Philippa Kaur

Elderflora: A Modern History of Ancient Trees Jared Farmer Basic (2022)About 45 million years ago, when the Arctic was ice-free, the world’s earliest known mummified trees flourished on what is now Axel Heiberg Island in Canada’s Qikiqtaaluk Region. In 1986, palaeobotanists identified the megaflora as members of Metasequoia occidentalis, an extinct redwood species. They had been buried in silt, then frozen, their wood preserved.The lead palaeontologist “celebrated his eureka by kindling a fire with 45-million-year-old twigs and boiling water for tea time,” writes historian Jared Farmer in Elderflora, his expansive global history of grand and venerable trees. Granted, these plants had been dead since the Eocene epoch. Nevertheless, as the author describes, the incident is part of a troubling pattern in which scientists rejoice at their discovery of the ‘oldest’ tree of their time — and then destroy it.In 1957, for example, Edmund Schulman at the University of Arizona in Tucson spent the summer seeking ancient bristlecone pines in California’s White Mountains. He found three more than 4,000 years old, and named them Alpha, Beta and Gamma. Then, in the interests of tree-ring science, he chose to “sacrifice” Alpha, taking snapshots as his nephew and a colleague sawed it down. When the University of Arizona issued a press release titled ‘UA Finds Oldest Living Thing’, Farmer writes, “they say nothing about the thing being dead”.Schulman’s aim was dendroclimatology — the reconstruction of climates using tree-ring data. That lofty motive cannot be ascribed to those who, in 1881, bored a tunnel into the 2,000-year-old Wawona tree in Yosemite National Park, allowing tourists to drive their cars through the 71.3-metre-high giant sequoia (Sequoiadendron giganteum), since toppled.Arboreal legendsAs Elderflora shows, big, old trees are objects of veneration and vandalism, appearing “in the oldest surviving mythologies and the earliest extant texts”. They were associated with gods and heroes, prophets and gurus: they had pivotal roles in the Mesopotamian Epic of Gilgamesh and in the Polynesian legend of Rātā, who fells a noble tree to carve a canoe. In more recent times, European settlers “dispossessed Indigenous peoples and cleared forests with abandon”. Research shows that, for 8,000 years after the glaciers of the last ice age retreated, forests in the Midwestern United States doubled in biomass (A. M. Raiho et al. Science 376, 1491–1495; 2022). Just 150 years of industrial logging and agriculture erased this carbon accumulation.
It takes a wood to raise a tree: a memoir
“Imperial conquests and industrial revolutions relied on timber,” Farmer writes. “Wood-stock long guns for capturing lands and peoples; naval vessels with mighty masts for transporting the enslaved and the harvests of their labor.” In New Zealand, European settlers decimated the majestic kauri trees, which can live for up to 2,000 years and that once covered 1.2 million hectares of land. The trees’ 50-metre-trunks became ships’ masts; their resin was made into varnish and linoleum.Like pines, firs, spruces, cedars, cypresses and redwoods, kauri (Agathis australis) is a gymnosperm. These flowerless plants with naked seeds tend to grow slower and live longer than angiosperms, flowering plants that bear fruit. About 25 plant species — most of them conifers — can live for more than a millennium without human assistance, surviving in restricted, vulnerable habitats.Farmer also offers a global survey of ancient trees that have been protected and exalted. They include olive trees of the Levant (Olea europaea); research published this year shows that these were domesticated about 7,000 years ago for their fruit and oil (D. Langgut and Y. Garfinkel Sci. Rep. 12, 7463; 2022). In Africa, the baobab (Adansonia sp.) is both the longest-lived tree and the largest, offering shade and shelter, foods, medicines and textiles. Enslaved Africans planted baobabs in the Caribbean; some survive still. Ginkgo biloba, a species that dates back 390,000 years, survived only in China, whence it was spread around the world in the past millennium. A grove of ginkgo trees survived the atomic bombing of Hiroshima in Japan in August 1945, pushing out new buds the following spring.The planet’s current tree cover, Farmer writes, includes 3 trillion large plants covering about 30% of all land. It is, in fact, expanding. But the new cover consists mostly of shelter belts (trees planted to protect crops or animals), temperate-zone timber crops and tropical plantations of eucalyptus and palm oil. A shrinking proportion of tree cover is made up of species-rich old-growth communities.Epic loss“What would humans and nonhumans stand to lose if these survivors all died prematurely? A world of things,” Farmer writes. “Old trees sustain forest communities” with their seeds and litter. Other plants grow on them, and animals live in them. Their roots share nutrients with other organisms via underground fungi. Groups of “Old Ones” are carbon sinks. Large-scale monocultures are shorter-lived and take less greenhouse gas out of circulation.But even bygone trees of the once-tropical Arctic might offer lessons for a warming world. Palaeobotanist Hope Jahren, in her 2016 memoir Lab Girl, describes how she spent three summers on Axel Heiberg Island, digging “through a hundred vertical feet of time”. Fir, cypress, larch, redwood, spruce, pine and hemlock trees populated this lush conifer forest, with an understory of angiosperms: maple, alder, birch, hickory, chestnut, beech, ash, holly, walnut, sweetgum, sycamore, oak, willow and elm. These plants thrived even through three months of winter darkness and three of constant summer light.“Here stood one of the great forests of all time,” Farmer writes. Today, as the Arctic warms nearly four times as fast as any other place on Earth, the genomes of species related to the trees of this mummified forest might be adaptable enough for the trees to flourish in a rewarmed planet, he says. Old trees have much to teach us: we would be wise to listen. More

Animal experimentsTeratoma formation experiments were performed at Iwate University. All surgical procedures and animal husbandry were performed in accordance with the international guidelines of the Animal Experiments of Iwate University and were approved by the university’s Animal Research Committee (approval number A201734).Chicken embryonic fibroblasts (Rhode Island Red) were obtained from a primary culture of chicken embryonic tissue provided by Prof. Atsushi Tajima, Tsukuba University. Chicken culture cells were obtained from chicken embryos, and the acquisition of these cells did not require approval. Mouse embryonic fibroblasts (CF-1 strain) were purchased from a manufacturer (CMPMEFCFL; DS Pharma Biomedical, Osaka, Japan). Approval was not required to obtain these cells.Somatic cells were obtained from wild animals (ex., Okinawa rail). The sampling details described below do not include the exact location of sampling to protect against poaching.Fibroblast cells from Okinawa rail and Japanese ptarmigan were obtained from dead animals, such as those killed by vehicles (Fig. 1A and Supplementary Fig. 1). Approval was not required to obtain these samples.Dead Okinawa rail were found on May 21, 2008, by the Okinawa Wildlife Federation, a nonprofit organization that focuses on the conservation of wild animals in the Okinawa area in the southwest region of Japan. The organization has permission from the Japanese Ministry of the Environment (MOE) to handle and perform first aid activities on endangered animals. The dead birds were transferred the following day to the National Institute for Environmental Studies (NIES). Primary cell culture was carried out from muscle tissue and skin of the dead birds (NIES ID: 715A).On July 8, 2004, tissues recovered from dead Japanese ptarmigan (e.g., skin and retina tissues) were also transferred to NIES from Gifu University Department of Veterinary Medicine. Primary cell culture from this tissue was performed (NIES ID: 22A).Somatic cells from Blakiston’s fish owl and Japanese golden eagle were obtained from emerging pinfeathers. Concerning the Blakiston’s fish owl, the MOE carries out bird banding, of wild birds with identification tags. The emerging pinfeathers we used had been accidentally release during banding. The banding had been performed by a veterinarian at the Institute for Raptor Biomedicine Japan (IRBJ) in the Hokkaido area on June 2, 2006. IRBJ is a private organization that primarily focuses on emergency medicine first aid and care for wild avians in Hokkaido region of Japan. IRBJ is contracted to MOE to handle and administer first aid for endangered animals. The MOE banding ring was 14C0242. Since banding was carried out with the permission of MOE for capturing wildlife, we did not require the approval to obtain these avian somatic cells. On July 8, 2006, Blakiston’s fish owl pinfeathers were transferred to from IRBJ to NIES, where primary cell culture was performed (NIES ID: 215A).Concerning the Japanese golden eagle, an emerging pinfeather accidentally fell off a bird during blood collection at the Yagiyama Zoo in Sendai, Japan on July 11, 2018. Dr. Yukiko Watanabe, an IRBJ veterinarian, collected the emerging pinfeather. The sample was shipped the following day to NIES where primary cell culture was performed (NIES ID: 5228).In addition to these birds, we obtained somatic cells emerging avian pinfeathers of Steller’s sea eagle, white-tail eagle, mountain hawk-eagle, northern goshawk, Taiga bean goose, and Latham’s snipe. These samples were provided by IRBJ.Concerning the Steller’s sea eagle, an injured individual was found in Hokkaido on July 11, 2006 (ID: 06-NE-SSE-1). The eagle was transferred to IRBJ. On December 4, 2006, IRBJ veterinarian Dr. Keisuke Saito collected fallen pinfeathers. Primary cell culture was performed at NIES on December 8, 2006 (NIES ID: 369A).Concerning the white-tailed eagle, an injured individual was found in Hokkaido, Japan, on July 12, 2007 (ID: 07-NE-WTE-4). The bird was transferred to IRBJ the same day for emergency treatment. On January 15, 2008, Dr. Saito collected fallen pinfeathers. Primary cell culture was performed on January 18, 2008 at NIES (NIES ID: 492A).Concerning the mountain hawk-eagle, an injured individual was found in the Hokkaido area on August 10, 2008 (ID: 08-Tokachi-HHE-2). The bird was transferred to IRBJ the same day. The bird was treated by an IRBJ veterinarian, but died on September 8, 2008. Emerging pinfeathers were collected from the dead bird by Dr. Saito. Primary cell culture was performed on September 11, 2008 at NIES (NIES ID: 847A).Concerning the Northern Goshawk, IRBJ accepted an injured bird for treatment on June 12, 2006. Following treatment and recovery, the bird was released into the wild in the Hokkaido area on August 1, 2006. During the treatment (July 4, 2006), Dr. Saito collected fallen pinfeathers. The primary cell culture was performed at NIES on July 6, 2006 (NIES ID: 222A).Concerning the Taiga bean geese, an injured individual was found in Hokkaido on September 15, 2016 (ID: 13B8005). The injured bird was transferred to IRBJ the same day for emergency treatment. On September 16, 2016, IRBJ veterinarian Dr. Yukiko Watanabe collected fallen emerging pinfeathers. Primary cell culture was performed on September 20, 2016 (NIES ID: 4420A).Finally, concerning the Latham’s snipe, fallen pinfeathers were collected during MOE approved bird banding performed on September 17, 2006, by Dr. Saito. Dr. Saito also collected fallen emerging pinfeathers (ID: 6A22598). The samples were transferred to NIES on September 20, 2006, for primary cell culture (NIES ID: 338A).All records are available at NIES.Cell culture and preservationOkinawa rail, Japanese ptarmigan, and Blakiston’s fish owl-derived fibroblasts were preserved in liquid nitrogen for 8–12 years (Fig. 1f). The preservation solution contained 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide. Cells were preserved at a cell density of 1 × 106–4 × 106 cell/mL. During the freezing period, the cells were maintained at minus The cells were frozen at a temperature of −135 °C. Japanese golden eagle fibroblasts were used without freezing.Avian-derived fibroblasts were cultured with Kuwana’s modified avian culture medium-1 (KAv-1), which is based on alpha-MEM containing 5% FBS and 5% chicken serum23. Mouse embryonic fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% antibiotic–antimycotic mixed stock solution (161–23181; Wako Pure Chemical Industries, Osaka, Japan). All avian and mouse cells were cultured at 37 °C under 5% CO2.Reprogramming vectorWe chemically synthesized an expression cassette that included seven reprogramming factors (MyoD-derived transactivation domain-linked Oct3/4, Sox2, Klf4, c-Myc, Klf2, Lin28, and Nanog; all genes derived from mice). The self-cleaving 2A peptide was inserted at the junction of the coding region (Fig. 1g). We transferred the complementary DNA (cDNA) insert from the shuttle vector to the PiggyBac transposon vector containing green fluorescent protein (PB-CAG-GFP). Although the original transposon vector drive the expression of cDNA with the elongation factor-1 (EF1) promoter (PJ547-17; DNA 2.0, Menlo Park, CA, USA), we replaced the EF1 promoter to CAG promoter in our previous study22,24. The reprogramming vector was designated PB-TAD-7F (Fig. 1g).In addition to the PB-TAD-7F reprogramming vector, we used the PB-DDR-8F reprogramming vector to establish Japanese golden eagle iPSCs. The complete coding sequence of DDR-8F (DDR-Oct3/4, Sox2, Klf4, c-Myc, Klf2, Nanog, Lin28, and Yap) was chemically synthesized. The expression cassettes containing the eight reprogramming factors were excised from the shuttle vector using restriction enzymes. The cDNA fragments were transferred to the PB-CAG-GFP PiggyBac transposon-based vector22,24. Detailed information regarding the PB-DDR-8F reprogramming vectors is shown in Fig. 10a.Establishment of iPSCsWe transfected PB-R6F or PB-TAD-7F reprogramming vectors into mouse, chicken, Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl-derived fibroblasts using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). After hygromycin selection (Wako Pure Chemical Industries), the cells were reseeded onto a mouse embryonic fibroblast (MEF) feeder layer. On days 14–32, we picked primary iPSC-like colonies and seeded them on new MEF feeder cell plates. The detailed protocol is shown in Fig. 1h.To establish Japanese golden eagle-derived iPSCs, we transduced PB-TAD-7F or PB-DDR-8F reprogramming vectors into Japanese golden eagle pinfeather-derived somatic cells. Transfection was performed using Lipofectamine 2000 transduction reagent (11668019; Thermo Fisher Scientific) according to the manufacturer’s instructions. After hygromycin selection (Wako Pure Chemical Industries), cells were seeded onto feeder culture plates. The golden eagle iPSCs were cultured in KAv-1-based medium5.The medium used to establish avian iPSCs was supplemented with 1000 × human Leukemia Inhibitory Factor (LIF) (125–05603; Wako Pure Chemical Industries), 4.0 ng/ml basic FGF (064–04541; Wako Pure Chemical Industries), 0.75 μM CHIR99021 glycogen synthase kinase-3 inhibitor (034–23103; Wako Pure Chemical Industries), 0.25 μM PD0325901 mitogen-activated protein kinase inhibitor (163–24001; Wako Pure Chemical Industries). In addition to those supplements, 0.25 μM thiazovivin (202–18011; Wako Pure Chemical Industries) was added in the media used to generate Okinawa rail, Japanese ptarmigan, Blakiston’s fish owl, and chicken iPSCs. In the medium used to generate mouse iPSCs, we added 1000 × LIF, 0.75 μM CHIR99021, and 0.25 μM PD0325901.iPSC culture conditionsTwo types of cell culture media were used: KAv-1 for avian iPSCs and DMEM for mouse iPSCs. The composition of KAv-1 for avian iPSCs was as follows: alpha-MEM containing 5% FBS and 5% chicken serum 1% antibiotic–antimycotic mixed solution, 1% nonessential amino acids (Wako Pure Chemical Industries), and 2 mM glutamic acid was added (Nacalai Tesque, Kyoto, Japan). The composition of DMEM for mouse was follows: DMEM supplemented with 15% SSR, 0.22 mM 2-mercaptoethanol (21438–82, Nacalai Tesque), 1% antibiotic–antimycotic mixed solution, 1% nonessential amino acids5,22. As a supplement to the iPSC medium, we used 1000 × human LIF (125–05603; Wako Pure Chemical Industries), 4.0 ng/ml basic FGF (064–04541; Wako Pure Chemical Industries), 0.75 μM CHIR99021 (034–23103; Wako Pure Chemical Industries), 0.25 μM PD0325901 (163–24001; Wako Pure Chemical Industries) for the media used to culture Okinawa rail, Japanese ptarmigan, Blakiston’s fish owl, Japanese golden eagle, and chicken-derived iPSCs. The supplements for media used to culture Okinawa rail and Japanese ptarmigan-derived iPSCs included 2.5 μM Gö6983 (074–06443, Wako Pure Chemical Industries). To analyze the cellular characteristics, we focused on the Janus kinase (JAK), FGF, ROCK, and glycolytic pathways, since the dependency of these pathways can indicate differences in cellular characteristics. We used 1–10 μM JAK inhibitor I (4200099; MERCK, Darmstadt, Germany), 0.5–4 μM of PD173074, which inhibits FGF receptor (FGFR) inhibitor (160–26831; Wako Pure Chemical Industries), 10 μM of Y27632, which inhibits ROCK (036–24023; Wako Pure Chemical Industries), and 2 or 4 mM 2-deoxyglucose (2DG, D0051; Tokyo Chemical Industry, Tokyo, Japan).AP and immunological staining of fibroblasts and iPSCsA red-color AP staining kit (AP100 R-1; System Bioscience, Palo Alto, CA, USA) was used to detect AP activity of iPSCs. iPSCs were stained for SSEA-1, SSEA-3, and SSEA-4 antibodies (Supplementary Table 2). To stain the iPSCs with the SSEA antibodies, the cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 3 min. Cells were permeabilized by 0.5% Triton X-100 (35501-15; Nacalai Tesque, Kyoto, Japan) for 60 min. After three washes with PBS, the iPSCs were blocked with 1% bovine serum albumin (BSA, 01863-06; Nacalai Tesque) for 45 min. iPSCs were incubated with a primary antibody overnight and then exposed to the corresponding fluorescent-labeled secondary antibodies for 60 min. Counterstaining was performed with a 4′,6-diamidino-2-phenylindole (DAPI) solution (Cellstain-DAPI solution, DOJINDO, Kumamoto, Japan).Japanese golden eagle and chicken-derived fibroblasts were seeded in 12-well cell culture plates for immunological staining. After 48 h of incubation, F-actin staining was performed using Alexa Fluor 568 phalloidin (A12380; Thermo Fisher Scientific) according to the manufacturer’s protocol. Double staining was performed with an anti-vimentin antibody (MA5-11883; Thermo Fisher Scientific) and Alexa Fluor 488-labeled secondary antibody (A-11001; Thermo Fisher Scientific) (Supplementary Table 2). The samples were counterstained with Cellstain-DAPI solution (DOJINDO) as described above.Detection of reprogramming vectors and internal control genes from iPSCsDNA was isolated using the EZ1 DNA Tissue Kit (953034; QIAGEN, Hilden, Germany). PCR was performed with 100 ng of template DNA. Primer sequences are listed in Supplementary Tables 3 and 4. We performed PCR assays using KOD FX Neo (KFX-201; TOYOBO, Osaka, Japan). PCR was conducted by predenaturation at 94 °C for 2 min, denaturation at 98 °C for 10 s, and extension at 68 °C for 30 s, with 40 cycles of denaturation and extension. PCR products were analyzed by electrophoresis on 2.0% agarose/Tris-acetate–ethylenediaminetetraacetic acid (EDTA) gels.Sequential passagingMouse, Okinawa rail, and Japanese ptarmigan-derived primary cells and iPSCs were seeded in six-well plates with feeder cells for analysis. When cell growth became confluent, all cells and the number of cells per dish was enumerated using a Countess cell counter (Thermo Fisher Scientific). The harvested and seeded cell numbers were used to calculate the PD time as an indicator of the speed of cell growth, using the formula PD = log2 (A/B), where A is the number of harvested cells at the end of each passage, and B is the number of seeded cells at the start25.Detection of mRNA expressionTotal RNA was isolated from iPSCs using an EZ1 RNA Tissue Mini Kit (959034; QIAGEN). cDNA was synthesized from total RNA using the PrimeScript reverse transcription (RT) reagent kit (Perfect Real Time, RR047A; TaKaRa Bio, Ohtsu, Japan). Real-time PCR was performed in a 12.5 μl volume containing 2 × KOD SYBR qPCR Mix (QKD-201; Toyobo), 10 ng of cDNA solution, and 0.3 μM of each primer. The primer sequences are listed in Supplementary Tables 5–10. The reaction was performed in duplicate. The cycling program was as follows: 98 °C for 120 s (initial denaturation), 98 °C for 10 s (denaturation), 58 °C for 10 s (annealing), and 68 °C for 32 s (extension) for 40 cycles. We normalized the expression levels of the target genes to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Mitochondria stainingMitochondria were stained by incubation with 50 nM MitoTracker Orange (M7510; Thermo Fisher Scientific) or 20 nM tetramethyl rhodamine ethyl ester perchlorate (TMRE, T669; Thermo Fisher Scientific) for 10 min. After staining, the solution was removed, and fresh medium was added for observation.EB formation and in vitro differentiationIn vitro differentiation of Okinawa rail, Japanese ptarmigan, Blakiston’s fish owl, and Japanese golden eagle iPSCs was performed. To generate EBs, iPSCs were seeded in low-binding dishes in KAv-1 medium. After 7–14 days, floating EBs were selected and seeded in 0.1% gelatin-coated 6-well plates with KAv-1 medium. To induce differentiation into neural cells, the floating EBs were cultured in 0.1% gelatin-coated plates containing KAv-1 supplemented with 10 μM ATRA and 4.0 ng/ml FGF for 7 days.Cells were immunochemically stained after in vitro differentiation using antibody to TUJ1, alpha-smooth muscle, or Gata4 (Supplementary Table 2). Differentiated cells were stained based on the immunological staining procedure of iPSCs described above.Teratoma formation and tissue sectioningThe Animal Committee of Iwate University approved the experimental protocol for teratoma formation (approval numbers A201734, A201737). For teratoma formation, 1 × 106 iPSCs were injected into the testes of SCID mice (C.B-17/Icr-scid/scidJcl; CLEA Japan, Tokyo, Japan). After 4–34 weeks post-injection, tumor tissues were excised from the mice. Each tumor tissue was fixed with 10% formaldehyde in PBS. Fixed tissue sections were stained with hematoxylin-eosin (HE) and observed by microscopy.Immunological staining was performed in addition to HE staining. For immunological staining, antibody to TUJ1, alpha-smooth muscle, or Gata4 was used (Supplementary Table 2). The paraffin block of each teratoma was sliced to produce a section 5 μm thick. After deparaffinization, the antigen was activated with citric acid buffer (SignalStain Citrate Unmasking Solution (10×), 14746; Cell Signaling Technology, Beverly, MA, USA) by microwaving for 10 min. To block endogenous peroxidase, tissue sections were incubated with 3% hydrogen peroxide (081–04215; Wako Pure Chemical). After washing with purified water, the tissue sections were incubated with 5% goat serum (555–76251; Wako Pure Chemical) in PBS. Next, the section were incubated in a solution containing a 1:100 dilution of primary antibody overnight at 4 °C. After washing with PBS, the tissue sections were incubated with horseradish peroxidase (HRP) conjugated secondary antibody (anti-IgG (H+L chain), mouse, pAb-HRP, code no. 330; MBL Co., Ltd., Nagoya, Japan) or anti-IgG (H+L chain, rabbit, pAb-HRP, code no. 458; MBL) for 1 h (Supplementary Table 2). After washing with PBS, the tissue sections were incubated with 3,3′-diaminobenzidine substrate solution (Histostar, code no. 8469; MBL) for 5–20 min. After washing with purified water, tissue sections were counterstained with hematoxylin for 1–2 min.DNA component analysisCultured cells fixed with 70% ethanol at least 4 h under −20 °C condition. The fixed cells stained with the Muse Cell Cycle Assay Kit (Merck Millipore Corporation, Darmstadt, Germany). The stained cells analyzed with Muse Cell Analyzer (Merck Millipore Corporation) were used for DNA content analysis.Karyotype analysisOur iPSCs were treated with 0.02 mg/ml colcemid. Those iPSCs exposed to a hypotonic solution and fixed with Carnoy’s fluid. We counted the chromosomal number in 50 cells and performed a G-banding analysis in 20 cells22.Production of interspecific chimeras and their immunological stainingTo evaluate whether iPSCs derived from Japanese ptarmigan could contribute to the generation of interspecific chimeras in chick embryos, iPSCs were stained with 10 μM CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, C7025; Thermo Fisher Scientific) for 30 min. Eggs of white leghorn chicken were purchased from a local farm (Goto-furanjyo, Gifu, Japan). We injected the labeled Japanese ptarmigan iPSCs into stage X chick blastoderms and cultured the embryos26. To confirm the contribution of chimera, fluorescence was observed after 72 h. To analyze the tissue-level contribution of chimera, embryos on day 5. The embryos were embedded in optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan), frozen in liquid nitrogen, and stored at −80 °C until use. Cryosections 20 μm in thickness were prepared using a cryostat, air-dried for 30 min at room temperature, and fixed with 4% paraformaldehyde for 2 min at room temperature. After washing three times with PBS, sections were incubated with PBS containing 5% FBS for 1 h. After blocking with FBS, the sections were incubated with an anti-hygromicin resistance gene antibody (anti-HPT2; Supplementary Table 2) overnight. After washing three times with PBS, the sections were incubated with secondary antibody (goat anti-mouse IgG, Alexa Fluor 568; Supplementary Table 2) and Cellstain- DAPI solution (DOJINDO) for 1 h.Detection of contribution of chimera from genomeWe injected Japanese ptarmigan iPSCs (without CellTracker Green CMFDA label) into a stage X chicken blastoderms. On day 5, the entire chicken embryos were collected. The genome of each embryo was collected using NucleoSpin Tissue (U0952S; MACHEREY-NAGEL, Düren, Germany). After collecting the chimeric genome, we detected the reprogramming vector cassette using genomic PCR analysis using 50 ng of template genome. To extend the target sequence, we used the KOD FX Neo (KFX-201; TOYOBO). Primer information is provided in Supplementary Table 11. This analysis was performed according to the manufacturer’s protocol. The cycling program comprised 45 cycles of 94 °C for 120 s (initial denaturation), 98 °C for 10 s (denaturation), and 68 °C for 50 s (annealing and extension). After PCR, 2% agarose gel electrophoresis was performed. Gels were stained with GelGreen (517–53333; Biotium, Inc., Fremont, CA, USA).Real-time PCR was also performed to detect the contribution of chimera. The fluorescence probe and primers designed to detect chimeric contributions are summarized in Supplementary Table 12. The template was a 30 ng genome. The analysis was performed using 1 × THUNDERBIRD Probe qPCR Mix (QPS-101; TOYOBO), 0.3 μM of each primer, 0.2 μM of probe, and 1 × Rox. Fifty cycle of 95 °C for 60 s (initial denaturation), 95 °C for 15 s (denaturation), and 60 °C for 60 s (annealing and extension) were used. The expression levels of the target genes were normalized to that of chicken Tsc-2.RNA preparation and sequencing for RNA-seq analysisTotal RNA from iPSCs, fibroblasts, and chicken embryo stage X was collected using NucleoSpin Tissue (740952.50; MACHEREY-NAGEL). Triplicate samples of all iPSCs, fibroblasts, and chicken embryo stage X were prepared. To prepare the library, we used the TruSeq Stranded mRNA LT Sample Prep Kit (RS-122-2101; Illumina, San Diego, CA, USA). The quality of the library was evaluated using the Qubit DNA Assay (Thermo Fisher Scientific) on a TapeStation with a D1000 screen tape (Agilent Technologies, Santa Clara, CA, USA). The cDNA samples were used for the sequencing reaction on an Illumina HiSeq X sequencing machine, resulting in more than 40 M reads with 150 bp ends for each sample, except chicken fibroblast No. 3, which displayed more than 40 M reads with 75 bp ends. To analyze the RNA-seq data, we used the CLC Genomic Workbench (CLC Bio, Aarhus, Denmark). In the trim read step, low-quality sequence with the quality score of the CLC workbench, 5′ end, 3′ end, and short sequences (shorter than 15 sequences) were removed. The trimmed sequence data were mapped onto the chicken reference genome. Gene expression data were obtained in this step. PCA was performed and a heat map created with CLC Genomic Workbench using gene expression data. In this step, normalization was automatically performed using TMM methods. To compare chicken cells, RNA-seq data from SRA (SRP115012 (GEO: GSE102353) and SRP087639 (GSE86592) were used. The RNA-seq data has been submitted to the DNA DataBank of Japan under accession number DRA013522 (Submission), PRJDB13093(BioProject), SAMD00444261–SAMD00444287 (BioSample).Statistics and reproducibilityNonparametric multiple comparison analysis used the Steal–Dwass test (Figs. 2e, 3 [Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl], 4d, 4f, 5b, 5d, 5f, 5h, 10i). For nonparametric independent two-group analysis, we used the Mann–Whitney U test (Fig. 3, for mouse and chicken, and 4b). Statistically significant differences are indicated by *(p  More

arising from J. Cheng et al. Nature Communications https://doi.org/10.1038/s41467-021-22087-2 (2021)Recently, Cheng J. et al.1 cited and simulated the relative percentage tree cover to interpret the ~3000–4000 years lag between tree cover and East Asian Summer Monsoon (EASM) rainfall. They concluded that vegetation feedback has caused a lagged ecosystem response to EASM rainfall during the Holocene (11.7–0 ka). Here, we question the feasibility of using the relative percentage tree cover to measure vegetation feedback to climate. First, the land cover in northern China includes forests, grasslands, and bare land2. Cheng J. et al.1 did not consider the role of bare land in climate feedback models. Absolute land cover, including forest, grassland, and bare land can accurately reveal feedback to climate3. Second, the biome reconstructions they cited represent changes in vegetation type only, whereas the relationship between vegetation type and vegetation cover is altered by many other factors3,4,5. Third, the paper they cited6 averaged the vegetation types on a millennium scale with an interval of 1000 years, so the view that vegetation has a ~3000–4000 years lag in EASM rainfall is not credible as the lag can be enlarged by data resolution. Therefore, absolute vegetation cover, not relative cover, is a prerequisite for studying ecosystem feedback.Our previous work3 was the first to reconstruct the absolute vegetation cover in northern China based on pollen concentrations in two well-dated sediment cores. Using a random forest method, the vegetation cover at Dali Lake in the forest-steppe transition in northern China was determined for the period from 19,000 cal. yr BP to the present with a resolution of approximately 200 years. Han et al.3 showed that tree cover peaked during the early Holocene and it has gradually declined since the middle Holocene. Pollen percentages are widely used in vegetation reconstruction, but they are challenging to analyze because they can be similar in composition, despite being produced by very different flora7,8. This means they represent the relative fractions of vegetation type and not the absolute vegetation cover (Fig. 1A, B). Han et al.3 suggested that pollen concentration data are suitable for the reconstruction of absolute vegetation cover, particularly in arid and semi-arid regions. Dali Lake represents a typical lake in the semi-arid area of northern China. The random forest model showed that the area had a high tree cover during the early Holocene, which suggests that tree cover was a timely response to Holocene monsoon rainfall and there was no time lag at this specific location. Therefore, the data at Dali Lake challenge the conclusion that tree cover has a ~3000–4000 years lag to EASM rainfall.Fig. 1: Trends in pollen percentage, absolute vegetation cover and fire history during the Holocene at Dali Lake, a typical lake in the semi-arid area of northern China.Changes in the percentages of arboreal and non-arboreal pollen (A)3. Reconstructed absolute tree and grass cover (B)3, with gray, yellow, and green shaded areas indicating the standard deviation of 1000 random forest model results for total cover, grass cover, and tree cover, respectively. Normalized fire activity index in the northern region of eastern monsoonal China (C)10. Z-score of transformed charcoal value showing fire activity trend in the temperate steppe of northern China (D)11.Full size imageMoreover, Cheng J. et al.1 used −17 °C as the threshold for tree and grass transition, but this could be an incorrect citation from Bonan et al.9. In the original text by Bonan et al.9, they showed that both temperate deciduous broadleaved trees and C3 grasses have a tolerance of −17 °C for the coldest month for their survival, but this temperature is not the favored threshold for the shift from grasses to trees. Actually, the trend in absolute vegetation cover was mainly driven by summer temperature, annual precipitation, and fire incidents, which is in line with the vegetation-climate relationships at Dali Lake3. That is, higher monsoon rainfall could increase the competitiveness of trees, while increased fire could increase the competitiveness of grasses as grasses are mostly annual and perennial, and they renew faster than trees after a fire. Between 10,000 and 8000 cal. yr BP, monsoon rainfall peaked and there were relatively few fires, which led to a significant increase in absolute tree cover. Since 6500 cal. yr BP, monsoon rainfall decreased and fire increased, resulting in stronger competition by grasses, which has led to an increased grass cover and reduced tree cover3 (Fig. 1B).The impact of secondary disturbances on vegetation dynamics requires careful consideration, particularly the impact of fires on vegetation cover in semi-arid areas of China, even though vegetation growth is strongly constrained by rainfall in this region. Both the normalized fire index in the northern region of eastern monsoonal China10 and the charcoal value in the temperate steppe of northern China11 show a clear antiphase relationship with the absolute forest cover of the Dali Lake region (Fig. 1B, C, D). However, Cheng J. et al.1 did not discuss fire incidence on vegetation evolution in northern China. Fire has occurred frequently through the Holocene10,12 and it plays an important role in vegetation dynamics based on observational evidence13,14. Particularly, fire is considered as a triggering disturbance that can reduce a forest’s resilience to drought under a drying climate during the mid to late Holocene. For Dali Lake, fire and drying climate has co-driven the evolution of vegetation cover since 6500 cal. yr BP3. Moreover, in northern China, data from Daihai Lake and Hulun Nuur Lake also suggest that fire has accelerated the decline of forest cover and the transition from forest to grass during the Holocene13,14. Unfortunately, Cheng J. et al.1 did not discuss the effect of fire when citing and interpreting the observation data, as scale-dependent fires could make the transition between forest and grassland and their interactions variable.In summary, we believe there are three flaws in the data interpretation of Cheng J. et al.1. (1) They contradict the observed evolution of absolute tree cover at Dali Lake in northern China, which is representative of the marginal area of EASM. Relative percentage tree cover cannot accurately reflect a forest’s response and feedback to past climate change. (2) Both temperate deciduous broadleaved trees and C3 grasses have a tolerance of −17 °C for the coldest month for their survival. Thus, −17 °C is not a correct threshold for the shift from grasses to trees. (3) They contradict the ecological theories of secondary disturbance on vegetation dynamics. Fire and other secondary disturbances may be crucial to the transition between forest and grassland. Interpretations of vegetation feedback might be biased if these important factors are not fully considered. Based on the evidence, their conclusion that vegetation feedback causes lagged ecosystem response to EASM rainfall during the Holocene could be problematic. More

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replying to Y. Cheng et al. Nature Communications https://doi.org/10.1038/s41467-022-33958-7 (2022)We welcome the comment from Matters arising from Cheng Y et al.1, which provides us an opportunity for further clarification of some of our points2. The Comment raised interesting and important issues about our paper, that undoubtably could enhance our understanding for the Holocene vegetation evolution in the northern China and its relationship with East Asian Summer Monsoon (EASM). In particular, the results from Dali lake pose the questions on the timing of the peak of tree cover, that invokes the further investigation to understand this complex tree changes over Holocene period. However, these comments do not impact the key result in our original study2, that is the periodical asynchronous evolutions between EASM and northern China ecosystem under specific conditions.Main points of our paper are: First, we propose that the EASM and its rainfall over northern China mainly followed the variation of the summer insolation and peaked in the early Holocene, while the relative tree cover of temperate deciduous broadleaf tree peaked in the mid-Holocene; the delayed tree cover peak is caused by the winter warming, and peak soil moisture also in the mid-Holocene, which could be related to a hydrological impact from vegetation shift from grass to tree and the positive feedback between this vegetation shift and soil wetting.Second, this asynchronous evolution between the EASM rainfall, which peaks in the early Holocene, and the northern China ecosystem, which peaks in the mid-Holocene, is caused mainly by the opposing effect of residual ice sheet retreat on the decreasing summer insolation. The declining summer insolation does cause a substantial decrease of EASM rainfall from the early to mid-Holocene. However, 2/3 of this rainfall decrease is canceled by the rainfall increase forced by the retreat of residual Laurentide ice sheet, resulting in a weak decreasing trend of rainfall over this period.Third, under this background of weak rainfall changes, winter warming, induced by increased winter insolation and ice sheet retreat, raised the coldest temperature to above −17 °C, the threshold for the survival of temperate deciduous broadleaf tree3, and then favored an increase in tree, meanwhile induced a decrease in grass for reasons of its lower competitiveness than that of tree. This vegetation shift then supported the wetting of northern China through its hydrological effect2. The vegetation shift and soil wetting could reinforce each other. Furthermore, the dominant effect of winter warming on vegetation from the early to mid-Holocene is supported by our sensitive experiments with an off-line land-vegetation model.As stated in Cheng Y’s Comment1, the land cover in northern China includes forests, grass and bare land. In our interpretation, the process of “the vegetation feedback to climate” is mentioned as a possible feedback that enhances this asynchronous response, but is not critically involved in the mechanism. As such, whether the absolute or relative vegetation cover is not a major issue in our discussion. It’s sure that the reconstructed absolute tree cover, which based on pollen concentration, could enrich our understanding of the vegetation changes over the Holocene period in northern China. Indeed, the hydrological impact of bare land (evaporation) had been considered in our hydrological analysis of northern China soil moisture, and the results indicated its impact is important but not critical to the Holocene long-term changes of soil moisture over this region. The relative tree cover, the percentage of cool mixed tree (COMX4) in fossil pollen which is consistent with that of temperate deciduous broadleaf tree in simulation2, that we cited4 is a synthesis of 31 records, which represented the general evolution of vegetation over a large part of northern China, and its main result is consistent with records from other part of northern China such as the 6 ka peak in Gonghai Lake5. In spite of its low time resolution, the general trend over the millennium scale seems to us clear.It’s true that the −17 °C of the coldest month temperature is the survival threshold for the temperate deciduous broadleaved tree. While, the temperature threshold for C3 grass and C3 arctic grass are complex, its direct impact on the changes of grass proposed in our paper is somewhat not strict. However, considering the different competitiveness between tree and grass, increased temperate deciduous broadleaved tree, which derived by the winter warming, could induce a decrease in the grass from the early to mid-Holocene. Indeed, summer temperature, annual rainfall and fire incidents are all the important factors determining the Holocene changes of vegetation over northern China, but series of sensitivity experiment proposed the key impact of winter temperature on the vegetation shift and soil moisture evolution, which is consistent with the results of transient coupled climate simulation and geological records. This grass-to-tree shift for this period is evidenced in the pollen percentages and well simulated by the climate model shown in our paper2.Fire is an important factor for the long-term changes of vegetation cover over semi-arid regions, and its emergence and impact on vegetation are already incorporated into our model6, then, in turn, the simulation. Future works could assess the impact of fire on the long-term changes of semi-arid vegetation through the combination of reconstruction and process-based simulation of fire7.Focusing on the contrary views of Holocene EASM within proxy records, we proposed an asynchronous evolution of EASM rainfall and northern China ecosystem for the period of early to mid-Holocene. Our proposal is based on a state-of-the-art transient climate simulation, which reproduced the diverse evolution of EASM proxies reasonably well. The mechanisms proposed for this asynchronous evolution appear to us consistent with the current evidences available. There are, however, uncertainties in models and proxies. Meanwhile, the northern China is a broad region with large gradient in rainfall and ecosystem, that could induce the possible diverse evolutions in climate and ecosystem under Holocene climate change. Therefore, we believe further studies using other models and new proxies are important to further improve our understanding of this issue. More