DOI: 10.5061/dryad.7m0cfxq61
title: CH4 production and emissions from the upper parts of tree trunks in cool-temperate upland forest

description: Trees, typically large trees in upland forests, emit CH4 produced in their
 trunk by methanogenic archaea. In this case, the spatial variability of
 emissions can be more complicated than when tree trunks act as a passive
 conduit for CH4 produced in the soil. However, due to poor accessibility,
 CH4 emissions from the trunk above 3 m, where a researcher cannot reach
 without a ladder, scaffold, or crane, have not been well studied. In this
 study, we investigated vertical patterns of CH4 emissions, internal CH4
 concentration, production, and copy number of the methanogenic archaeal
 gene mcrA at 6 different heights up to 12 m above ground, in six
 trees of three species in a cool-temperate upland forest.
description: We targeted 6 mature trees from 3 species in a cool-temperate
 upland mixed forest. We measured variables at 6 different heights per 1
 individual. Trunk CH4 flux was measured by the dynamic
 closed chamber method. A polypropylene chamber, with a measuring area of
 37 cm2 on average and a volume of 695 mL, including the cell of the
 analyser and tubing, was attached to the trunk with neutral seal putty. A
 lid connected to a portable trace gas analyser (Li-7810, LICOR, Lincoln,
 USA) with two 6-mm-diameter, 2-m-long PTED tubes was used to close the
 chamber during the measurement. The trunk CH4 flux was calculated from the
 changes in the CH4 molar fraction in the closed system measured every
 second during four minutes of chamber closure. Trunk CH4 flux was
 expressed not only in per unit area (nmol m-2 s-1), but also in the entire
 trunk circumference per unit length of trunk (nmol 0.1 m-1 s-1) to compare
 it to internal CH4 production rate. CH4 concentration
 inside the trunk was measured after the CH4 flux measurement. The chambers
 were removed, and the trunk at each measuring location was bored with an
 increment borer (5-mm internal diameter, Haglof Sweden) to the pith. After
 the removal of the wood core, the hole left by the borer was enlarged to a
 diameter of 10 mm with a drill. A stainless-steel tube (10-mm outer
 diameter) was inserted into the hole, driven by a hammer to stop 7.5 cm
 before the pit. The outside of the tube was covered with silicon sealant
 prior to insertion to prevent any air from leaking along the contact area
 between the tube and the wood. The tube was flushed with pure N2 to expel
 as much O2 as possible before the open end of the tube was capped with a
 rubber septum (Asone, Butyl W Plug, Osaka, Japan). As the inner 5 or 7.5
 cm part of the hole was not covered with the stainless-steel tube, gases
 inside the central trunk can diffuse into the cavity of the tubing and
 reach equilibrium. Five days later, 0.1 mL of the gas inside the tube was
 sampled with a syringe penetrating the septum and immediately injected
 through the septum of the small volume sample kit (Li 7810-110, LICOR,
 Lincoln, USA) connected to the analyser. The molar fraction of gas
 injected was calculated from the volume of the closed system, including
 the sampling kit and injected sample, and the CH4 molar fraction in the
 system before and after the injection (Mochidome & Epron, 2024,
 Trees, 38: p625–636).  CH4 production potential was
 measured by laboratory incubation of wood cores sampled. The cores were
 cut into up to three segments of 7 cm in length, depending on the radius
 at the sampling location. The borer was sterilized with 70% ethanol every
 time after boring. Within five minutes after sampling, each of the 7-cm
 segments was placed into 12-mL glass vials separately. Within two hours
 after the sampling, the vials were flushed first with N2 for three minutes
 to expel CH4 and O2 from the vial and then with the incubation mixture
 (N2, 10% CO2, and 1% H2) for 1.5 minutes to supply substrates to
 methanogenic archaea. Soon after flushing, 0.2 mL of the gas inside the
 vials was drawn from the vial through the septum into a syringe, and the
 CH4 molar fraction in the vials was measured as described above for trunk
 internal [CH4]. The second gas samples were collected six hours after
 flushing. The vials were kept inside an opaque, insulated box between gas
 samples. CH4 production potential was calculated as an increase in CH4
 amount in the vial headspace divided by duration and dry weight of the
 wood core. The wood core was then weighed before and after drying and
 scaled before drying to calculate the bulk density and moisture content of
 the wood. For DNA extraction and subsequent
 quantitative PCR to target the mcrA gene, one individual was selected from
 each of the three species that encode the methyl-coenzyme M reductase α
 subunit of methanogenic archaea. Only the innermost wood core segments at
 every sampling height, for a total of 18 cores (1 depth × 6 heights × 3
 trees), were used. After the anaerobic incubation experiments, the wood
 cores were stored in a freezer at -20 °C. The samples were then
 freeze-dried for at least 48 hours in a freeze dryer (FDU-1200, EYELA,
 Tokyo, Japan) and ground via a ball mill (MM-400, Retsch, Düsseldorf,
 Germany) for 3 minutes. DNA was extracted from a 40-mg ground wood sample
 via the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, USA)
 following the manufacturer’s instructions. The DNA samples were further
 purified with a OneStep PCR Inhibitor Removal Kit (Zymo Research, Irvine,
 USA) before PCR amplification. DNA extraction was performed in duplicate,
 and the reported values are the average of the duplicate samples.
 Real-time PCR analysis of the mcrA genes was performed via the Thermal
 Cycler Dice Real Time System (TaKaRa, Kusatsu, Japan) and primers MLf/MLr
 (Luton et al., 2002). Each 25-µL mixture contained TB Green Premix Ex Taq
 (TaKaRa), MLf/MLr primers (0.2 µmol/L each), 0.25 µL of bovine serum
 albumin (BSA, 0.2 g/L), 2 µL of template DNA, and sterilized ultrapure
 water. The PCR thermal cycle was as follows: 95 °C for 10 s
 (denaturization); 45 cycles of 95 °C for 40 s (denaturization), 55 °C for
 30 s (annealing), and 72 °C for 60 s (elongation). An increase in the PCR
 product was detected by the fluorescent signal, and its threshold cycles
 (Ct values) were determined via the second derivative maximum method. The
 amplification of nonspecific DNA fragments was checked via dissociation
 curve analysis. The copy number of the mcrA gene was calculated via a
 standard curve generated from a series of dilutions (1.14×108 to 1.14×101
 copies/µL) of the standard sample.
description: # CH4 production and emissions from the upper parts of tree trunks in
 cool-temperate upland forest Dataset DOI:
 [10.5061/dryad.7m0cfxq61](10.5061/dryad.7m0cfxq61) ## Description of the
 data and file structure All the experiments were conducted in a
 cool-temperate upland mixed forest in Ashiu Experimental Forest of Kyoto
 University (35.34 N, 135.76 E)  in Kyoto, Japan. 6 mature trees of 3
 species were selected, and variables were measured at 6 heights per
 individual. Using a crane truck, we investigated vertical patterns of CH4
 emissions, internal CH4 concentration, and production and copy number of
 the methanogenic archaeal gene *mcrA*. Trunk CH4 flux was measured by the
 dynamic closed chamber method. A polypropylene chamber was attached to the
 trunk with neutral seal putty. A lid connected to a portable trace gas
 analyser (Li-7810, LICOR, Lincoln, USA) was used to close the chamber
 during the measurement. The trunk CH4 flux was calculated from the changes
 in the CH4 concentration in the closed system measured every second during
 four minutes of chamber closure. Trunk CH4 flux was expressed not only per
 unit area (nmol m-2 s-1) but also in the entire trunk circumference per
 unit length of trunk (nmol 0.1m-1 s-1) to compare it to the internal CH4
 production rate. CH4 concentration inside the trunk was measured after CH4
 flux measurement. The chambers were removed, and the trunk at each
 measuring location was bored and sampled with an increment borer (5-mm
 internal diameter, Haglof Sweden) to the pith. A stainless-steel tube
 (10-mm outer diameter) was inserted into the hole left, driven by a hammer
 to stop 7.5 cm before the pith. The tube was capped for five days, and 0.1
 mL of the gas inside the tube was sampled with a syringe penetrating the
 septum and immediately measured by the analyzer with the small volume
 sample kit.  CH4 production potential was measured by laboratory
 incubation of wood cores sampled. The cores were cut into up to one to
 three segments of 7 cm in length and then placed into 12-mL glass vials
 separately. The vials were incubated with the incubation mixture (N2, 10%
 CO2, and 1% H2) in an opaque, insulated box. CH4 production potential was
 calculated based on the increase of CH4 amount in vial headspace during 6
 hours, divided by duration and dry weight of the wood core. The wood core
 was then weighed before and after drying and scaled before drying to
 calculate the bulk density and moisture content of the wood. For DNA
 extraction and subsequent quantitative PCR to target the *mcrA* gene, one
 individual was selected from each of the three species. Only the innermost
 wood core segments at every sampling height, for a total of 18 cores (1
 depth × 6 heights × 3 trees), were used. After the incubation experiments,
 the wood cores were stored in a freezer at -20 °C and then freeze-dried
 for at least 48 hours in a freeze dryer (FDU-1200, EYELA, Tokyo, Japan)
 and ground via a ball mill (MM-400, Retsch, Düsseldorf, Germany) for 3
 minutes. DNA was extracted from a 40-mg ground wood sample via the FastDNA
 SPIN Kit for Soil (MP Biomedicals, Santa Ana, USA) and further purified
 with a OneStep PCR Inhibitor Removal Kit (Zymo Research, Irvine, USA).
 Real-time PCR analysis of the mcrA genes was performed via the Thermal
 Cycler Dice Real Time System (TaKaRa, Kusatsu, Japan) and primers MLf/MLr.
 See the main article for the details of RT-PCR. ### Files and variables
 #### File: main.csv The 2nd row of the table indicates the unit for each
 quantity. **Description:** This table includes data related to each
 sampling height of the tree. In total, 36 data (6 heights x 6 trees) are
 available for each variable. ##### Variables * tree: tree id consists of
 species id + individual number. It stands for Aesculus turbinata, Cj
 stands for Cryptomeria japonica, and Fc stands for Fagus crenata. Three
 trees (#1 and #2) were used for each species. * height: sampling height
 above the ground (soil) in m. * diameter: diameter of trunk at the
 sampling height in m. * ch4flux: trunk CH4 flux expressed per unit area of
 trunk surface. * ch4emission: trunk CH4 emission from the entire
 circumference per unit length (10cm) of the trunk (= ch4flux x
 circumference x 0.1m). * ch4production: trunk CH4 production inside the
 entire trunk volume per unit length (10cm) of the trunk. This column
 contains NA value where total CH4 production at that trunk height was not
 be able to be calculated due to a big empty space in the middle of the
 trunk. * ch4concentration: CH4 molar fraction at the centre of the trunk,
 expressed in ppb. #### File: sub.csv The 2nd row of the table indicates
 the unit for each quantity. **Description:** This table includes data on
 each single wood core (1 to 3 depths x 6 heights x 6 individuals).  #####
 Variables * tree: tree id consists of species id + individual number. At
 stands for Aesculus turbinata, Cj stands for Cryptomeria japonica, and Fc
 stands for Fagus crenata. Three trees (#1 and #2) were used for each
 species. * height: sampling height above the ground (soil) in m. *
 radial_depth: relative depth from the trunk surface to the pith. When the
 trunk radius is enough large, wood cores at 3 depths (outermost, middle,
 innermost) were sampled, but otherwise, only 2 or 1 depths were sampled. *
 depth_****m: depth from the trunk surface in m. * prod.rate: increase rate
 of CH4 amount in the headspace of laboratory incubation vial for
 approximately 6 hours, divided by the duration and dry mass of the wood
 core. Several measurement was missing due to human error during the
 experiment (shown as NA). * copy.mcrA: copy number of *mcrA* gene per dry
 mass of the wood core. We targeted the innermost wood core of only 1
 individual per 1 specie, and we were always able to obtain the copy number
 from the targeted samples. Untargetted wood samples were shown as NA. *
 MC: dry-base moisture content.  * BD: bulk density. Several measurement
 was missing due to human error during the experiment (shown as NA).