E ﬀ ects of Light Intensity and Girdling Treatments on the Production of Female Cones in Japanese Larch ( Larix kaempferi (Lamb.) Carr.): Implications for the Management of Seed Orchards

: To ensure sustainable forestry, it is important to establish an e ﬃ cient management procedure for improving the seed production capacity of seed orchards. In this study, we evaluated the e ﬀ ects of girdling and increasing light intensity on female cone production in an old L. kaempferi (Lamb.) Carr. seed orchard. We also evaluated whether there is a genotype-speciﬁc reproductive response to these factors among clones. The results showed that female cone production was augmented by girdling and increasing light intensity. There was a di ﬀ erence in the e ﬀ ectiveness of girdling treatment levels, and the probability of producing female cones increased markedly at higher girdling levels. At light intensities where the relative photosynthetic photon ﬂux density was higher than 50%, more than half of the trees tended to produce female cones, even in intact (ungirdled) trees, and the genotype-speciﬁc response to light intensity was more apparent in less-reproductive clones. These ﬁndings suggested that girdling less-reproductive trees combined with increasing light intensity was an e ﬀ ective management strategy for improving cone production in old seed orchards.


Introduction
Japanese larch (Larix kaempferi) is a major plantation species in central and northern Japan [1]. The species has been introduced widely in China, Europe and North America, because of its rapid growth and sparse branching characteristics. Larix kaempferi often shows superior growth compared to other larch species (e.g., L. decidua Mill. and L. laricina (Du Roi) K. Koch) and has therefore been widely used in breeding programs involving hybrid breeding [2,3]. As a result of these efforts, hybrids between L. kaempferi and other larches have been used commercially in North America [4], Europe [3] and Japan [5,6].
In Japan, a breeding program for L. kaempferi was initiated in the 1950s. As part of the program, more than 500 first-generation plus trees were selected and used to establish clonal seed orchards [1]. The selection of second-generation plus trees started in the 2010s, and this is ongoing [1,7]. Compared to traditional seed sources, the superior growth of seedlings derived from the clonal seed orchards of the plus trees has been demonstrated [1], and significant variations in growth and wood property traits among families were reported [7,8]. Data analyses based on several provenance tests clarified that genotype × environment (G × E) interactions in several of these growth traits were not small [9].

Light Intensity
The photosynthetic photon flux density (PPFD) was used as an indicator of light intensity in this study. Measurements were conducted on a cloudy day in July using LI-250 light meters (LI-COR, Lincoln, Dearborn, MI, USA). The measurements were performed four times (in four directions) around the crown of each tree at the approximate midpoint of the height of the crown (5-6 m). The mean relative PPFD (rPPFD) was calculated for each planting position, as follows: rPPFD = PPFD above the crown of each tree/PPFD above forest canopy (i.e., open sky).

Tree size and Reproductive Status
We scored the reproductive status of each tree based on the extent of female cone production, as follows: trees that did not produce female cones (hereafter referred to as index 1), trees with female cones that were very sparsely distributed within the crown (index 2), trees with female cones that were sparsely distributed within the crown, or trees produced numerous female cones on a few branches only (index 3) and trees producing cones abundantly on several branches (index 4).
The number of female cones produced per stem was counted manually, and all counts were performed in triplicate. To investigate the relationship between reproductive status and tree size, all living tree stems (trunks) within the orchard were mapped and their diameter at breast height (DBH), tree height and crown radius was measured.

Data Analysis
To analyze the reproductive performance of L. kaempferi, we used generalized linear mixed-effect models [38], by using R 3.2.5 [39]. Based on the reproductive score data, the effects of light intensity and girdling on the probability of female cone production were analyzed using ordered logit and binomial logit functions. The fixed-effect explanatory variables were rPPFD and girdling treatment; the rPPFD was used as a covariate, while the girdling was treated as a main factor. We treated "block within site" as a random-effect factor, to account for spatial pseudo-replication. To test whether the genotype-specific reproductive response to light intensity varied among clones, we included "clone" and the interaction with rPPFD (i.e., "clone:rPPFD") as random effects.
The relationship between female cone production and tree size traits was analyzed by ANCOVA-like linear mixed models. The DBH, height, crown radius and height/crown radius ratio for tree stems were treated as fixed-effect covariates. We also included "clone" and the interaction terms with traits (e.g., "clone:DBH") as random effects. In this study, each trait was analyzed separately because of the multicollinearity among size traits.

Status of the L. kaempferi Orchard
There was a marked variation in the light environment in the study orchard, and the mean rPPFD was 50.1 ± 15.2% ( Figure 1A). A total of 526 stems belonging to 44 clones were investigated. Of these stems, 31.3% (164/526) produced female cones ( Figure 1B). Of the 164 trees that reproduced, reproductive scores of 76.8% (126) and 15.2% (25) were obtained for indices 2 and 3, respectively, while only 7.9% (13) was obtained for index 4. In total, 18,114 cones were produced in the orchard, with the mean and maximum numbers of cones per stem being 34.4 and 5960, respectively ( Figure 1C). The mean (±SD) DBH, height and crown radius were 38.6 ± 6.2 cm, 10.3 ± 2.2 m and 4.3 ± 0.9 m, respectively. Of these stems, 31.3% (164/526) produced female cones ( Figure 1B). Of the 164 trees that reproduced, reproductive scores of 76.8% (126) and 15.2% (25) were obtained for indices 2 and 3, respectively, while only 7.9% (13) was obtained for index 4. In total, 18,114 cones were produced in the orchard, with the mean and maximum numbers of cones per stem being 34.4 and 5960, respectively ( Figure  1C). The mean (±SD) DBH, height and crown radius were 38.6 ± 6.2 cm, 10.3 ± 2.2 m and 4.3 ± 0.9 m, respectively. Fruiting index of each tree. Index 1: trees that did not produce female cones, index 2: trees with female cones that were very sparsely distributed within the crown, index 3: trees with female cones that were sparsely distributed within the crown, or trees produced numerous female cones on a few branches only and index 4: trees that produced female cones abundantly on several branches. (C) Number of female cones produced per tree. Red circles indicate girdled trees and black circles indicate intact trees.

Relationship between Female Cone Production and Light Intensity or Girdling Treatments
The probability of female cone production varied markedly in response to light intensity (rPPFD) across different girdling treatments ( Figure 2, Table 1). In girdling level 0 (i.e., intact stems), the probability of producing female cones (green: index 2 and orange: indices 3 and 4) increased markedly with increasing light intensity. In the situation where rPPFD was greater than approximately 0.5 (i.e., 50% sunlight), the probability of producing female cones (green and orange) was larger than that of not producing cones (blue: index 1). Two out of 12 trees in girdling level 3 died, but none of the trees in girdling levels 0-2 died.
In darker environments, such as where rPPFD = 0.2, fewer than 20% of trees in the control group (i.e., intact stems) produced female cones (Figue 2). On the other hand, in the girdling level 3 group, more than 70% of trees produced female cones, even at similar light intensities. (B) Fruiting index of each tree. Index 1: trees that did not produce female cones, index 2: trees with female cones that were very sparsely distributed within the crown, index 3: trees with female cones that were sparsely distributed within the crown, or trees produced numerous female cones on a few branches only and index 4: trees that produced female cones abundantly on several branches. (C) Number of female cones produced per tree. Red circles indicate girdled trees and black circles indicate intact trees.

Relationship between Female Cone Production and Light Intensity or Girdling Treatments
The probability of female cone production varied markedly in response to light intensity (rPPFD) across different girdling treatments ( Figure 2, Table 1). In girdling level 0 (i.e., intact stems), the probability of producing female cones (green: index 2 and orange: indices 3 and 4) increased markedly with increasing light intensity. In the situation where rPPFD was greater than approximately 0.5 (i.e., 50% sunlight), the probability of producing female cones (green and orange) was larger than that of not producing cones (blue: index 1). Two out of 12 trees in girdling level 3 died, but none of the trees in girdling levels 0-2 died. Table 1. Summary of generalized linear mixed-effect model for estimating the probability of reproduction in Japanese larch. Relative photosynthetic photon flux density (rPPFD) was used as a fixed-effect covariate, while the effect of girdling treatment was used as a fixed-effect main factor.

Coef.
S.E. In darker environments, such as where rPPFD = 0.2, fewer than 20% of trees in the control group (i.e., intact stems) produced female cones ( Figure 2). On the other hand, in the girdling level 3 group, more than 70% of trees produced female cones, even at similar light intensities.  Figure 3 shows the genotype-specific reproductive response to light conditions. Gray lines indicate response curves estimated for each of the 44 clones. The among-clone variation in the probability of female cone production was more apparent in darker areas, and less apparent under more brightly lit conditions (rPPFD > 0.8).

Z-Value p-Value
The probability of female cone production varied markedly in response to light intensity (rPPFD) across different girdling treatments ( Figure 2, Table 1). In girdling level 0 (i.e., intact stems), the probability of producing female cones (green: index 2 and orange: indices 3 and 4) increased markedly with increasing light intensity. In the situation where rPPFD was greater than approximately 0.5 (i.e., 50% sunlight), the probability of producing female cones (green and orange) was larger than that of not producing cones (blue: index 1). Two out of 12 trees in girdling level 3 died, but none of the trees in girdling levels 0-2 died.
In darker environments, such as where rPPFD = 0.2, fewer than 20% of trees in the control group (i.e., intact stems) produced female cones (Figue 2). On the other hand, in the girdling level 3 group, more than 70% of trees produced female cones, even at similar light intensities. Probability of fruiting    Figure 3 shows the genotype-specific reproductive response to light conditions. Gray lines indicate response curves estimated for each of the 44 clones. The among-clone variation in the probability of female cone production was more apparent in darker areas, and less apparent under more brightly lit conditions (rPPFD > 0.8). Based on the coefficient of variances (Table 2), among-clone variances in the probability of producing cones were much evident under conditions of lower light intensities and girdling levels, while the variation among clones decreased with increasing light intensities.  Based on the coefficient of variances (Table 2), among-clone variances in the probability of producing cones were much evident under conditions of lower light intensities and girdling levels, while the variation among clones decreased with increasing light intensities.

Relationship between Female Cone Production and Tree Size
When examining the relationships between female cone production and traits of the trees (size and shape), a significant negative relationship was observed between female cone production and the height/crown radius ratio (p < 0.05; bold black line in Figure 4D), and smaller trees with a relatively wider crown radius tended to produce more female cones. No significant relationships were observed between female cone production and the other traits ( Figure 4A-C).

Relationship between Female Cone Production and Tree Size
When examining the relationships between female cone production and traits of the trees (size and shape), a significant negative relationship was observed between female cone production and the height/crown radius ratio (p < 0.05; bold black line in Figure 4D), and smaller trees with a relatively wider crown radius tended to produce more female cones. No significant relationships were observed between female cone production and the other traits ( Figure 4A-C).

Discussion
In northern conifer species including Japanese larch, mast seeding, i.e., low frequent abundant fruiting events, is a limiting factor for sustainable seed production, and numerous efforts have been made to overcome this limitation [12,13]. Several trials for increasing cone production have been conducted on larch species, such as L. kaempferi, L decidua and L. laricina, and some success has been reported for treatments involving girdling [18][19][20][21][22][23], gibberellins [40], nitrogen fertilizer [19], drought [24] and branch bending [41]. However, the results obtained from yet other studies using similar treatments have often been inconsistent, such as ineffective stimulation by gibberellins [42][43][44], nitrogen fertilizer [15] and root pruning [24]. These disparities could be attributed to differences in the age and growing conditions (natural stands, seed orchards or pots in greenhouses, etc.). In old seed orchards containing trees that are not well managed, restoring the reproductive capacity could be considered to be relatively difficult. However, the findings of our study showed that female cone production of old L. kaempferi trees can be enhanced by girdling and increasing the light intensity.
As in previous studies [18][19][20][21][22][23], our study confirmed that girdling was effective for improving female cone production, even in the old L. kaempferi orchard. In an orchard of 42-year-old L. kaempferi, more than 90% (19/20) of the girdled trees totally produced about 8000 cones, while only 25% (5/20) of the intact trees yielded 42 cones [22]. Similarly, female cone production in girdled trees increased more than ten times in natural stands of 70- [45] and 90-year-old [19] western larch, compared to intact trees. However, when girdling was conducted on young trees (17 years old), the effects of girdling on the proportion of trees which produced cones and the cone production per tree were less apparent [40], suggesting that age-dependent sexual maturity may affect the efficiency of girdling. Although the efficiency of girdling at different ages has not yet been clarified, our study found that girdling is a low-cost method that can be used to efficiently restore the cone production capacity of old L. kaempferi seed orchards.
Girdling has been regarded as a cost-efficient and useful technique for disrupting phloem transport while limiting detrimental effects [46]. However, it has also been reported that the positive effects of girdling are relatively limited in duration, and repeated girdling might adversely affect tree vigor and decrease total reproductive output [47]. In our study, there was considerable variation in

Discussion
In northern conifer species including Japanese larch, mast seeding, i.e., low frequent abundant fruiting events, is a limiting factor for sustainable seed production, and numerous efforts have been made to overcome this limitation [12,13]. Several trials for increasing cone production have been conducted on larch species, such as L. kaempferi, L decidua and L. laricina, and some success has been reported for treatments involving girdling [18][19][20][21][22][23], gibberellins [40], nitrogen fertilizer [19], drought [24] and branch bending [41]. However, the results obtained from yet other studies using similar treatments have often been inconsistent, such as ineffective stimulation by gibberellins [42][43][44], nitrogen fertilizer [15] and root pruning [24]. These disparities could be attributed to differences in the age and growing conditions (natural stands, seed orchards or pots in greenhouses, etc.). In old seed orchards containing trees that are not well managed, restoring the reproductive capacity could be considered to be relatively difficult. However, the findings of our study showed that female cone production of old L. kaempferi trees can be enhanced by girdling and increasing the light intensity.
As in previous studies [18][19][20][21][22][23], our study confirmed that girdling was effective for improving female cone production, even in the old L. kaempferi orchard. In an orchard of 42-year-old L. kaempferi, more than 90% (19/20) of the girdled trees totally produced about 8000 cones, while only 25% (5/20) of the intact trees yielded 42 cones [22]. Similarly, female cone production in girdled trees increased more than ten times in natural stands of 70- [45] and 90-year-old [19] western larch, compared to intact trees. However, when girdling was conducted on young trees (17 years old), the effects of girdling on the proportion of trees which produced cones and the cone production per tree were less apparent [40], suggesting that age-dependent sexual maturity may affect the efficiency of girdling. Although the efficiency of girdling at different ages has not yet been clarified, our study found that girdling is a low-cost method that can be used to efficiently restore the cone production capacity of old L. kaempferi seed orchards.
Girdling has been regarded as a cost-efficient and useful technique for disrupting phloem transport while limiting detrimental effects [46]. However, it has also been reported that the positive effects of girdling are relatively limited in duration, and repeated girdling might adversely affect tree vigor and decrease total reproductive output [47]. In our study, there was considerable variation in the magnitude of the girdling effect among the different girdling treatment levels (see the coefficients in Table 1: larger coefficient values indicate a larger positive effect); the probability of producing cones was significantly increased as the treatment level was higher ( Figure 2). However, 2 out of 12 girdled trees in the level 3 group died, while none of the girdled trees in the level 1 and 2 groups died. These results suggest that level 2 girdling might be better for balancing tree vigor and reproduction, while level 3 might be too severe. Although the sample size of severe girdling levels in the present study may be slightly small and the long-term effects of girdling on cone production are still unclear, the short-term effect on increasing female cone production of L. kaempferi was confirmed in the old seed orchard.
In the present study, improvements in light intensity also had a positive influence on the probability of producing cones. Previous studies similarly reported that trees on southern slopes that received higher levels of insolation tended to produce abundant cones, and the branches in a crown that received full sunlight achieved the highest cone production [48][49][50]. In our estimates, even in intact (ungirdled) trees, more than half of the trees tended to produce cones when the light conditions reached rPPFD > 50%. In a study on L. gmelinii var. japonica, Uchiyama et al. [14] recommended that light intensity at over 50% rPPFD in an orchard is optimal. Since there was a large spatial variation in light intensity in our studied orchard, thinning the darker areas to reduce the stem density appears to be an efficient means of improving the reproductive status of L. kaempferi trees and for improving the seed production capacity of the seed orchards.
Moreover, our quantitative estimates demonstrated the existence of a genotype-specific response to increases in light intensity. When light intensity was sufficient, all of the clones had similar reproductive potentials. However, among-clone differences in reproductive potential were much evident under conditions of insufficient light intensity, and less-reproductive L. kaempferi clones were susceptible to light conditions. Uchiyama et al. [14] also reported a significant G × E interaction among eight L. gmelinii var. japonica clones. Large among-clone variation in fecundity often makes it difficult to achieve panmixia, as seed orchards are designed on the premise of an equal reproductive contribution of each constitutive clone [51]. In some cases, less than half of the mother trees were responsible for more than half of the parentage in the seed orchard, resulting in an uneven genetic contribution among seedlings [52,53]. It has been reported that treatments often have a greater effect on less-reproductive trees, improving their genetic contribution across an orchard [52]. Improving light conditions by thinning can therefore be an effective method for ensuring that mating in a seed orchard more closely approximates panmixia through increasing the participation of clones in reproduction. In this context, improving the light conditions and collecting information on genotype-specific responses to light intensity may be useful for optimizing management regimes for improving seed production capacity of old seed orchards, not only in terms of seed quantity but also in quality.
In an experiment on young L. kaempferi trees [41], bending the upright branches so they were oriented downward had the effect of increasing cone bud initiation, especially on the lower surfaces of the horizontal shoots. In the early stages of orchard management, top pruning (cutting the main trunk and upright leader shoots) is typically conducted at a height of 3-4 m. However, in the less-intensively managed old seed orchards of L. kaempferi, it has been found that upright branches often re-sprouted dense adventitious shoots from the cut-off position, and the tree height was recovered. Based on our findings, ensuring that trees have a wide crown radius and relatively low height could be a better management strategy for improving female cone production in old orchards.

Conclusions
This study quantitatively evaluated the effects of girdling and increasing light intensity on female cone production in an L. kaempferi seed orchard. The findings showed that female cone production was augmented by both girdling and increasing light intensity. The probability of producing cones was markedly increased when higher girdling levels were applied. When the light intensity reached rPPFD > 50%, more than half of the mother trees tended to produce cones, even intact (ungirdled) trees,