also contribute to explain the observed pattern. Pax and Muller40 found hermaphroditic colonies of P. axinellae in the Adriatic Sea, while observed that colonies of P. axinellae from the British Isles (North Atlantic) are gonochoric41. Moreover, Previati et al.42 observed that the “Stocky” morphotype collected in the Ligurian Sea, where the two morphotypes live in sympatry, exhibited both asexual and sexual reproduction . Although not much information is available on the ecological and biological traits of this morphotype, it seems to be defined by a lower dispersal capacity compared to the “Slender” morphotype in this area.Despite the limited geographical distribution compared to the “Slender” morphotype, the “Stocky” morphotype shows genetic structure between northeastern Tyrrhenian populations  and the northwestern ones (Banyuls -sur -mer and Alassio) with a break around Portofino. Portofino area represents a barrier to gene flow for several species of sessile invertebrates (Antipathozoanthus43). These Authors found that, despite being highly variable compared to mitochondrial markers (due to presence of indels), ITS marker can leave some uncertainties regarding the specific status of different morphotypes. ITS markers are therefore unable to fully resolve the phylogenetic relationships of this group43, which coincides with what was already described for other groups of cnidarians (for instance Eunicella44). Hypervariable ITS sequences can lead to alignment challenges (difference in both length and nucleotide identity), and intragenomic variation, that are widely acknowledged as being the primary obstacles to successfully using these sequences in phylogenetic inference in Zoanthidea45. These difficulties stress the need to increase the number of analyzed individuals to detect even small differences that can unlock the relationships among species, particularly in Zoanthidae phylogenetic studies46.Sardinia samples (codes SRY and SRO) present an intermediate pattern of differentiation, with some samples from both “Slender” and “Stocky” morphs sharing the same sequence with the morphologically distinct phenotype. These identities on ITS-rDNA sequences were already observed within another genus within Parazoanthidae . This confirms once more that uncovering the origin of the “Stocky” morph in the western Mediterranean is a challenge. Future research using an integrative taxonomy approach13 could complement the results of the present study regarding the status of the Mediterranean Parazoanthus genus. In fact taxonomy using only molecular tools should not be a replacement of classical taxonomy, but findings derived from one approach should be used as a driver for further investigation for the other. A multidisciplinary approach should include (1) reproductive biology of both morphotypes in sympatry, (2) field experiments to evaluate substrate specificity26, (3) studies of chemodiversity49, (4) naturalistic studies16 and (5) more variable molecular markers  and/or sequencing of the complete mitochondrial genome51.Despite these molecular issues, from the phylogenetic point of view, our results are in accordance with the latest accepted phylogeny of Parazoanthidae52. Finally, our results highlight occurence of hidden diversity within the flag-genus Parazoanthus, even in well-studied geographical areas, calling for a carefull taxonomic reevaluation of other key-species of the fragile endemic Mediterranean coralligenous ecosystem53.The results of this work stress the importance of studies on species delimitation and connectivity for the implementation of effective plans for the conservation of coralligenous species. In fact, other studies have observed that in closely related species, with similar biological features, differences in the phylogeographic patterns can occurParazoanthus was done by SCUBA diving in Portofino where, during the same dive, the “Slender” morphotype was observed in the deeper and in the shallower layers of a cliff, while the “Stocky” morphotype was found at an intermediate depth. Based on these preliminary results we developed a sampling design at a Mediterranean scale identifying sites where the occurrence on Parazoanthus was recorded , 4 µl MgCl 25 mM, 0.5 µl dNTPs 10 mM, 0.5 µl of each primer (10 mM), and one unit of GO Taq G2 Flexi DNA polymerase (Promega) and filled with nuclease free water to reach the volume. Amplifications were conducted on a GeneAmp 2,700 thermal cycler (Applied Biosystems) under the following conditions: a hold at 94° C for 3′ followed by 30 cycles of denaturation at 94 °C for 45″, annealing at a primer specific temperature (59 °C for COI and 50° C for ITS) for 1′ and extension at 72 °C for 2′, finishing with a final extension at 72° for 7′.DNA was extracted from single polyps using EuroClone EuroGold tissue DNA mini kit. Mitochondrial COI fragment was amplified using species-specific designed primers COIpaxFwd (sequence 5′–3′: CGGTATGATAGGAACAGC), and COIpaxRev (sequence5′-3′:CGGGGTCAAAGAAGGTAGTG). A fragment of the nuclear DNA including 18S, ITS-1, 5,8Sa, ITS-2, and 28S (hereafter ITS) was amplified using zoanthid-specific primers described inPCR products were checked in 1.5% agarose gel stained with Gelred (BIOTIUM) 1% after a 20′ 120 V electrophoresis. They were then sent to Macrogen Europe Inc. for purification and sequencing.Field and experimental protocols were approved by the University of Bologna, Italy and were performed in accordance with its relevant guidelines and regulations. No permit was required for the collection of the species.55 through phyloch R package56 (see below for more detail).Each sequence was checked for quality in MEGA v.6 and good quality sequences were aligned using MAFFT (Multiple Alignment using Fast Fourier Transform)57. Haplotype networks for COI and ITS markers were built using pegas R package35 in Rstudio (Version 1.1.453)58. Haplotypes were reconstructed with haploNet function using an infinite site model based on Hamming distances of DNA sequences. As there was an overlap between genetically approximate haplotypes on the Fig. 59 to better clarify Morphotype/Haplotype/Sampling locations correlations, and Inkscape (version 0.92.4) was used to integrate the plots and finalize graph compilation.The number of haplotypes (H) and haplotype and nucleotide diversity (Hd and π respectively), were calculated for each sample and marker in DNAsp60 to infer links between the most similar haplotypes based on a previously computed distance matrix, and to visualize the number of mutations between them. This was done by performing a Multidimensional Scaling (MDS) analysis on Hamming distances computed between haplotypes, using the show.mutation = T option to show the number of mismatches between linked haplotypes. The results were presented as a two-dimensional MDS plot.A Minimum Spanning Tree was computed as inST based on haplotype frequencies and molecular divergence) and its significance determined using a permutation test  for each marker in Arlequin v. 3.561. Significance values were corrected for multiple comparisons following FDR correction method62.Genetic differentiation between samples  was estimated using Φ statistics , Tyrrhenian Sea , Ionian and South Adriatic , and North Adriatic (Chioggia and Rovinj).Hierarchical Analysis of Molecular Variance (AMOVA) was carried out for each marker using (1) morphotype as grouping factor (two levels: “Slender” and ”Stocky”) and (2) biogeographic areas as grouping factorFu and Li33 and Tajima63′s neutrality tests were performed in DNAsp for each locus and morphotype across the study area. Subsequently, the demographic history was also assessed by performing a mismatch distribution analysis, in which the frequencies of pairwise nucleotide differences between individuals were compared with the expected values under a sudden expansion model34 using Arlequin. The best fit was tested by evaluating both the sum of squared deviation (SSD) and the Harpending’s raggedness (HRI) indexes with a total of 1,000 permutations.To study the demographic history of the two morphotypes We retrieved COI and ITS sequences belonging to the family Parazoanthidae from Genbank. Full details on the species, accession numbers, approximate geographic origin, and original reference are given in Supplementary Table 64). For each marker, the retrieved sequences were aligned together with our sequences using multiple sequence aligner MAFFT v7.310 (Multiple Alignment using Fast Fourier Transform)55 through phyloch R package56. P. axinellae + Genbank COI sequences were aligned with default parameters, whereas L-INS-i algorithm with 1,000 iterations  was used to align P. axinellae + Genbank ITS sequences due to a presence of gaps between individuals showing high sequence divergence.We decided to perform all downstream analyses at the taxonomic level of family due to large uncertainties regarding the phylogeny and systematics within Parazoanthidae at lower taxonomic categories, which led to significant and recent taxonomic modifications for these cnidarians , b2 = 0.9, (the minimum number of sequences for a flank position), b4 = 2,  and b5 = "h", to remove gaps present in > 50% of individuals.Gblocks Version 0.91bphangorn67 to decide on the model of nucleotide evolution that best fits COI and ITS markers, and the best nucleotide substitution model was identified based on minimum AICc values for each marker separately.Likelihood ratio test was performed with modelTest function in 68 and Maximum Likelihood (ML) trees with UPGMA algorithm and clustering dendrograms were used to resolve phylogenetic situation of P. axinellae species complex within Parazoanthidae family based on COI and ITS gene markers. Distance matrices were calculated using the suggested F81 nucleotide substitution model and Maximum Likelihood (ML) trees with 1,000 bootstrap iterations were estimated from the obtained distance matrices using the UPGMA algorithm. This was done in phangorn67 by computing the likelihood of a given tree with the function pml, and with the function optim.pml, which was used to optimize tree topology and branch length for F81 model of nucleotide evolution. Tree plotting was done using plotBS function, with haplotypes presented on the tree nodes in roman numerals, and bootstrap support values  shown on tree branches with a cutoff value of p = 50 (%). To further clarify “haplotype-sampling location” associations, clustering dendrograms were reconstructed for each gene marker in RAWGraphs59 and combined with ML trees in Inkscape (version 0.92.4). Hierarchically, the clustering dendrograms are composed out of 2 levels for sequences downloaded from GenBank  and 3 levels for individuals sampled in our study . Sequences originating from our samples were colored in yellow and orange for “Slender” and “Stocky” morphotypes, respectively, while all sequences downloaded from GenBank are in grey.Then, Bayesian inference (BI) in MrBayes v. 3.2.6Supplementary Information.Supplementary data 1.Supplementary data 2.


Robust activation of glial cells has been reported to occur particularly during the pathogenesis of bone cancer pain (BCP). Researchers from our group and others have shown that histone deacetylases (HDACs) play a significant role in modulating glia-mediated immune responses; however, it still remains unclear whether HDACs are involved in the activation of glial cells during the development of BCP.BCP model was established by intra-tibia tumor cell inoculation (TCI). The expression levels and distribution sites of histone deacetylases (HDACs) in the spinal dorsal horn and dorsal root ganglia were evaluated by Western blot and immunofluorescent staining, respectively. Suberoylanilide hydroxamic acid (SAHA), a clinically used HDAC inhibitor, was then intraperitoneally and intrathecally injected to rescue the increased expression levels of HDAC1 and HDAC2. The analgesic effects of SAHA administration on BCP were then evaluated by measuring the paw withdrawal thresholds (PWTs). The effects of SAHA on activation of glial cells and expression of proinflammatory cytokines  in the spinal dorsal horn and dorsal root ganglia of TCI rats were further evaluated by immunofluorescent staining and Western blot analysis. Subsequently, the effects of SAHA administration on tumor growth and cancer cell-induced bone destruction were analyzed by hematoxylin and eosin (HE) staining and micro-CT scanning.TCI caused rapid and long-lasting increased expression of HDAC1/HDAC2 in glial cells of the spinal dorsal horn and dorsal root ganglia. Inhibiting HDACs by SAHA not only reversed TCI-induced upregulation of HDACs but also inhibited the activation of glial cells in the spinal dorsal horn and dorsal root ganglia, and relieved TCI-induced mechanical allodynia. Further, we found that SAHA administration could not prevent cancer infiltration or bone destruction in the tibia, which indicated that the analgesic effects of SAHA were not due to its anti-tumor effects. Moreover, we found that SAHA administration could inhibit GSK3β activity in the spinal dorsal horn and dorsal root ganglia, which might contributed to the relief of BCP.Our findings suggest that HDAC1 and HDAC2 are involved in the glia-mediated neuroinflammation in the spinal dorsal horn and dorsal root ganglia underlying the pathogenesis of BCP, which indicated that inhibiting HDACs by SAHA might be a potential strategy for pain relief of BCP. Cancer is one of the major types of disease that seriously threatens public health. Given the current trend of an increase in survival rates and survival time of patients with advanced cancer, it is predicted that there will be 22.2 million new cancer cases and 13.2 million cancer-related deaths in 2030 [Histone deacetylases (HDACs) have been reported to be closely correlated with the aberrant transcriptional responses in the periphery and central nervous system during chronic pain –6. SerieTo test our hypothesis, BCP models were established via intra-tibia tumor cell inoculation (TCI). Then we compared the distribution sites of HDACs (HDAC1 and HDAC2) in the spinal dorsal horn and dorsal root ganglia during BCP and neuropathic pain induced by spinal nerve ligation (SNL) to evaluate the different roles of HDACs in BCP and SNL. Subsequently, suberoylanilide hydroxamic acid (SAHA), a type of HDACi clinically approved by the Food and Drug Administration, was intraperitoneally injected to explore whether inhibiting HDACs could sufficiently downregulate the activation of glial cells to alleviate BCP. Given that glycogen synthase kinase-3 beta (GSK3β) was a key point of convergence of many signaling pathways to modify neuroinflammation, we further explored whether inhibiting HDACs could inhibit GSK3β activities to alleviate BCP.Female Sprague-Dawley rats 180–200 g) were provided by the Laboratory Animal Center of the Fourth Military Medical University (FMMU). Five to six adult female rats were housed per cage under specific pathogen-free conditions with soft bedding under a controlled temperature 22 ± 2 °C). Before the experiments, all animals were adapted to the experimental circumstances for 5–7 days. All surgeries were performed under anesthesia with sodium pentobarbital . Animals were randomly divided into fourteen groups. For each group of experiments, the animals were age- and body weight-matched. SAHA and AR-A014418 were purchased from Selleckchem . The drug doses were selected based on previous reports [0 g were  ± 2 °C. 7 cells/mL) cancer cells were injected into the abdominal cavities of female rats. Seven to ten days later, 2 mL of extracted ascitic fluid was centrifuged at 1500 rpm for 3 min. Then the pellet was washed with phosphate-buffered saline (PBS) and resuspended in 1 mL of PBS. Subsequently, the cells were diluted with PBS to achieve a final concentration of 5 × 107 cells/mL. The cell suspension was kept on ice until intra-tibia injection. In the Sham group, the same procedures were followed, except that heat-killed carcinoma cell suspension with equal volume and density was administered instead of normal carcinoma cells.Walker 256 rat mammary gland carcinoma cells were purchased from American Type Culture Collection . As described previously , 24, 0.55 cells) was slowly injected into the intramedullary cavity of the right tibia. The injection site was immediately sealed using bone wax when the syringe was removed. In the Sham group, the same procedures were followed, except that an equal volume of heat-treated carcinoma cells was administered instead of normal carcinoma cells. Sham rats on postoperative day (POD) 7 were used as the control for Western blot and immunofluorescent staining.As described in previously reported procedure , 24, aftSix rats were anesthetized and placed in a prone position. Then, the left L6 transverse process was carefully removed with a small rongeur to expose the L4 and L5 spinal nerves. The L5 spinal nerve was then carefully isolated and tightly ligated with a 6–0 silk thread 2–5 mm distal to the dorsal root ganglia. Finally, the wound and surrounding skin were sutured. Animals showing obvious mechanical allodynia were used for the subsequent immunofluorescent staining. The surgical procedures for the Sham group were identical to those of the model group, except that the spinal nerves were not ligated. Sham rats on POD 7 were used as the control for immunofluorescent staining.The intrathecal implantation was performed as described in our previously published study . Briefly3) on an elevated mesh floor. After 30 min of acclimation, mechanical thresholds were tested using von Frey filaments  by experimenters who were blinded with respect to the group assignments. The ipsilateral hind paws were pressed upwards using Von Frey filaments  with gradually increasing stiffness  to cause a slight bend and left for 5–6 s. Each filament was applied ten times, and the minimal value that caused at least six responses was recorded as the paw withdraw threshold (PWT). Acute withdrawal, biting, licking, or shaking of the ipsilateral hind limb and vocalization were considered as positive signs of withdrawal.The rats were habituated to the testing environment for 5–7 days before baseline testing. Baseline nociceptive tests were carried out for three consecutive days prior to the TCI. Animals were discarded in the present study when the difference of the baseline before and after surgery was greater than 4 g. In the test, each rat was put individually under inverted plastic boxes  after analgesia and were exposed to an X-ray source  for 1/20 s at 40 KVP. The X-ray film was developed by a film developer .On POD 21, rats were anesthetized with an overdose of pentobarbital  and were perfused with 150 mL of 0.9% normal saline. The tibial bones ipsilateral to the TCI were then removed and decalcified in 10% ethylenediaminetetraacetic acid for 3 weeks. The bones were rinsed, dehydrated, and then embedded in paraffin, cut into 7-μm cross-sections using a rotary microtome , and stained with hematoxylin and eosin (HE) to visualize the extent of tumor infiltration and bone destruction. Finally, the stained specimens were observed under a bright-field microscope.3) according to the current guidelines [The tibia bone was carefully harvested and fixed in 4% paraformaldehyde until they were scanned with a high-resolution microCT . The scans were performed in the long axis of the diaphysis, with the following basic scan parameters: voltage, 80 kVp; current, 80 μA; exposure time, 3000 ms; total rotation angle, 360°; and rotation angle of increment, 0.4°. Analyses were performed using the Micview V2.1.2 software. The 3D datasets were low-pass filtered and segmented with a fixed threshold filter  containing 4% paraformaldehyde and 2% picric acid. After perfusion, the L4–5 spinal segment and dorsal root ganglia (L4 or L5) were immediately removed and then cryoprotected for 24 h at 4 °C in 0.1 M phosphate buffer containing 30% sucrose. Transverse frozen spinal sections (25 μm in thickness) were then cut with a cryostat  and collected serially into several dishes.The sections were incubated for 1 h at room temperature and overnight at 4 °C with primary antibodies  was used to conduct all statistical analyses. Nociceptive behavioral tests over time among groups were tested with two-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni’s post hoc tests. Differences in Western blot values over time for each group were tested using one-way ANOVA with a Student-Newman-Keuls (SNK) post hoc test. All data are presented as the mean ± SEM. The criterion for statistical significance was a X-ray radiograph showed that there were visibly radiolucent lesions in the proximal epiphysis of the tibias in the TCI group as compared with Sham group on POD 21 . Western blot analysis showed that TCI induced continuous and significant increase in the expression levels of HDAC1 in the spinal dorsal horn from POD 14 to POD 28  results showed that the mRNA expression levels of To explore the roles of HDAC1 and HDAC2 in the spinal dorsal horn during BCP, we further investigated the expression and distribution of HDAC1 and HDAC2 at various time points  following TCI. Rats with SNL (Sham and POD 14) were included in the present study to identify different roles of HDACs in rat models of BCP and neuropathic pain. Immunofluorescent staining showed that the distributions of HDAC1-like immunoreactivities (green fluorescence) were observed in the spinal dorsal horn. Following either TCI or SNL, the immunofluorescent intensity of HDAC1 in the spinal dorsal horn was markedly increased Fig. a. DoubleImmunofluorescent staining showed that the distributions of HDAC2-like immunoreactivities (green fluorescence) were observed in the spinal dorsal horn. In accordance with the Western blot results, confocal images showed that the immunofluorescent intensity of HDAC2 in the spinal dorsal horn markedly increased following TCI or SNL Fig. a. DoubleSimilarly, immunofluorescent staining showed that the immunofluorescent intensities of HDAC1 in the dorsal root ganglia increased following both SNL and TCI Fig. a. The doAs for HDAC2, immunofluorescent staining also showed that the immunofluorescent intensities of HDAC2 in the dorsal root ganglia increased following SNL and TCI Fig. a. The doTo test whether inhibiting HDACs could ameliorate BCP, 50 mg/kg of SAHA, a widely and clinically used HDAC inhibitor, was i.p. injected daily for 21 consecutive days Fig. a. ComparThe immunofluorescent staining and Western blot analysis showed that i.p. administration of SAHA significantly inhibited the TCI-induced upregulation of HDAC1, HDAC2, GFAP, and Iba-1 in the spinal dorsal horn on POD 21 Fig. c–e. HoweTo exclude the possibility that SAHA may work through outside of spinal dorsal horn and dorsal root ganglia, the analgesic effects of i.t. administrated SAHA on BCP were further analyzed. Consistently, i.t. administrated SAHA significantly elevated the PWTs of the TCI rats from POD 6 to POD 14  is a key point of convergence of many signaling pathways that modify neuroinflammation, we further explored whether SAHA administration alleviate BCP by inhibiting GSK3β activities. The Western blot analysis showed that the expression levels of p-GSK3β  in the spinal dorsal horn and dorsal root ganglia significantly decreased on POD 21 following TCI. When SAHA was i.p. injected, the expression levels of p-GSK3β in the spinal dorsal horn and dorsal root ganglia increased significantly. In addition, the expression levels of total GSK3β were not affected by SAHA administration  was used to block GSK3β activity. Compared with the TCI + vehicle group, i.p. AR-A014418 treatment significantly elevated the PWTs of the ipsilateral hind paws, the effects of which started on POD 5 and lasted until the final detection day Fig. a, b. GivIn the present study, we found that the expression levels of HDAC1 and HDAC2 in glial cells of the spinal dorsal horn increased sharply in the rat model of BCP, which was quite different from their distributions in the SNL rats. Meanwhile, our data also showed that HDAC1 and HDAC2 in the satellite glial cells of dorsal root ganglia increased persistently following TCI. Inhibition of HDACs by SAHA, a clinically used HDAC inhibitor, could significantly alleviate BCP, reverse TCI-induced upregulation of HDACs, and inhibit the activation of glial cells and accompanying neuroinflammation in the spinal dorsal horn and dorsal root ganglia. We also confirmed that the analgesic effects of SAHA were not due to its anti-tumor effects. Moreover, we found that the administration of SAHA could significantly upregulate the expression levels of p-GSK3β in the spinal dorsal horn and dorsal root ganglia to inhibit GSK3β activities, which may exert analgesic effects on the BCP. Collectively, these findings suggest that suppression of HDACs by SAHA administration may exert analgesic effects on BCP via inhibiting glia cell-mediated neuroinflammation in the spinal dorsal horn and dorsal root ganglia.When cancer cells metastasize in bone, both cancer cells and their associated stromal cells generate pain by releasing algogenic substances, such as protons and bradykinin , 28. TheAn epigenetic point of view of pain may reveal a new paradigm for developing pain management strategies , 32. It Accumulative evidence has indicated that HDACs played important roles in central sensitization of BCP, and the underlying mechanisms mainly focus on HDAC-mediated effects on neuroplasticity. For example, Hou’s and our previous researches have both indicated that HDACi could restore MOR or KCC expressions in a rat , 35. HowIt has been reported that glial activation can be observed in both BCP and SNL, but glial cells are activated more robustly in the rat model of BCP than in the rat model of SNL , 12. In Emerging evidences have indicated that HDACi administration can exert analgesic effects on neuropathic pain and inflammatory pain , 20, 44.The effects of HDACi on the activation of microglia and astrocytes still remain controversial. Some studies have demonstrated that HDACi can induce anti-inflammatory activities via the suppression of microglial activation , 45–47. Though it is irrefutable that i.p. administrated SAHA not only reversed TCI-induced HDAC upregulation in SDH and DRG but also inhibited the activation of glial cells, but it still exist the possibility that SAHA exerts its role in pain relieving through inhibiting HDACs outside of SDH and DRG, then inhibits glial activation and HDAC expression due to pain relieving. To rule out this possibility, i.t. injection with SAHA was especially performed. Consistently, our data showed that i.t. administrated SAHA could not only attenuate the mechanical allodynia of the ipsilateral hind paws of TCI but also strongly reverse TCI-induced upregulation of HDACs in the dorsal horn and dorsal root ganglia. These finding strongly indicated that the analgesia of SAHA was derived from its inhibitory effects on glial activation in the spinal dorsal horn and dorsal root ganglia.SAHA was licensed in 2006 for the treatment of cutaneous T cell lymphoma following the Food and Drug Administration approval . AlthougGlycogen synthase kinase-3 beta (GSK3β) has been considered as a crucial regulator of the balance between pro- and anti-inflammatory responses in both the peripheral and central nervous systems . It has Collectively, our data suggested that the upregulation of HDAC1 and HDAC2 was implicated in the over-activation of glial cells in the spinal dorsal horn and dorsal root ganglia during pathogenesis of BCP. Inhibition of HDACs by SAHA reversed the glial activation and relieved pain behavior following TCI. Meanwhile, the analgesic effects of SAHA on BCP were not due to its anti-tumor effects. Moreover, the activity of GSK3β might be a potential target regulated by HDACs that participates in pain management during BCP. Our findings indicate that HDACs may be involved in development of BCP via glia-mediated neuroinflammation and suggest the inhibition of HDACs as a novel strategy for treating BCP.Additional file 1 Figure S1. Gene expression of HDAC1~HDAC6 in the spinal dorsal horn at various time points  following TCI. Relative mRNA expression levels of HDAC1 (a), HDAC2 (b), HDAC3 (c), HDAC4 (d), HDAC5 (e) and HDAC6 (F) in the spinal dorsal horn of TCI rats (n = 3). Data are expressed as the mean ± SEM. *p < 0.05, and ***p < 0.001 versus the Sham group.Additional file 2 Figure S2. The effects of i.t. administrated SAHA on TCI-induced mechanical allodynia and upregulation of HDACs. (a) Experimental paradigms. (b) The effects of i.t. administrated SAHA on mechanical allodynia of TCI rats (n = 6 for each group). (c and d) Representative bandsand quantitative analysis of HDAC1 and HDAC2 in the spinal dorsal horn of the TCI + Vehicle and the TCI + SAHA group (n = 4). (e and f) Representative bands and quantitative analysis of HDAC1 and HDAC2 in the dorsal root ganglia of the TCI + Vehicle and the TCI + SAHA group (n = 4). Data are expressed as mean ± SEM. **p < 0.01 ***p < 0.001 versus the TCI + Vehicle group.Additional file 3 Figure S3. The effects of i.t. administrated AR-A014418 on TCI-induced mechanical allodynia. (a) Experimental paradigms. (b) The effect of i.t. administration of AR-A014418 on mechanical allodynia of TCI rats (n = 6 for each group). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 ***p < 0.001 versus the TCI + Vehicle group.Additional file 4. Table S1. Primer sequences used in this study.