Effects of Light Intensity and Girdling Treatments on The

Article Eﬀects 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

1, , 2, 1 1 Michinari Matsushita * †, Hiroki Nishikawa †, Akira Tamura and Makoto Takahashi 1 Forest Tree Breeding Center, Forestry and Forest Products Research Institute, 3809-1 Ishi, Juo, Hitachi, Ibaraki 319-1301, Japan; akirat@affrc.go.jp (A.T.); makotot@affrc.go.jp (M.T.) 2 Fujiyoshida Examination Garden, Yamanashi Forest Research Institute, 1-18-2 Shinnishihara, Fuji-Yoshida, Yamanashi 403-0017, Japan; [email protected] * Correspondence: matsushita@affrc.go.jp Both authors contributed equally to this work. † Received: 7 September 2020; Accepted: 15 October 2020; Published: 19 October 2020

Abstract: To ensure sustainable forestry, it is important to establish an efficient management procedure for improving the seed production capacity of seed orchards. In this study, we evaluated the effects 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 difference in the effectiveness 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 effective management strategy for improving cone production in old seed orchards.

Keywords: breeding; genotype environment interaction; mast seeding; seed production; thinning ×

1. 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]. × ×

Forests 2020, 11, 1110; doi:10.3390/f11101110 www.mdpi.com/journal/forests Forests 2020, 11, 1110 2 of 10

However, genotype-specific responses in the reproductive traits (e.g., cone production) to environmental conditions have not yet been clarified in detail for L. kaempferi. Reforestation using L. kaempferi has increased over the last decade in Japan, and L. kaempferi is now the second most important forestry species for plantation in Japan and occupies about 25% of newly planted forest areas [10]. Owing to its high juvenile growth performance, the demand for improved seeds and seedlings of L. kaempferi has been increasing [11]. However, the clonal seed orchards of the second-generation plus trees are currently too young to produce enough seeds. On the other hand, the clonal seed orchards of the first-generation plus trees, which were established mainly in the 1960s and 1970s, are now more than 50 years old. Due to the high density of old stems and branches, these old seed orchards are too tall to be managed efficiently, and too dark to produce sufficient quantities of good-quality seeds. In many conifers, mast seeding is a limiting factor for sustainable seed production [12,13]. To overcome this limitation, numerous efforts have been made to enhance seed production, e.g., improving light intensity [14–17], girdling [18–23], fertilizer [19] and drought [24,25]. To establish an efficient management system for the old seed orchards of Japanese larch, it is necessary to quantitatively evaluate such treatment effects on seed production. Clear reproductive responses to changes in light intensity have been reported in forest plants [26–28]. Previous studies that examined the effect of increasing light intensity found that thinning operations often offer increased flowering [14,29,30]. Trees located at well-lit sites tended to have more flowers, and the quality and quantity of their seeds were positively correlated with magnitudes of flowering [31]. Previous studies on larch orchards have also reported that increasing light intensity improved cone production [14–17]. Uchiyama et al. [14] quantified a relationship between light levels and female cone production in an old orchard of L. gmelinii var. japonica. However, quantitative estimation of the effect of light intensity is still limited for old seed orchards of L. kaempferi. In horticulture, girdling is commonly used to promote flower production [32] and improve fruit quality [33] and yield [32,34]. Girdling interrupts phloem transport by removing a part of the bark and cambium without affecting xylem transport [35]. When the main stem (trunk) of a tree is girdled, the translocation of assimilates to the below-ground parts of the tree is interrupted, resulting in a super-abundance of assimilates in the above-ground parts above the girdle [36,37]. Girdling has therefore been used to control the resource allocation, and to promote cone production [18–23]. It is, however, rarely quantified how different girdling levels (severity) affect the cone production of L. kaempferi. To ensure the sustainability of forestries, establishing an efficient management procedure for improving the seed production in seed orchards is necessary. In this context, studies on the effects of light intensity and girdling manipulation on the reproductive performance of mast seeding conifer species, such as Larix and Picea, have attracted the interest of silviculturists and forest managers. In this study, we quantitatively evaluated the effects of girdling manipulation and increasing light intensity on female cone production in an old L. kaempferi seed orchard. We also evaluated genotype-specific responses in reproduction among L. kaempferi clones after making changes in light intensity. Finally, we examined the relationship between female cone production and tree sizes.

2. Materials and Methods

2.1. Study Site

The study site was the Fujisan Seed Orchard (35.42◦ N, 138.74◦ E; 1320–1350 m a.s.l.; total area 10 ha), managed by Yamanashi prefecture. The orchard, located on the northeastern slope of Mount Fuji, is divided into several plots and the largest plot (#9; 2.4 ha) was used for this study. The orchard was established between 1961 and 1962 and contains 526 stems (stem density: 223.8 stems/ha in 2017) comprising 44 ﬁrst-generation L. kaempferi clones that were selected mainly from forests in Yamanashi and Nagano prefectures. The mean annual temperature and precipitation were 10.6 ◦C and 1568 mm, Forests 2020, 11, 1110 3 of 10 respectively. The soil of the area is comprised primarily of weathered volcanic sediments derived from Mount Fuji.

2.2. Girdling Treatments We examined whether girdling could be used to enhance the reproductive performance of L. kaempferi. Girdling is a procedure that involves removing a 2 cm-wide semilunar ring of bark and cambium at a height of approximately 80–100 cm on the main stem (trunk) using a knife (Figure A1). We clariﬁed the eﬀects of the following four types of girdling treatments: one semilunar ring of bark and cambium was removed (referred as to level 1); two semilunar rings of bark and cambium were removed, with each ring oriented in opposite directions (level 2); three semilunar rings of bark and cambium with the same orientation were removed (level 3); and no girdling was performed (level 0; i.e., intact). We randomly assigned 8, 14 and 12 trees to levels 1, 2 and 3, respectively, and all of the remaining trees were assigned to level 0. Girdling was conducted in May 2016. As two of 12 trees from the level 3 treatment group were dead in autumn, these trees were removed from the analysis.

2.3. 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).

2.4. 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.

2.5. 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. Forests 2020, 11, 1110 4 of 10

3. Results

3.1. Status of the L. kaempferi Orchard Forests 2020, 11, x FOR PEER REVIEW 4 of 11 There was a marked variation in the light environment in the study orchard, and the mean rPPFD Ofwas these 50.1 stems,15.2% 31.3% (Figure (164/526)1A). A produced total of 526 female stems cones belonging (Figure to 1B). 44 clones Of the were 164 trees investigated. that reproduced, Of these ± reproductivestems, 31.3% (164scores/526) of produced 76.8% (126) female and cones 15.2% (Figure (25) were1B). Of obtained the 164 treesfor indices that reproduced, 2 and 3, respectively, reproductive whilescores only of 76.8% 7.9% (126)(13) was and obtained 15.2% (25) for were index obtained 4. In total for, indices18,114 cones 2 and were 3, respectively, produced whilein the onlyorchard, 7.9% with(13) wasthe mean obtained and for maximu index 4.m Innumbers total, 18,114 of cones cones per were stem produced being 34.4 in theand orchard, 5960, respectively with the mean (Figure and 1C).maximum The mean numbers (±SD) ofDBH, cones height per stem and beingcrown 34.4 radius and were 5960, 38.6 respectively ± 6.2 cm, (Figure10.3 ± 2.21C). m Theand mean4.3 ± 0.9 ( SD)m, ± respectively.DBH, height and crown radius were 38.6 6.2 cm, 10.3 2.2 m and 4.3 0.9 m, respectively. ± ± ± B) Index 1 Index 3 C) A) 0.0 1.0 Index 2 Index 4

5 Col within Col within orchard

0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Row within orchard

Figure 1. Within the orchard, spatial variation in (A) relative photosynthetic photon flux density. Figure 1. Within the orchard, spatial variation in (A) relative photosynthetic photon flux density. (B) (B) Fruiting index of each tree. Index 1: trees that did not produce female cones, index 2: trees with Fruiting index of each tree. Index 1: trees that did not produce female cones, index 2: trees with female female cones that were very sparsely distributed within the crown, index 3: trees with female cones that cones that were very sparsely distributed within the crown, index 3: trees with female cones that were were sparsely distributed within the crown, or trees produced numerous female cones on a few branches 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 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. female cones produced per tree. Red circles indicate girdled trees and black circles indicate intact 3.2. Relationshiptrees. between Female Cone Production and Light Intensity or Girdling Treatments The probability of female cone production varied markedly in response to light intensity 3.2. Relationship between Female Cone Production and Light Intensity or Girdling Treatments (rPPFD) across different girdling treatments (Figure2, Table1). In girdling level 0 (i.e., intact stems), the probabilityThe probability of producing of female female cone conesproduction (green: varied index markedly 2 and orange: in response indices 3 to and light 4) increased intensity (rPPFD)markedly across with different increasing girdling light intensity. treatments In the (Figure situation 2, Table where 1). rPPFD In girdling was greater level 0 than(i.e., approximatelyintact stems), the0.5 (i.e.,probability 50% sunlight), of producing the probability female cones of producing (green: index female 2 conesand orange: (green andindices orange) 3 and was 4) largerincreased than markedlythat of not with producing increasing cones (blue: light intensity.index 1). Two In outthe of situation 12 trees in where girdling rPPFD level was 3 died, greater but none than of approximatelythe trees in girdling 0.5 (i.e. levels, 50% 0–2 sunlight), died. 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, Tablebut none 1. Summaryof the trees of in generalized girdling levels linear 0– mixed-e2 died.ff ect model for estimating the probability of Inreproduction darker environments, in Japanese such larch. as Relative where rPPFD photosynthetic = 0.2, fewer photon than flux 20% density of trees (rPPFD) in the control was used group (i.e., intactas a fixed-e stems)ffect produced covariate, female while the cones effect (Figue of girdling 2). On treatment the other was hand, used asin athe fixed-e girdlingffect main level factor. 3 group, more than 70% of trees produced female cones, even at similar light intensities. Coef. S.E. Z-Value p-Value Intercept 1.84 0.45 4.05 <0.001 Treatment Treatment− Treatment− Treatment GirdlingLevel0 level 1Level1 1.25 0.78Level 1.612 0.109Level3 Girdling level 2 2.39 0.71 3.39 <0.001

1.0 Girdling level 31.0 Index3&4 3.09 1.121.0 Index3&4 2.771.0 0.006Index3&4 rPPFD 1.73 0.79 2.20 0.028

0.8 Index2 0.8 0.8 0.8 Index2

In darker environments,0.6 such as where0.6 rPPFD = 0.2,0.6 fewerIndex2 than 20%0.6 of treesIndex2 in the control group (i.e., intact stems) produced female cones (Figure2). On the other hand, in the girdling level 3 group,

0.4 pred.3_t1 0.4 pred.3_t2 0.4 pred.3_t3 0.4 more than 70% of trees produced female cones, even at similar light intensities.

0.2 0.2 0.2 0.2

Probability fruiting Probability of Index1 Index1 Index1 Index1

0.0 0.0 0.0 0.0

0.0 0.4 0.8 0.0 0.4 0.8 0.0 0.4 0.8 0.0 0.4 0.8 Relative photosynthetic photon flux density

Forests 2020, 11, x FOR PEER REVIEW 4 of 11

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.

B) Index 1 Index 3 C) A) 0.0 1.0 Index 2 Index 4 5 10152025 Col within Col within orchard

0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Row within orchard

Figure 1. Within the orchard, spatial variation in (A) relative photosynthetic photon flux density. (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.

3.2. 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 Forests 2020, 11, 1110 5 of 10 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, Figurebut none3 shows of the the trees genotype-speciﬁc in girdling levels reproductive 0–2 died. response to light conditions. Gray lines indicate ForestsresponseIn 20 darker20, curves11, x FORenvironments, estimated PEER REVIEW for such each as of where the 44 rPPFD clones. = The0.2, fewer among-clone than 20% variation of trees in the the control probability group5 of 11 of (i.e.,female intact cone stems) production produced was female more apparent cones (Figue in darker 2). On areas, the other and less hand, apparent in the undergirdling more level brightly 3 group, lit Figure 2. Relationship between fruiting probability of trees and light intensity (relative photosynthetic moreconditions than 70% (rPPFD of trees> 0.8). produced female cones, even at similar light intensities. photon flux density) for different girdling treatment levels. Blue: fruiting index 1, trees not producing female cones. Green: fruitingTreatment index 2, treesTreatment producing femaleTreatment cones sparselyTreatment within their crown. Orange: fruiting index 3,Level0 trees producing abundantLevel1 female conesLevel2 within their crown.Level3

Table 1. Summary of generalized linearIndex3&4 mixed-effect modelIndex3&4 for estimatingIndex3&4 the probability of reproduction in Japanese larch.Index2 Relative photosynthetic photon flux density (rPPFD) was used as a fixed-effect covariate, while the effect of girdlingIndex2 treatment was used as a fixed-effect main factor. Index2 Index2 Coef. S.E. Z-Value p-Value Intercept −1.84 0.45 −4.05 <0.001 Girdling level1 1.25 0.78 1.61 0.109

Probability Probability fruiting of Index1 Index1 Index1 Index1 Girdling level2 2.39 0.71 3.39 <0.001

Girdling0.0 0.2 0.4 0.6 level3 0.8 1.0 0.0 0.23.09 0.4 0.6 0.8 1.0 1.120.0 0.2 0.42.77 0.6 0.8 1.0 0.00.006 0.2 0.4 0.6 0.8 1.0 0.0rPPFD 0.40.8 0.01.73 0.4 0.80.79 0.0 0.42.20 0.8 0.00.028 0.4 0.8 Relative photosynthetic photon flux density Figure 3 shows the genotype-specific reproductive response to light conditions. Gray lines indicateFigure response 2. Relationship curves between estimated fruiting for probability each of the of trees 44 clones. and light The intensity among (relative-clone photosynthetic variation in the photon ﬂux density) for diﬀerent girdling treatment levels. Blue: fruiting index 1, trees not producing probability of female cone production was more apparent in darker areas, and less apparent under female cones. Green: fruiting index 2, trees producing female cones sparsely within their crown. more brightly lit conditions (rPPFD > 0.8). Orange: fruiting index 3, trees producing abundant female cones within their crown.

Treatment Treatment Treatment Treatment Level0 Level1 Level2 Level3

1.0 1.0 1.0 1.0

0.8 0.8 0.8 0.8

0.6 0.6 0.6 0.6

0.4 0.4 0.4 0.4

pred.1_t1[1, ] pred.1_t1[1, ] pred.1_t2[1, ] pred.1_t3[1,

0.2 0.2 0.2 0.2 Probability of fruiting of Probability

0.0 0.0 0.0 0.0 0.0 0.4 0.8 0.0 0.4 0.8 0.0 0.4 0.8 0.0 0.4 0.8 Relative photosynthetic photon flux density

Figure 3. Genotype-specific relationship between fruiting probability and light intensity Figure 3. Genotype-specific relationship between fruiting probability and light intensity (relative (relative photosynthetic photon flux density) across different girdling treatment levels. Each gray line photosynthetic photon flux density) across different girdling treatment levels. Each gray line indicates indicates each L. kaempferi genotype (clone). each L. kaempferi genotype (clone). Based on the coefficient of variances (Table2), among-clone variances in the probability of 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, producing cones were much evident under conditions of lower light intensities and girdling levels, while the variation among clones decreased with increasing light intensities. while the variation among clones decreased with increasing light intensities. 3.3. Relationship between Female Cone Production and Tree Size Table 2. The coefficient of variance (CV) for among-clone differences in the probability of producing When examining the relationships betweenfemale cones. 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 < rPPFD:0.05; bold 0.2black rPPFD: line in 0.5 Figure rPPFD:4D), and 0.8 smaller trees with a relatively wider crownGirdling radius tendedlevel0 to produce26.4% more female13.1% cones. No2.9% significant relationships were observed between femaleGirdling cone level1 production 16.8% and the other traits7.4% (Figure4A–C).1.4% Girdling level2 8.3% 3.3% 0.6% Girdling level3 4.8% 1.8% 0.3%

Forests 2020, 11, 1110 6 of 10

Forests 2020, 11, x FOR PEER REVIEW 6 of 11 Table 2. The coeﬃcient of variance (CV) for among-clone diﬀerences in the probability of producing 3.3.female Relationship cones. between Female Cone Production and Tree Size

When examining the relationships betweenrPPFD: female 0.2 rPPFD: cone production 0.5 rPPFD: and 0.8 traits of the trees (size and shape), a significant negative relationship was observed between female cone production and Girdling level 0 26.4% 13.1% 2.9% the height/crown radiusGirdling ratio level (p < 10.05; bold 16.8% black line 7.4% in Figure 4D), 1.4% and smaller trees with a relatively wider crownGirdling radius leveltended 2 to produce 8.3% more female 3.3% cones. 0.6%No significant relationships were observed betweenGirdling female level cone 3 production 4.8% and the other 1.8% traits (Figure 0.3% 4A–C).

B) 5000 A) 5000 5000 C) 5000 D)

. . . .

個個個個

500 500 500 500

......

50 50 50 50

着果総数着果総数着果総数着果総数

5 5 5 5 log(y) = 2.35 + 0.02 x log(y) = 4.15 − 0.08 x log(y) = 2.37 + 0.22 x log(y) = 4.46 − 0.45 x

1 1 1 1

20 30 40 50 60 4 6 8 10 12 14 16 2 4 6 8 1.5 2.5 3.5 4.5

No. of per cones of tree female No. DBH (cm) Height (m) Crown radius (m) Height / Crown radius

FigureFigure 4. Relationship4. Relationship between between number number of of femalefemale cones per tree tree and and (A (A) )tree tree diameter, diameter, (B ()B tree) tree height, height, (C)(C crown) crown radius radius and and (D ()D height) height/crown/crown radius radius ratio. ratio. In panel panel D, D, the the thick thick black black line line indicates indicates a a significantsignificant regression regression relationship relationship across across all genotypes all genotypes (p < 0.05), (p while< 0.05), gray while lines grindicateay lines relationships indicate forrelationships each genotype. for each There genotype. were no Theresignificant were relationships no significant between relationships female between cone production female cone and theproduction other traits and (A– theC). other traits (A–C). 4. Discussion 4. Discussion In northernIn northern conifer conifer species species including including Japanese Japanese larch,larch, mast seeding, seeding, i.e., i.e., low low frequen frequentt abundant abundant fruitingfruiting events, events, is is a limitinga limiting factor factor for for sustainable sustainable seedseed production, and and numerous numerous efforts efforts have have been been mademade to toovercome overcome this this limitation limitation [ 12 [12,13].,13]. SeveralSeveral trials for increasing cone cone production production have have been been conductedconducted on on larch larch species, species, such as as L.L. kaempf kaempferieri, L, deciduaL decidua andand L. laricinaL. laricina, and, some and somesuccess success has been has beenreported reported for treatments for treatments involving involving girdling girdling [18–23], [18 gibberellins–23], gibberellins [40], nitrogen [40], fertilizer nitrogen [19], fertilizer drought [19 ], drought[24] and [24 branch] and branch bending bending [41]. However, [41]. However, the results the obtained results obtained from yet fromother yet studies other using studies simi usinglar similartreatments treatments have have often often been been inconsistent, inconsistent, such such as ineffective as ineffective stimulation stimulation by by gibberellins gibberellins [42 [42–44–],44 ], nitrogennitrogen fertilizer fertilizer [15 [15]] and and root root pruning pruning [ 24[24].]. TheseThese disparitiesdisparities could could be be attributed attributed to to differences differences in in thethe age age and and growing growing conditions conditions (natural (natural stands, stands, seedseed orchardsorchards or pots in in greenhouses, greenhouses, etc.). etc.). In Inold old seedseed orchards orchards containing containing trees trees that that are are not not well well managed,managed, restoring the the reproductive reproductive capacity capacity could could be consideredbe considered to to be be relatively relatively di difficult.fficult. However, However, thethe findingsfindings of our our study study showed showed that that female female cone cone productionproduction of of old oldL. L. kaempferi kaempferitrees trees can can be be enhanced enhanced byby girdlinggirdling andand increasing the the light light intensity. intensity. AsAs in in previous previous studies studies [18 [1–823–23],], our our study study confirmedconfirmed that girdling girdling was was effective effective for for improving improving femalefemale cone cone production, production, even even in in the the old oldL. L. kaempferikaempferi orchard. In In an an orchard orchard of of 42 42-year-old-year-old L. L.kaempferi kaempferi, , more than 90% (19/20) of the girdled trees totally produced about 8000 cones, while only 25% (5/20) 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 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 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 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 girdlingapparent on the[40], proportion suggesting of that trees age which-dependent produced sexual cones maturity and themay cone affect production the efficiency per treeof girdling. were less apparentAlthough [40 the], suggesting efficiency of that girdling age-dependent at different sexual ages has maturity not yet maybeen aclarified,ffect the our effi studyciency found of girdling. that Althoughgirdling the is a e lowfficiency-cost method of girdling that atcan di befferent used agesto efficiently has not yetrestore been the clarified, cone production our study capacity found thatof girdlingold L. iskaempferi a low-cost seed method orchards. that can be used to efficiently restore the cone production capacity of old L. kaempferiGirdlingseed hasorchards. been regarded as a cost-efficient and useful technique for disrupting phloem transportGirdling while has been limiting regarded detrimental as a cost-e effectsfficient [46]. andHowever, useful it technique has also been for disrupting reported that phloem the positive transport whileeffects limiting of girdling detrimental are relatively effects limited [46]. However, in duration, it hasand alsorepeated been girdling reported might that theadversely positive affect effects tree of girdlingvigor areand relativelydecrease total limited reproductive in duration, output and repeated[47]. In our girdling study, mightthere was adversely considerable affect treevari vigoration in and

Forests 2020, 11, 1110 7 of 10 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 Table1: larger coefficient values indicate a larger positive effect); the probability of producing cones was significantly increased as the treatment level was higher (Figure2). 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 [48–50]. 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.

5. 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, Forests 2020, 11, 1110 8 of 10 and theForests genotype-specific 2020, 11, x FOR PEER REVIEW response to light intensity was more apparent in less-reproductive8 of 11 clones. A significant negative relationship was observed between female cone production and the height/crown less-reproductive clones. A significant negative relationship was observed between female cone radiusproduction ratio. Taken and together, the height/crown these findings radius suggestedratio. Taken that together, a management these findings procedure suggested that that combines a girdlingmanagement less-reproductive procedure treesthat combine and improvings girdling light less-reproductive intensity could trees be and used improving to optimize light intensity and improve the conecould production be used to optimize capacity and of oldimproveL. kaempferi the coneseed production orchards. capacity of old L. kaempferi seed orchards.

AuthorAuthor Contributions: Contributions:M.M., M.M., H.N., H.N., A.T. A.T. and and M.T.M.T. conceivedconceived and and designed designed the the research. research. M.M., M.M., H.N. H.N.and A.T. and A.T. performedperformed the fieldthe field survey. survey. H.N. H.N. managed managed the the study study site.site. M.M. M.M. conducted conducted data data analyses analyses and andwrote wrote the original the original draft.draft. A.T. A.T. and and M.T. M.T. conducted conducted supervision supervision and and project project administration. administration. M.M.M.M. and M.T.; and funding M.T.; funding acquisition. acquisition. All All authorsauthors have have readread and agreed to to the the published published version version of the of manuscript. the manuscript. Funding:Funding:This This research research was supportedwas supported by grants by grants from from the Project the Project of the of NARO the NARO Bio-oriented Bio-oriented Technology Technology Research AdvancementResearch Advancement Institution (the Institution special (the scheme special project scheme on project regional on regional developing developing strategy; strategy; Forestry Forestry C105) C105) and JSPS KAKENHIand JSPS Grant KAKENHI Number Grant 17K15291. Number 17K15291. Acknowledgments:Acknowledgments:We We thank thank the the sta staffff of of the the Fujisan Fujisan OrchardOrchard for for their their assistance assistance with with field field investigations. investigations. ConflictsConflicts of Interest: of Interest:The The authors authors declare declare no no conflict conflict ofof interest. Appendix A Appendix A

Girdling level 1 Girdling level 2 Girdling level 3

FigureFigure A1. A1.Example Example photosphotos for for girdling girdling levels levels 1–3. 1–3.

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