Factors Affecting the Number of Pollen Grains Per Male Strobilus in Japanese Cedar (Cryptomeria Japonica)

Article Factors Affecting the Number of Pollen Grains per Male Strobilus in Japanese Cedar (Cryptomeria japonica)

Hiroyuki Kakui 1 , Eriko Tsurisaki 2, Rei Shibata 2 and Yoshinari Moriguchi 1,*

1 Graduate School of Science and Technology, Niigata University, Niigata City, Niigata 950-2181, Japan; [email protected] 2 Faculty of Agriculture, Niigata University, Niigata City, Niigata 950-2181, Japan; [email protected] (E.T.); [email protected] (R.S.) * Correspondence: [email protected]; Tel.: +81-25-262-6861

Abstract: Japanese cedar (Cryptomeria japonica) is the most important timber species in Japan; however, its pollen is the primary cause of pollinosis in Japan. The total number of pollen grains produced by a single tree is determined by the number of male strobili (male ﬂowers) and the number of pollen grains per male strobilus. While the number of male strobili is a visible and well-investigated trait, little is known about the number of pollen grains per male strobilus. We hypothesized that genetic and environmental factors affect the pollen number per male strobilus and explored the factors that affect pollen production and genetic variation among clones. We counted pollen numbers of 523 male strobili from 26 clones using a cell counter method that we recently developed. Piecewise Structural Equation Modeling (pSEM) revealed that the pollen number is mostly affected by genetic variation,

male strobilus weight, and pollen size. Although we collected samples from locations with different environmental conditions, statistical modeling succeeded in predicting pollen numbers for different clones sampled from branches facing different directions. Comparison of predicted pollen numbers Citation: Kakui, H.; Tsurisaki, E.; Shibata, R.; Moriguchi, Y. Factors revealed that they varied >3-fold among the 26 clones. The determination of the factors affecting Affecting the Number of Pollen pollen number and a precise evaluation of genetic variation will contribute to breeding strategies to Grains per Male Strobilus in Japanese counter pollinosis. Furthermore, the combination of our efﬁcient counting method and statistical Cedar (Cryptomeria japonica). Plants modeling will provide a powerful tool not only for Japanese cedar but also for other plant species. 2021, 10, 856. https://doi.org/ 10.3390/plants10050856 Keywords: pollen; pollinosis; cell counter; genetic variation; piecewise Structural Equation Modeling (pSEM); model prediction Academic Editor: Rosalyn B. Angeles-Shim

Received: 29 March 2021 1. Introduction Accepted: 19 April 2021 Published: 23 April 2021 Pollen grain number is an important trait in plants. From an evolutionary perspective, the number of pollen grains produced has a direct effect on the number of offspring,

Publisher’s Note: MDPI stays neutral and is known to vary among mating systems. For example, outcrossing plants tend to with regard to jurisdictional claims in produce more pollen grains compared to closely related selfing species [1–4]. Among published maps and institutional affil- outcrossing plants, wind-pollinators tend to produce more pollen grains than animal- iations. pollinated species [5,6]. From an agricultural perspective, while domesticated crop species such as rice or wheat tend to produce fewer pollen grains, effective pollination with abundant pollen grains is sometimes needed for purposes such as breeding hybrid crops or artificial pollination [7–12]. Pollen is also important from a medical standpoint, because it triggers an allergic reaction called pollinosis around the world [13,14]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Species in the family Cupressaceae are distributed worldwide [15]. While the family This article is an open access article includes many economically-valuable species used for timber or ornamental purposes, distributed under the terms and some species, such as cypress (Chamaecyparis obtusa), mountain cedar (Juniperus ashei), and conditions of the Creative Commons Japanese cedar (Cryptomeria japonica) have been linked to serious pollinosis [16]. Japanese Attribution (CC BY) license (https:// cedar is the primary source of building materials in Japan, and comprises ~43% of plan- creativecommons.org/licenses/by/ tation stands (~4.4 million ha) [17]. Trees with more desirable traits (e.g., timber quality, 4.0/). growth rates, disease tolerance, environmental tolerances, etc.) have been selected and

Plants 2021, 10, 856. https://doi.org/10.3390/plants10050856 https://www.mdpi.com/journal/plants Plants 2021, 10, 856 2 of 15

propagated using cuttings or seedlings [18]. However, the pollen of Japanese cedar is the primary cause of pollinosis in Japan, and in 2008, this species caused pollinosis in 26.5% of the population, a drastic increase from 16.2% in 1998 [19]. Moreover, Japanese cedar pollinosis tends to be more serious in Tokyo: the Bureau of Social Welfare and Public Health of Tokyo Metropolitan Government recently reported that 45.6% of patients with pollinosis from Japanese cedar were in Tokyo [20]. As such, pollen production per tree is among the most important considerations in breeding strategies for Japanese cedar. Generally, the total number of pollen grains produced by a single tree is the product of the number of pollen grains per male strobilus (male flower) and the number of male strobili per tree. For phenotyping, it is relatively simple to quantify the number of male strobili, as they are macroscopic and can be easily counted. More than 140 clones of Japanese cedar are classified as low pollen clones [21]; however, these were defined only by the number of male strobili, and pollen production per male strobilus was not considered. The number of pollen grains per male strobilus also affects the total pollen production of a single tree, but little is known about this trait as it is not visible to the naked eye, and methods for counting pollen grains are not well-established. Microscopes have conventionally been used for counting pollen grains, and have been used to quantify pollen production in Japanese cedar [22–24]. However, this approach may be time consuming and/or inefficient. For example, the microscope method typically requires approximately 17 min to count ~200 pollen grains [25]. Recently, we developed a pollen counting method for Japanese cedar that uses mesh columns and a cell counter [26]. The cell counter method can count approximately 20,000 particles per measurement within five minutes (i.e., >3 times faster and counts > 100 times the number of particles). Here, we focus on pollen grain production per male strobilus (hereafter “pollen number”). We counted pollen numbers from 26 clones of Japanese cedar and used piecewise Structural Equation Modeling (pSEM) to identify factors that affect pollen number. pSEM, which has gained attention in recent years in the fields of ecology and evolution, can reveal complex relationships among various factors by constructing a path diagram [27–31]. Because trees grow densely in breeding stands, it can be difficult to sample male strobili from branches of the same height and facing the same direction. Thus, we collected male strobili representing different directions and heights. We used statistical modeling to obtain a corrected pollen number that controls for the effects of these differences in environmental conditions, thus facilitating comparisons among clones.

2. Materials and Methods 2.1. Plant Materials and Sampling Conditions We collected 523 male strobili from 26 Japanese cedar clones in the Niigata Prefectural Forest Research Institute (38◦16022.700 N 139◦30035.400 E). We collected male strobilus samples in February 2020 because the pollen grains are developmentally fully mature but the male strobilus has not yet opened at this time [32]. All data are shown in Supplementary Table S1 and summary data are provided in Table1. Male strobili were always collected from the open-air side of the tree (the side where sunlight is not blocked by other trees; Figure1a,c) because sunlight positively affects the growth of male strobili [ 33]. Male strobili from Wogon-sugi and Iwafune-15 were collected from multiple directions (Table1, Figure1c), and male strobili from Wogon-sugi, Iwafune-2, and Iwafune-16 were collected at different heights (above or below 5 m; Table1, Figure1b,d). At least two inﬂorescences were collected per clone (Figure1e,f, Table1). Pollen number was estimated by counting at least six male strobili per clone (Table1). Plants 2021, 10, x FOR PEER REVIEW 3 of 16 Plants 2021, 10, x FOR PEER REVIEW 3 of 16 Plants 2021,, 10,, xx FORFOR PEERPEER REVIEWREVIEW 3 of 16 Plants Plants 20212021,, 1010,, xx FORFOR PEERPEER REVIEWREVIEW 33 ofof 1616

Plants 2021, 10, x FOR PEER REVIEW 3 of 16 Plants 2021,, 10,, xx FORFOR PEERPEER REVIEWREVIEW 3 of 16 Plants 2021, 10, x FOR PEER REVIEW 3 of 16

Plants 2021,, 10,, xx FORFOR PEERPEER REVIEWREVIEW Table 1. Summary of male strobilus samples in this study. 3 of 16 Plants 2021, 10, x FOR PEER REVIEW Table 1. Summary of male strobilus samples in this study. 3 of 16 Plants 2021, 10, x FOR PEER REVIEW Table 1. Summary of male strobilus samples in this study. 3 of 16 Plants 2021,, 10,, xx FORFOR PEERPEER REVIEWREVIEW Table 1. Summary of male strobilus samples in this study. 3 of 16 Plants 2021, 10, x FOR PEER REVIEW † ‡ 3 of§ 16 Plants 2021Clone, 10, x NameFOR PEER REVIEWDirection * Height ** Inflorescence No. † Male Strobilus No. ‡ Symbol3 of § 16 Plants 2021,, 10,, xx FORFOR PEERPEER REVIEWREVIEW Table 1. Summary of male strobilus samples in† this study. ‡ 3 of§ 16 Clone Name Direction * Height ** Inflorescence No. Male Strobilus No. Symbol Plants 2021 10 Clone, , 856 Name DirectionTable 1.* SummaryHeight of ** male strobilusInflorescence samples No in. † †this study.Male Strobilus No. ‡‡ Symbol3 § of 15 Clone Name DirectionTable 1.* SummaryHeight of ** male Inflorescencestrobilus samples No in.. † this study.Male Strobilus No.. ‡ Symbol §

Clone Name Direction * Height ** Inflorescence No. Male Strobilus No. Symbol

Table 1. Summary of male strobilus samples in this study. Wogon-sugi TableN 1. Summary>5 m of male strobilus samples2 in this study. 10

Wogon-sugi TableN 1. Summary>5 m of male strobilus samples2 in† this study. 10 ‡ § CloneWogon Name-sugi DirectionN * Height>5 m ** Inflorescence2 No. †† Male Strobilus10 No. ‡‡ Symbol §§ Wogon-sugi N >5 m 2 † 10 ‡ § CloneWogon Name-sugi DirectionTableN 1.* SummaryHeight>5 m of ** male Inflorescencestrobilus samples2 No in. this study.Male Strobilus10 No. Symbol Clone Name Direction * Height ** InflorescenceInflorescence No.. † Male Strobilus No.. ‡ Symbol § WogonClone Name-sugi DirectionN * Height>5 m ** Inflorescence2 No. Male Strobilus10 No. Symbol Table 1. Summary of male strobilus samples in this study. Clone Name Direction * Height ** Inflorescence No. Male Strobilus No. Symbol Clone Name Direction * Height ** Inflorescence No. Male Strobilus No. Symbol Wogon-sugi N <5 m 4 12 Wogon-sugi TableN 1. Summary<5 m of male strobilus samples4 in this study. 12 Table 1. Summary of male strobilus samples in† this study. ‡ §

CloneWogon Name-sugi DirectionN * Height<5 m ** Inflorescence4 No. †† Male Strobilus12 No. ‡‡ Symbol §§ CloneWogon Name-sugi DirectionTableN 1.* SummaryHeight<5 m of ** male Inflorescencestrobilus samples4 No in.. † this study.Male Strobilus12 No.. ‡ Symbol § Table 1. Summary of male strobilus samples in this study. WogonWogon--sugisugi NN >5<5 mm 24 1012 Clone Name Direction * Height ** Inflorescence No. Male Strobilus No. Symbol

CloneWogon Name-sugi DirectionN * Height<5 m ** Inflorescence4 No. Male Strobilus12 No. Symbol Wogon--sugi N >5 m 2 10

† ‡ § Wogon-sugi NTable 1. Summary>5 m of male strobilus2 samples in this study. 10

Wogon-sugi E >5 m 4 19 † ‡ § † ‡ § CloneWogon Name-sugi DirectionN * Height>5 m ** Inflorescence2 No. † Male Strobilus10 No. ‡ Symbol §

† ‡ § CloneWogon Name-sugi DirectionE * Height>5 m ** InflorescenceInflorescence4 No.. † Male Strobilus19 No.. ‡ Symbol §

CloneWogon Name--sugi DirectionNE * Height>5 m ** Inflorescence24 No. Male Strobilus1019 No. Symbol CloneWogon Name-sugi DirectionE * Height>5 m ** Inflorescence4 No. Male Strobilus19 No. Symbol Clone Name Direction * Height ** Inflorescence No. Male Strobilus No. Symbol

WogonWogon--sugisugi NE <5>5>5 mm 424 121019 † ‡ § Wogon-sugi N >5 m 2 10

Wogon-sugi NE <5>5 m 4 † 1219 ‡ § † ‡ § Wogon-sugi N <5 m 4 12 Wogon-sugi N <5 m 4 12

Wogon-sugi N >5<5 m 24 † 1012 ‡ § CloneWogon Name-sugi DirectionS * Height>5 m ** Inflorescence3 No. Male Strobilus13 No. Symbol Wogon-sugi S >5 m 3 13 Clone Name Direction * Height ** Inflorescence No. Male Strobilus No. Symbol§ CloneWogon Name-sugi DirectionN * Height<5 m ** Inflorescence4 No.. † Male Strobilus12 No..‡ Symbol

Clone Name Direction * Height ** Inﬂorescence No. Male Strobilus No. Wogon--sugi NS >5 m 23 1013 Symbol

Wogon--sugisugi NS <5>5 m 423 121013

Wogon-sugi NS >5 m 32 1310 WogonWogon--sugisugi NES >5<5>5 mm 43 191213 Wogon-sugi NS >5<5 m 324 131012 Wogon-sugi E >5 m 4 19 Wogon--sugi E >5 m 4 19 Wogon-sugi N <5 m 4 12 Wogon-sugi NE <5>5 m 4 1219 Wogon-sugi S <5 m 4 16 Wogon-sugi S <5 m 4 16

Wogon-sugi E >5 m 4 19 Wogon-sugi N >5 m 2 10 Wogon-sugi NE >5 m 24 1019

Wogon-sugi N >5 m 2 10 Wogon--sugi NS <5 m 4 1216

Wogon-sugi N >5 m 2 10

Wogon--sugi N <5 m 4 12 Wogon--sugi NES >5<5 m 4 191216 Wogon-sugi NS <5 m 4 1612

Wogon-sugi E >5 m 4 19 Wogon-sugi ES >5 m 34 1319 Wogon-sugi SE >5 m 34 1319 WogonWogon--sugisugi NS <5<5 mm 44 1216 Wogon-sugi E >5 m 4 19 Wogon-sugi S <5 m 4 16 Wogon-sugi S >5 m 3 13 Wogon--sugi S >5 m 3 13

Wogon-sugi E >5 m 4 19 Wogon-sugi WES >5 m 34 111913 Wogon-sugi S >5 m 3 13 Wogon-sugi NS <5>5 m 43 1213

Wogon-sugiWogon-sugi NN <5<5 m m 4 412 Wogon--sugi WE >5 m 43 1911

Wogon--sugisugi WES >5 m 34 131911 Wogon-sugi E >5 m 4 19

WogonWogon--sugisugi WS <5>5>5 mm 433 161311 Wogon-sugi E >5 m 4 19 Wogon-sugi ES >5 m 43 1913 Wogon-sugi WS <5>5 m 43 1611 Wogon--sugi S <5 m 4 16

Wogon-sugi S >5 m 3 13

Wogon-sugi S >5 m 3 13 Wogon-sugi WS <5 m 4 2016 Wogon-sugi ES >5<5 m 4 1916

Wogon-sugi E >5 m 4 19 Wogon-sugi E >5 m 4 19 Wogon-sugiWogon-sugi EE >5>5 m m 4 4 19 Wogon--sugi WS >5<5 m 34 1320

Wogon--sugi S >5 m 3 13 Wogon--sugi WS <5>5 m 43 162013 Wogon-sugi S <5 m 4 16 WogonWogon--sugisugi WWS >5<5 mm 34 11131620

Wogon-sugi S <5 m 4 16 Wogon-sugi W <5 m 4 20 Wogon--sugi W >5 m 3 11 Wogon-sugi WS <5>5 m 43 1611

Iwafune-2 W >5 m 3 13 Iwafune-2 W >5 m 3 13

Wogon-sugi S >5 m 3 13 Wogon-sugi WS >5 m 3 1311

Wogon-sugi S >5 m 3 13 Wogon-sugi S >5 m 3 13 WogonIwafune-sugi-2 WS <5>5 m 43 1613

WogonIwafune--sugisugi-2 WS >5<5 m 34 111613

WogonIwafune-sugi-2 WS >5<5 m 34 1316

Wogon-sugi W >5 m 3 11 WogonIwafune-sugi-2 WW <5>5 mm 43 201113 Wogon-sugi S <5 m 4 16 Wogon-sugiWogon-sugi SWS <5<5>5 m m 43 416 11

Iwafune-2 W >5 m 3 13 Wogon-sugi W <5 m 4 20

Wogon--sugi W <5 m 4 20 Wogon-sugi W >5 m 3 11 WogonIwafune-sugi-2 W <5 m 34 1420 Iwafune-2 W <5 m 3 14

Wogon-sugi WS <5 m 4 1620 Wogon-sugi W <5 m 4 20

Wogon-sugi S <5 m 4 16 Wogon-sugi S <5 m 4 16 WogonIwafune-sugi-2 W >5<5 m 3 1114

Wogon--sugi W >5 m 3 11 WogonIwafune-sugi-2 W <5>5 m 43 201114 WogonIwafune-sugi-2 W <5>5 m 3 1411

Wogon-sugi W <5 m 4 20 WogonIwafuneIwafune-sugi--22 WW >5<5 mm 343 132014 Wogon-sugiWogon-sugi WW >5>5 m m 3 311 Wogon-sugi W >5 m 3 11 WogonIwafune-sugi-2 W <5 m 34 1420 Iwafune-2 W >5 m 3 13 IwafuneIwafune--2 W >5 m 3 13

Wogon-sugi W <5 m 4 20 WogonIwafune-sugi-2 W <5>5 m 43 2013

Iwafune-15 SE >5 m 4 18 Iwafune-15 SE >5 m 4 18

Iwafune-2 W >5 m 3 13 Wogon-sugi W >5 m 3 11 Iwafune-2 W >5 m 3 13

Wogon-sugi W >5 m 3 11 Wogon-sugi W >5 m 3 11 WogonIwafune-sugi-15 WSE <5>5 m 4 2018

Wogon-sugi W <5 m 4 20 WogonIwafuneIwafune-sugi--152 SEW >5<5 m 34 132018

Iwafune-2 W >5 m 3 13 Iwafune-2 W >5 m 3 13 WogonIwafuneIwafune-sugi--152 WSE <5>5 mm 34 14201318 Wogon-sugi W <5 m 4 20 Wogon-sugiIwafune-2 WW <5>5 m m 3 4 2013

IwafuneIwafune--152 SEW <5>5 m 34 1418

Iwafune-2 W <5 m 3 14 Iwafune-2 W <5 m 3 14

Iwafune-2 W >5<5 m 3 1314 Iwafune-15 NW >5 m 3 6 Iwafune-15 NW >5 m 3 6

WogonIwafune-sugi-2 W <5 m 43 2014 Wogon-sugi W <5 m 4 20 IwafuneIwafune--152 NWW >5 m 3 136

IwafuneIwafuneIwafune---152 NWW <5>5 m 3 14136 IwafuneIwafune--152 NWW >5 m 3 136

Iwafune-2IwafuneIwafuneIwafune---15152 WNWSEW >5>5<5>5 m mm 433 31814 136 Iwafune-2 W >5 m 3 13 IwafuneIwafune--152 NWW >5<5 m 3 13146 Iwafune-15 SE >5 m 4 18 IwafuneIwafune--15 SE >5 m 4 18

Iwafune-2 W <5 m 3 14 IwafuneIwafune--152 SEW <5>5 m 34 1418 Iwafune-16 W >5 m 3 8 Iwafune-16 W >5 m 3 8

Iwafune-15 SE >5 m 4 18 Iwafune-2 W >5 m 3 13 IwafuneIwafune--152 SEW >5 m 34 1318 Iwafune-2 W >5 m 3 13 IwafuneIwafune--162 W <5>5 m 3 148

IwafuneIwafune--2 W <5 m 3 14 Iwafune-2IwafuneIwafuneIwafune---15162 WSEW <5>5<5 m m 43 318 148 IwafuneIwafune--162 W >5<5 m 3 148

Iwafune-15 SE >5 m 4 18 Iwafune-15 NWSE >5 m 34 186 Iwafune-1516 SEW >5 m 43 188 Iwafune-2 W <5 m 3 14 Iwafune-15 SE >5 m 4 18 Iwafune-16 W >5 m 3 8 Iwafune-15 NW >5 m 3 6 IwafuneIwafune--15 NW >5 m 3 6

Iwafune-15 SE >5 m 4 18 Iwafune-1615 NWSEW <5>5 m 43 20186 Iwafune-15 NW >5 m 3 6 IwafuneIwafune--152 NWW <5>5 m 3 146

Iwafune-2 W <5 m 3 14 IwafuneIwafune--1516 SEW >5<5 m 4 1820

Iwafune-15IwafuneIwafune--1516 SENWSEW >5>5<5 m m 34 41820 6 Iwafune-15 SE >5 m 4 18

IwafuneIwafune--161516 NWWW >5<5 mm 34 2086 Iwafune-15 SE >5 m 4 18 Iwafune-15 NWSE >5 m 43 186 Iwafune-16 W >5<5 m 34 208 IwafuneIwafune--16 W >5 m 3 8

Iwafune-15 NW >5 m 3 6 Iwafune-15 NW >5 m 3 6 Iwafune-16 W >5 m 3 8 Iwafune-1 SE >5 m 4 16 Iwafune-1516 SEW >5 m 43 188

Iwafune-15 SE >5 m 4 18 Iwafune-15 SE >5 m 4 18 Iwafune-15 SE >5 m 4 18 Iwafune-15IwafuneIwafune--151 NWNWSE >5>5 m m 34 3166

IwafuneIwafuneIwafune---16151 NWSEW >5 m 34 1686

IwafuneIwafune-1615-1 NWWSE <5>5>5 mm 434 201686 Iwafune-15 NW >5 m 3 6 Iwafune-16 W >5 m 3 8

Iwafune-1 SE >5 m 4 16 Iwafune-16 W <5 m 4 20 IwafuneIwafune--16 W <5 m 4 20 Iwafune-16 W >5 m 3 8 Iwafune-16 W <5 m 4 20 Iwafune-16Iwafune-4 WSW >5<5m m 4 320 8 Iwafune-4 SW <5m 4 20

Iwafune-16 W <5 m 4 20 Iwafune-15 NW >5 m 3 6 Iwafune-1516 NWW >5<5 m 34 206 Iwafune-15 NW >5 m 3 6 IwafuneIwafune--164 SWW >5<5m m 34 208 IwafuneIwafuneIwafune---164 SWW <5>5<5m m 43 208

IwafuneIwafune--164 SWW >5<5m m 43 208 Iwafune-16 W <5 m 4 20 IwafuneIwafuneIwafune---1614 SWSEW >5<5<5m m 44 1620 Iwafune-16 W >5<5 m 34 208

Iwafune-4 SW <5m 4 20 Iwafune-1 SE >5 m 4 16 IwafuneIwafune--1 SE >5 m 4 16 Iwafune-16 W <5 m 4 20 IwafuneIwafune--161 SEW <5>5 m 4 2016 Iwafune-5 SE <5 m 3 14 Iwafune-5 SE <5 m 3 14

Iwafune-1 SE >5 m 4 16 IwafuneIwafune--161 SEW >5 m 34 168

Iwafune-16 W >5 m 3 8

Iwafune-16 W >5 m 3 8 IwafuneIwafune--165 WSE <5 m 43 2014

IwafuneIwafuneIwafuneIwafune----1615 SEW >5<5 m 43 162014 IwafuneIwafune--165 SEW <5 m 34 1420

Iwafune-1 SE >5 m 4 16 IwafuneIwafune--415 SWSE >5<5<5m m 43 201614 Iwafune-16 W <5 m 4 20 IwafuneIwafune--161 SEW <5>5 m 4 2016

Iwafune-45 SWSE <5<5m m 43 2014 IwafuneIwafune--4 SW <5m 4 20

Iwafune-1 SE >5 m 4 16 Iwafune-1 SE >5 m 4 16 Iwafune-1Iwafune-4 SESW >5<5m m 4 4 1620 Iwafune-8 SW >5 m 4 20

IwafuneIwafune--164 SWW <5<5m m 4 20

Iwafune-16 W <5 m 4 20 Iwafune-16 W <5 m 4 20

IwafuneIwafuneIwafune---1618 SWSEW >5<5 m 4 1620

Iwafune-1 SE >5 m 4 16 IwafuneIwafune--418 SWSE >5<5m m 4 2016 Iwafune-81 SWSE >5 m 4 2016

IwafuneIwafune--5418 SWSWSE <5>5<5m mm 344 14201620 Iwafune-1 SE >5 m 4 16 Iwafune-4 SW <5m 4 20

Iwafune-8 SW >5 m 4 20 Iwafune-5 SE <5 m 3 14 IwafuneIwafune--5 SE <5 m 3 14 Iwafune-4 SW <5m 4 20 Iwafune-45 SWSE <5<5m m 43 2014 Iwafune-9 SE >5 m 3 11 Iwafune-9 SE >5 m 3 11

Iwafune-15 SE >5<5 m 43 1614

Iwafune-1 SE >5 m 4 16 IwafuneIwafune--49 SWSE >5<5m m 43 2011

IwafuneIwafune--549 SWSE <5>5<5m m 34 142011

Iwafune-94 SWSE >5<5m m 34 1120 Iwafune-5 SE <5 m 3 14 IwafuneIwafune--859 SWSE >5<5>5 mm 43 201411 Iwafune-5Iwafune-45 SESWSE <5<5<5m m m 43 320 14

Iwafune-89 SWSE >5 m 43 2011 IwafuneIwafune--8 SW >5 m 4 20

Iwafune-58 SWSE <5>5 m 34 1420 Iwafune-11 SE <5 m 3 13 Iwafune-11 SE <5 m 3 13

Iwafune-8 SW >5 m 4 20 Iwafune-4 SW <5m 4 20 Iwafune-48 SW >5<5m m 4 20

Iwafune-4 SW <5m 4 20 IwafuneIwafune--115 SE <5 m 3 1413

IwafuneIwafuneIwafune---1185 SWSE >5<5 m 43 201413 IwafuneIwafune--115 SE <5 m 3 1314

Iwafune-89 SWSE >5 m 43 2011 Iwafune-98 SWSE >5 m 34 1120 Iwafune-8IwafuneIwafune--115 SWSE >5<5 m m 3 414 2013 Iwafune-58 SWSE <5>5 m 34 1420 Iwafune-11 SE <5 m 3 13 Iwafune-9 SE >5 m 3 11 IwafuneIwafune--9 SE >5 m 3 11

Iwafune-8 SW >5 m 4 20 IwafuneIwafune--1289 SWNESE >5 m 34 20118 Iwafune-9 SE >5 m 3 11 Iwafune-59 SE <5>5 m 3 1411

Iwafune-5 SE <5 m 3 14 IwafuneIwafune--1285 SWNESE >5<5 m 43 20148

Iwafune-9IwafuneIwafuneIwafune---1298 SESWNESE >5>5 m m 34 31120 8 Iwafune-8 SW >5 m 4 20

IwafuneIwafuneIwafune---111298 SWNESE <5>5>5 mm 343 1311208 Iwafune-89 SWSE >5 m 43 2011 Iwafune-1112 NESE <5>5 m 3 138 IwafuneIwafune--11 SE <5 m 3 13

Iwafune-9 SE >5 m 3 11 Iwafune-9 SE >5 m 3 11 Iwafune-1711 SE >5<5 m 23 138 IwafuneIwafune--118 SWSE >5<5 m 43 2013

Iwafune-8 SW >5 m 4 20 Iwafune-11Iwafune-8 SESW <5>5 m m 4 320 13 Iwafune-8 SW >5 m 4 20 IwafuneIwafune--179 SE >5 m 32 118

IwafuneIwafune--9 SE >5 m 3 11 IwafuneIwafuneIwafune---11179 SE <5>5 m 32 13118

Iwafune-11 SE <5 m 3 13 IwafuneIwafuneIwafune---1211179 NESESE >5<5>5 mm 32 111388

Iwafune-11 SE <5 m 3 13 Iwafune-17 SE >5 m 2 8 IwafuneIwafune--12 NE >5 m 3 8 Iwafune-1112 NESE <5>5 m 3 138

Higashikanbara-1 NW <5 m 3 14 Higashikanbara-1 NW <5 m 3 14

Iwafune-12 NE >5 m 3 8 Iwafune-12IwafuneIwafune--129 NENESE >5>5 m m 3 3118

Iwafune-9 SE >5 m 3 11 Iwafune-9 SE >5 m 3 11 HigashikanbaraIwafune-11 -1 NWSE <5 m 3 1314

HigashikanbaraIwafuneIwafune--1211 -1 NWNESE >5<5 m 3 13148

HigashikanbaraIwafune-11 -1 NWSE <5 m 3 1413

Iwafune-12 NE >5 m 3 8 Iwafune-1712 NESE >5 m 23 8 Higashikanbara-1 NW <5 m 3 14 Iwafune-1112 NESE <5>5 m 3 138

HigashikanbaraIwafune-17 -1 NWSE >5<5 m 23 148

Iwafune-17 SE >5 m 2 8 Iwafune-17 SE >5 m 2 8 Iwafune-12 NE >5 m 3 8 HigashikanbaraIwafune-17 -5 NWSE >5 m 42 208 Higashikanbara-5 NW >5 m 4 20

Iwafune-1117 SE <5>5 m 32 138 Iwafune-17 SE >5 m 2 8

Iwafune-11 SE <5 m 3 13 Iwafune-11 SE <5 m 3 13 HigashikanbaraIwafune-12 -5 NWNE >5 m 34 208

IwafuneIwafune--12 NE >5 m 3 8 HigashikanbaraIwafune-1712 -5 NWNESE >5 m 234 208 HigashikanbaraIwafune-12 -5 NWNE >5 m 43 208

Iwafune-17 SE >5 m 2 8 HigashikanbaraHigashikanbaraIwafune-17 --15 NWNWSE <5>5 mm 324 14208 Iwafune-12 NE >5 m 3 8 Iwafune-12 NE >5 m 3 8 HigashikanbaraIwafune-17 -5 NWSE >5 m 42 208 Higashikanbara-1 NW <5 m 3 14 Higashikanbara--1 NW <5 m 3 14

Iwafune-17 SE >5 m 2 8 HigashikanbaraIwafune-17 -1 NWSE >5<5 m 23 148

Higashikubiki-3 NE >5 m 3 7 Higashikanbara-1Higashikubiki-3 NWNE <5>5 m m 3 3 147 HigashikanbaraIwafune-12 -1 NWNE >5<5 m 3 148 HigashikanbaraIwafune-12 -1 NWNE >5<5 m 3 148

Iwafune-12 NE >5 m 3 8 HigashikubikiIwafune-17 -3 NESE >5 m 23 87

IwafuneIwafune--17 SE >5 m 2 8 HigashikanbaraHigashikubikiIwafune-17 --31 NWNESE <5>5 m 32 1487 HigashikubikiIwafune-17 -3 NESE >5 m 32 78

Higashikanbara-1 NW <5 m 3 14 Higashikanbara-1 NW <5 m 3 14 Higashikanbara-51 NW >5<5 m 43 2014 HigashikubikiIwafune-17 -3 NESE >5>5 mm 23 87 Higashikanbara-1 NW <5 m 3 14

HigashikanbaraHigashikubiki-3-5 NWNE >5 m 43 207

Higashikanbara-5 NW >5 m 4 20 Higashikanbara-5 NW >5 m 4 20

Higashikanbara-5HigashikanbaraHigashikubiki-5-15 NWNW >5<5>5 m m 43 42014 Higashikubiki-5 NW <5 m 4 20

HigashikanbaraIwafune-17 -5 NWSE >5 m 24 208 Iwafune-17 SE >5 m 2 8

HigashikanbaraHigashikubiki--51 NW <5 m 34 1420

HigashikanbaraHigashikubiki--5-51 NW >5<5 m 43 2014 HigashikanbaraHigashikubiki-5-1 NW <5 m 43 2014

HigashikanbaraHigashikubikiHigashikubiki--3-55 NWNWNE >5<5 mm 344 20207 Higashikanbara-1 NW <5 m 3 14 Higashikanbara-15 NW <5>5 m 34 1420 Higashikubiki-5 NW <5 m 4 20 Higashikubiki-3 NE >5 m 3 7 Higashikubiki--3 NE >5 m 3 7 Higashikubiki-3 NE >5 m 3 7

Higashikanbara-5 NW >5 m 4 20

Higashikanbara-5 NW >5 m 4 20 HigashikubikiKashiwazaki-2-3 NWNE <5>5 m 43 207

HigashikanbaraHigashikubiki-3-1 NWNE <5>5 m 3 147 HigashikanbaraHigashikubiki-3-1 NWNE >5<5 m 3 147 Higashikanbara-1 NW <5 m 3 14

HigashikanbaraKashiwazaki-2- 51 NW >5<5 m 43 2014

Higashikanbara--5 NW >5 m 4 20 HigashikanbaraHigashikubikiKashiwazaki--23- 5 NWNE >5<5 m 34 207

Higashikubiki-3 NE >5 m 3 7 Higashikubiki-5HigashikanbaraHigashikubikiKashiwazaki--25-3 5 NWNWNWNE <5<5>5<5 m mm 434 420 207

Higashikubiki-3 NE >5 m 3 7 HigashikubikiKashiwazaki--2-5 NW <5 m 4 20

MinamikanbaraHigashikubiki-3-53 NWNESE >5<5 m 34 11207

HigashikanbaraHigashikubiki-5-5 NW >5<5 m 4 20

Higashikanbara-5 NW >5 m 4 20 Kashiwazaki-2MinamikanbaraHigashikubiki-3-3 NWNESE <5>5 m m 3 4 20117

MinamikanbaraHigashikubiki--53-3 NWNESE <5>5 m 43 20117

MinamikanbaraHigashikubiki-3-3 NESE >5 m 3 117

MinamikanbaraHigashikubikiKashiwazaki--25- 3 NWSE <5>5 mm 43 2011

MinamikanbaraHigashikubikiKashiwazaki-2-3-5 3 NWNESE <5>5 m 43 20117

HigashikubikiKashiwazaki--2-5 NW <5 m 4 20 Minamikanbara-3HigashikubikiKashiwazakiMishima-1 --235 SENWNE >5>5<5 m m 34 320 117

Higashikubiki-3 NE >5 m 3 7 HigashikubikiMishima-1 -35 NWNE >5<5 m 34 207

Higashikubiki--5 NW <5 m 4 20 HigashikubikiKashiwazakiMishima-1 -2-5 NW <5>5 m 4 20 Kashiwazaki-2 NW <5 m 4 20 MinamikanbaraHigashikubikiKashiwazakiMishima-1 --25- 3 NWNWSE >5<5>5 mm 344 112020 KashiwazakiMishima-1 -2 NW >5<5 m 4 20 MinamikanbaraMishima-1--3 NWSE >5>5 m m 3 411 20 MinamikanbaraKashiwazaki-2- 3 NWSE <5>5 m 43 2011

Murakami-3 NE <5 m 2 10 Murakami-3 NE <5 m 2 10 MinamikanbaraHigashikubiki-5-3 NWSE <5>5 m 43 2011

Higashikubiki-5 NW <5 m 4 20 Higashikubiki-5 NW <5 m 4 20 KashiwazakiMurakami-3-2 NWNE <5 m 42 2010 MinamikanbaraKashiwazakiMurakami-3--2 - 3 NWNESE >5<5 m 342 112010

Minamikanbara-3 SE >5 m 3 11 MinamikanbaraKashiwazakiMurakamiMishima-1-3 -2 - 3 NWNESE >5<5 mm 432 201110 MinamikanbaraMurakami-3Kashiwazaki-2- 3 NENWSE <5<5>5 m m 43 220 1011

Murakami-3 NE <5 m 2 10 Mishima-1 NW >5 m 4 20

Mishima--1 NW >5 m 4 20 Minamikanbara-3 SE >5 m 3 11 MurakamiMishima--14 NW <5>5 m 4 20 Murakami-4 NW <5 m 4 20

KashiwazakiMishima-1 -2 NW <5>5 m 4 20 Mishima-1 NW >5 m 4 20

Kashiwazaki-2 NW <5 m 4 20 MinamikanbaraMurakami-4 -3 NWSE >5<5 m 34 1120

Minamikanbara--3 SE >5 m 3 11 MinamikanbaraMurakamiMishima-1-4 -3 NWSE >5<5 m 43 2011 MinamikanbaraMurakami-4 -3 NWSE <5>5 m 43 2011

Murakami-4Mishima-1 NWNW <5>5 m m 4 420 MurakamiMurakamiMishima--1-34 NWNE <5>5<5 mm 24 1020 Minamikanbara-3 SE >5 m 3 11 Minamikanbara-3 SE >5 m 3 11 MurakamiMishima--14 NW <5>5 m 4 20 Murakami-3 NE <5 m 2 10 Murakami--3 NE <5 m 2 10

Mishima-1 NW >5 m 4 20 MurakamiMishima-1-3 NWNE >5<5 m 42 2010

Nakakanbara-1 SE <5 m 4 18 Nakakanbara-1 SE <5 m 4 18

Murakami-3 NE <5 m 2 10 Minamikanbara-3 SE >5 m 3 11 Murakami-3 NE <5 m 2 10

Minamikanbara-3 SE >5 m 3 11 Minamikanbara-3 SE >5 m 3 11 NakakanbaraMishima-1 -1 NWSE >5<5 m 4 2018

Mishima-1 NW >5 m 4 20 Nakakanbara-1NakakanbaraMurakamiMishima-1-3 - 1 SENWNESE <5<5>5 m m 24 4102018

Murakami-3 NE <5 m 2 10 Murakami-3 NE <5 m 2 10 NakakanbaraMurakamiMishima-1-43 - 1 NWNESE <5>5<5 mm 424 201018 Mishima-1 NW >5 m 4 20 Murakami-3 NE <5 m 2 10

NakakanbaraMurakami-4- 1 NWSE <5 m 4 2018

Murakami-4 NW <5 m 4 20 Murakami-4 NW <5 m 4 20

Murakami-34 NWNE <5 m 24 1020 Nishikanbara-1 NW >5 m 4 20 Nishikanbara-1 NW >5 m 4 20

MurakamiMishima-1-4 NW >5<5 m 4 20 Mishima-1 NW >5 m 4 20 NishikanbaraMurakami-3- 1 NWNE <5>5 m 24 1020 Nishikanbara-1 NW >5 m 4 20

NishikanbaraMurakami--43- 1 NWNE <5>5 m 42 2010 NishikanbaraMurakami-3- 1 NWNE >5<5 m 42 2010

NakakanbaraNishikanbaraMurakami-4-- 11 NWNWSE <5>5 mm 44 182020 Murakami-3 NE <5 m 2 10 NishikanbaraMurakami-34- 1 NWNE >5<5 m 42 2010 Nakakanbara-1 SE <5 m 4 18 Nakakanbara--1 SE <5 m 4 18

Murakami-4 NW <5 m 4 20 NakakanbaraMurakami-4- 1 NWSE <5 m 4 2018 Nakakubiki-2 NW >5 m 4 20 Nakakubiki-2Nakakubiki-2 NWNW >5>5 m m 4 420

Nakakanbara-1 SE <5 m 4 18 Murakami-3 NE <5 m 2 10 NakakanbaraMurakami-3- 1 NESE <5 m 24 1018 Murakami-3 NE <5 m 2 10 NakakubikiMurakami-4-2 NW <5>5 m 4 20

Murakami--4 NW <5 m 4 20 NakakanbaraNakakubikiMurakami--4-2 1 NWSE <5>5 m 4 1820 NakakubikiMurakami--42 NW >5<5 m 4 20

Nakakanbara-1 SE <5 m 4 18 NishikanbaraNakakanbara-1 NWSE >5<5 m 4 2018 Nakakanbara-1 SE <5 m 4 18 NakakubikiMurakami-4-2 NWNW <5>5 mm 44 2020 Nakakanbara-1 SE <5 m 4 18 Nakakubiki-2 NW >5 m 4 20 Nishikanbara-1 NW >5 m 4 20 Nishikanbara--1 NW >5 m 4 20

Nakakanbara-1 SE <5 m 4 18 Nakakubiki-7NakakanbaraNishikanbaraNakakubiki-7-1 NENWNESE <5<5>5 m m 34 31418 20 Nishikanbara-1 NW >5 m 4 20 NishikanbaraMurakami-4- 1 NW <5>5 m 4 20

Murakami-4 NW <5 m 4 20 NakakanbaraNakakubiki--71 NESE <5 m 43 1814

NishikanbaraNakakanbaraNakakubiki-7--1 NWNESE >5<5 m 43 201814 Nakakanbara-1 SE <5 m 4 18

NishikanbaraNakakubikiNakakubiki--2-71 NWNE >5<5 mm 43 2014 Nakakanbara-1 SE <5 m 4 18 NakakanbaraNishikanbara-1 NWSE <5>5 m 4 1820 Nakakubiki-27 NWNE >5<5 m 43 2014 Nakakubiki--2 NW >5 m 4 20

Nishikanbara-1 NW >5 m 4 20 Nishikanbara-1 NW >5 m 4 20 Ryotsu-1NakakubikiRyotsu-1 -2 SWNWSW >5>5 m m 4 419 20 NakakanbaraNakakubiki-2-1 NWSE <5>5 m 4 1820 Nakakanbara-1 SE <5 m 4 18 Nakakanbara-1 SE <5 m 4 18 NishikanbaraRyotsu-1 -1 NWSW >5 m 4 2019

NishikanbaraNakakubikiRyotsu-1 -2--1 NWSW >5 m 4 2019 NishikanbaraNakakubikiRyotsu-1 -72-1 NWNESW <5>5>5 mm 344 142019

Nakakubiki-2 NW >5 m 4 20 Ryotsu-1 SW >5 m 4 19 Nakakubiki--7 NE Total<5 m 3 120 52314 Nakakubiki-27 NWNE >5<5 m 43 2014

Total 120 523 Total 120 523

NishikanbaraNakakubiki-7-1 NWNE >5<5 m 43 2014 Nishikanbara-1 NW >5 m 4 20 Nakakubiki -2 NW Total>5 m 1204 52320 Nakakubiki --72 NWNE Total<5>5 m 12034 5231420

Nakakubiki -72 NWNE Total<5>5 m 12034 5231420 NakakubikiRyotsu-1 -7 SWNE >5<5 m 43 1914 *Nakakubiki Direction of collected-27 male strobilusNWNE sample (i.e.,>5Total<5 Directionm of open-air12043 side, Figure1a,c).; ** Height523 of2014 collected strobilus sample

Ryotsu-1 SW >5 m 4 19 Total 120 523 * DirectionRyotsu of-- 1collected male strobilusSW sample >5(i.e., m Direction of open-4air side, Figure 1a,c).; ** Height19 of collected strobilus * DirectionNakakubikiRyotsu of- 1collected -7 male strobilusNESW sample <5>5(i.e., m Direction of open-34air side, Figure 1a,c).; ** Height1419 of collected strobilus

* DirectionNakakubikiRyotsu of-1 collected† -2 male strobilusNWSW sample >5(i.e.,‡ m Direction of open4-air side, Figure§ 1a,c).; ** Height2019 of collected strobilus Nakakubiki-27 NWNE >5<5 m 43 2014

(Figure1b,d).; Collected inﬂorescence number.; Collected inﬂorescence number.; The symbol corresponding to Figure 6b and * Direction of collected† male strobilus sample (i.e., Direction‡ of open-air side, Figure 1a,c).; ** §Height of collected strobilus * DirectionNakakubikiRyotsu of- 1collected --7 male strobilusSWNE sample >5<5(i.e., m Direction of open-43air side, Figure 1a,c).; ** Height1914 of collected strobilus * DirectionNakakubiki of collected-7 male strobilusNE sample <5(i.e., m Direction of open3-air side, Figure 1a,c).; ** Height14 of collected strobilus sample (Figure 1b,d).; Collected inflorescence number.; Collected inflorescence number.; The symbol corresponding sample* DirectionNakakubikiRyotsu (Figure of- 1collected -1b7 ,d).; †male Collected strobilusSWNE inflorescence sample Total>5<5(i.e., number m Direction .; ‡ Collected of open120-43 airinflorescence side, Figure number 1a,c).; **.; §Height The5231914 symbol of collected corresponding strobilus NakakubikiRyotsu -1 -7 NESW Total<5>5 m 12034 5231419 † Total ‡ 120 § 523 sampleSupplementaryRyotsu (Figure -1 Figure 1b,d).; S1. † Collected SW inflorescence Total>5 numberm .; ‡ Collected1204 inflorescence number.; § The52319 symbol corresponding sampleNakakubiki (Figure - 1b7 ,d).; †† Collected NE inflorescence Total<5 number m .; ‡‡ Collected1203 inflorescence number.; §§ The52314 symbol corresponding tosample NakakubikiFigureRyotsu (Figure 6b and-1 - 1b7 Supplementary ,d).; CollectedSWNE Figure inflorescence S1. >5<5 numberm .; Collected43 inflorescence number.; The1914 symbol corresponding to Figure 6b and Supplementary Figure S1.

*sampleto Direction FigureRyotsu (Figure 6b of -and- 1collected 1b Supplementary,d).; male Collected strobilusSW Figure inflorescence sample S1. Total>5(i.e., number m Direction .; Collected of open120-4 airinflorescence side, Figure number 1a,c).; **.; Height The52319 symbol of collected corresponding strobilus *to* Direction FigureRyotsu 6b of -and 1collected Supplementary male strobilusSW Figure sample S1. Total>5(i.e., m Direction of open120-4-air side, Figure 1a,,c).;.; **** Height52319 of collected strobilus *toto Direction FigureFigureRyotsu 6b6b of and-and 1collected SupplementarySupplementary† male strobilusSW FigureFigure sample SS11.. >5(i.e., m Direction ‡ of open-4air side, Figure 1a,c).; ** §Height19 of collected strobilus sample* DirectionRyotsu (Figure of- 1collected 1b,d).; † †male Collected strobilusSW inflorescence sample Total>5(i.e., number m Direction .; ‡‡ Collected of open120-4 airinflorescence side, Figure number 1a,c).; **.; §§Height The52319 symbol of collected corresponding strobilus sample* Direction (Figure of collected 1b,,d).;.; † male Collected strobilus inflorescence sample Total(i.e., number Direction .;.; ‡ Collected of open120-- airinflorescence side, Figure number 1a,,c).;.; **.;.; §Height The523 symbol of collected corresponding strobilus to* Direction Figure 6b of and collected Supplementary† male strobilus Figure sample S1. (i.e., Direction‡ of open-air side, Figure 1a,c).; ** §Height of collected strobilus tosampleto Figure (Figure 6b and 1b Supplementary,d).; †† Collected Figure inflorescence S1.. Total number .; ‡‡ Collected120 inflorescence number.; §§ The523 symbol corresponding sample*to* Direction Figure (Figure 6b of and collected 1b Supplementary,,d).;.; † male Collected strobilus Figure inflorescence sample S1. Total(i.e., number Direction .;.; ‡ Collected of open120-- airinflorescence side, Figure number 1a,,c)c).;.; **.;**.; §Height The523 symbol of collected corresponding strobilusstrobilus to*sample Direction Figure (Figure 6b of and collected 1b Supplementary,,d).;.; male Collected strobilus Figure inflorescence sample S1. (i.e., number Direction.;.; Collected of open- airinflorescence side, Figure number 1a,,c).;.; **.;.; Height The symbol of collected corresponding strobilus to Figure 6b and Supplementary†† Figure S1. ‡‡ §§ *tosampletosample Direction Figure (Figure(Figure 6b of and collected 1b1b Supplementary,,d).;.; †male Collected strobilus Figure inflorescence sample S1.. (i.e., number Direction.;.; ‡ Collected of open- airinflorescence side, Figure number 1a,c).; **.;.; §Height The symbol of collected corresponding strobilus *sampleto Direction Figure (Figure 6b of and collected 1b Supplementary,,d).;.; †male Collected strobilus Figure inflorescence sample S1.. (i.e., number Direction.;.; ‡ Collected of open-- airinflorescence side, Figure number 1a,,c).;.; **.;.; §Height The symbol of collected corresponding strobilus sampletoto Figure (Figure 6b and 1b Supplementary,d).; †† Collected Figure inflorescence S1.. number.; ‡‡ Collected inflorescence number.; §§ The symbol corresponding sampleto Figure (Figure 6b and 1b Supplementary,,d).;.; Collected Figure inflorescence S1.. number.;.; Collected inflorescence number.;.; The symbol corresponding toto Figure 6b and Supplementary Figure S1..

Plants 2021, 10, 856 4 of 15 Plants 2021, 10, x FOR PEER REVIEW 4 of 16

Figure 1. Sample collection. Male strobilus samples were always collected from the open-air side (a) (seeFigure also (1.c)). Sample Tree height collection. was Male measured strobilus using samples a measuring were always pole (b collected) and direction from the was open confirmed-air side with(a) a(see field also map (c)). and Tree compass. height (wasc,d) measured Conceptual using explanations a measuring of direction pole (b) ( cand) and direction height ( dwas). Samples con- fromfirmed Clones with 1 anda field 8 were map collectedand compass. from two(c,d) differentConceptual directions explanations (Clone of 1: direction N and W, (c Clone) and 8:height E and (d). Samples from Clones 1 and 8 were collected from two different directions (Clone 1: N and W, S). Clones 2, 3, 6, and 7 were sampled from only one direction (Clones 2 and 3: N, Clones 6 and 7: Clone 8: E and S). Clones 2, 3, 6, and 7 were sampled from only one direction (Clones 2 and 3: N, S). Clones 4 and 5 were not sampled because they had no open-air side. Wogon-sugi (“W”) was Clones 6 and 7: S). Clones 4 and 5 were not sampled because they had no open-air side. Wogon- sampledsugi (“W” in all) was directions sampled because in all directions it was solitary because in theit was field. solitary (e,f) Inflorescences in the field. (e from,f) Inflorescences Iwafune-5 (e ) andfrom Higashikubiki-3 Iwafune-5 (e) (andf). Several Higashi malekubiki strobili-3 (f). developSeveral male from strobili a single develop inflorescence. from a Arrowssingle inflo- indicate malerescence. strobili Arrows (e, right; indicate see also male Figure strobili2a–c). (e Scale, right; bar see = also 1 cm. Figure 2a–c). Scale bar = 1 cm. 2.2. Male Strobili Measurement 2.2. Male Strobili Measurement Male strobili were weighed using a digital precision scale (ViBRA-HT, Shinko Denshi, Male strobili were weighed using a digital precision scale (ViBRA-HT, Shinko Tokyo, Japan). Images of samples were taken under a stereomicroscope (SZ-11, Olympus, Denshi, Tokyo, Japan). Images of samples were taken under a stereomicroscope (SZ-11, Japan) using a digital camera (WRAYCAM-EL310, WRAYMER, Osaka, Japan). Pollen Olympus, Japan) using a digital camera (WRAYCAM-EL310, WRAYMER, Osaka, Japan). grain size was measured using ImageJ and Fiji software [34]. Representative photos from Pollen grain size was measured using ImageJ and Fiji software [34]. Representative photos

Plants 2021 10 Plants 2021, 10, 856x FOR PEER REVIEW 5 5of of 16 15

fromsmall, small, medium, medium and, largeand large male male strobili strobili areshown are shown in Figure in Figure2a–c, 2a respectively–c, respectively (also (also see Section 3.1). Figure 2 see Section 3.1). a Iwafune-11 b Minamikanbara-3 c Nakakubiki-2

1mm 1mm 1mm

Iwafune-11

d60

e b

e l

c 20

a P

0 5 10 15 20 25 30 35 40 45 50 Diameter (μm) Minamikanbara-3

e60

r e

b 40

e l

c 20

a P

0 5 10 15 20 25 30 35 40 45 50 Diameter (μm) Nakakubiki-2

f 60

r e

b 40

l 20

a P

0 5 10 15 20 25 30 35 40 45 50 Diameter (μm) Overlay

g60

r e

b 40

l c

i 20

a P

0 5 10 15 20 25 30 35 40 45 50 Diameter (μm)

Figure 2. 2. VariationVariation in in male male strobili, strobili, pollen pollen number, number, and and pollen pollen size. size. Representative Representative samples samples from Iwafune from Iwafune-11-11 (a), Min (aa-), mMinamikanbara-3ikanbara-3 (b), and (b), Nakakubiki and Nakakubiki-2-2 (c). Scale (c). Scale bars bars= 1 mm. = 1 mm.Particle Particle distribution distribution at Iwafune at Iwafune-11-11 (d), (Mid),nam Minamikanbara-3ikanbara-3 (e), (ande), andNakakubiki Nakakubiki-2-2 (f), and (f), overlay and overlay view view(g). The (g). x- Theaxis xrepresents-axis represents particle particle diameter diameter (μm) and (µ m)theand y-axis the representsy-axis represents particle number. particle number. 2.3. Measurement of Pollen Number and Size Using a Cell Counter

Plants 2021, 10, 856 6 of 15

2.3. Measurement of Pollen Number and Size Using a Cell Counter Pollen number and size were measured as previously described [26]. Briefly, a single male strobilus was gently crushed with a pestle and suspended in water. The pollen suspension was then filtered through two mesh filters (100 and 20 µm) to remove debris and small particles. The cleaned suspension was mixed with an isotonic cell counter buffer, and pollen number and pollen size (diameter) were assessed using a cell counter (CASY cell counter, OMNI Life Science, Bremen, Germany). Representative results for the samples shown in Figure2a–c are presented in Figure2d–f. Data were analyzed using the CASY application (OMNI Life Science) and plots were constructed in R [35].

2.4. Evaluation for the Hypothesized and Best Fit Pathways Using Statistical Modeling (pSEM) We used pSEM to examine the direct and indirect effects of environmental conditions and other factors on pollen number. First, we constructed a full pathway model, shown in Figure 3a, which included all hypothesized causal relationships. We then fitted a linear mixed effect model (LMM) for each of three response variables; male strobilus weight and pollen size were modeled as a function of direction and height, whereas pollen number was modeled as a function of male strobilus weight and pollen size. Direction was converted into a numerical variable in which −1 represented north, −0.5 represented northwest and northeast, 0 represented west and east, 0.5 represented southwest and southeast, and 1 represented south. Collection height was converted to a dummy variable with 0 indicating collection below 5 m and 1 indicating collection above 5 m. Before fitting the LMMs, male strobilus weight and pollen number were log-transformed to meet assumptions of normality. We included genetic contributions (i.e., variations associated with clone) as random effects in the LMMs as these clearly affected response variables. Inflorescence was also included as a random effect to account for the bias associated with the nested sampling structure (i.e., the effects of collecting multiple samples from a single inflorescence), but this is not discussed further as it was not our focus. Male strobilus weight and pollen size were clearly correlated, but the direction of causality was not clear; these two variables were therefore treated as correlated errors. The three abovementioned LMMs and the two correlated errors were integrated into the full hypothesized pathway model (hypothetical model). We then simplified the hypothetical model by removing less significant paths to improve overall goodness of fit. Goodness of fit was compared among models using the Akaike information criterion (AIC), and the removal of a path was accepted if it decreased the AIC value [36]. We selected the pSEM with the lowest AIC value as the best fit model (best model). For both the hypothetical model and best model, we extracted the standardized effect size of all paths to determine path strength. We also calculated the variance (R2) explained by each predictor and by genetic contributions, to compare their individual importance in the LMMs [37]. The pSEM models and LMMs were constructed using the piecewiseSEM and lmerTest R packages, respectively [27,38].

2.5. Corrected Pollen Number by Statistical Modeling (LMM) Using the three LMMs included in the best model, we predicted the expected pollen number for each clone after correcting for the effects of sampling direction. The effect of sampling height was disregarded because it was not a component of the best model (Figure 3b). First, we extracted the random effects of clone, which represent within-group variation among the response variables (i.e., male strobilus weight, pollen size, and pollen number), from the LMMs. We then extracted the predicted mean and 95% conﬁdence intervals (CIs) for male strobilus weight and pollen size for each clone, assuming that the effects of sampling direction on response variables were zero, i.e., that sampling direction was east or west. Finally, we extracted the predicted mean pollen number and 95% CIs for each clone, assuming that values of the predictors (i.e., male strobilus weight and pollen size) were the predicted mean ± 95% CIs of each clone predicted above. Plants 2021, 10, 856 7 of 15

3. Results and Discussion 3.1. Male Strobilus Characteristics and Genetic/Environmental Conditions We assessed a total of 523 male strobili collected from 26 clones. Male strobili were weighed and photographed before pollen was counted. Male strobili varied considerably in weight and size (Supplementary Table S1, Figure2a–c). Male strobilus weight and area were strongly correlated (Supplementary Figure S1, r = 0.892). Hereafter we discuss only male strobilus weight, as it is a trait that can be easily determined and it is considered more precise than area (area only contains information on two dimensions of the male strobilus, whereas weight includes information about the entire sample). Cell counter results indicated that larger/heavier male strobili tended to have more pollen grains (Figure2), and that pollen size varied among samples (Figure2d–g). We analyze and discuss the factors affecting pollen number in more detail in the following sections.

3.2. Hypothetical and Best Models To explore the factors affecting pollen number, we first specified five input conditions: pollen number per male strobilus (“pollen number”); pollen grain diameter (“pollen size”); male strobilus weight (“male strobilus weight”); sampling direction (“direction”); and sampling height (“height”). We also incorporated a factor (“clone”) to account for the genetic variation. The hypothetical model was constructed using pSEM, and is shown in Figure3a. In this hypothetical model, direction had a significant effect on pollen number, whereas the effects of height were non-significant. This result was unexpected as we had initially assumed that both direction and height would affect the growth of male strobili due to their effects on light received. Our findings indicate that direction is more significant than height because we always collected samples from the open-air side of the tree (Figure1a–d). In the open-air, the total amount of sunlight may not differ by height, but the amount of sunlight per year clearly differs by direction [39]. Although direction significantly affected male strobilus weight, effects on pollen size were non-significant (Figure3a). We then constructed the best model (Figure3b) by simplifying the hypothetical model. Comparison of AIC values indicated that the best model was better than the hypothetical model (Figure3). The best model revealed that pollen number is explained well by male strobilus weight, clone, and pollen size (total contribution 85.2%). Effect size (black arrow) revealed that male strobilus weight positively affects the pollen number, while pollen size negatively affects the pollen number. Male strobilus weight is affected by a genetic contribution (clone) and by direction as an environmental condition. Pollen size is mainly affected by the genetic contribution, and mutually affects male strobilus weight. Overall, pSEM revealed which factors significantly affect other factors and successfully determined the contributions. We analyze and discuss each factor in more detail in the following sections.

3.3. Factors Affecting Pollen Number The best model indicated that pollen number was significantly affected by male strobilus weight and pollen size, and was influenced by genetic contributions (Figure3b ). Figure4a presents a bar plot of pollen number by clone; pollen number varied from approximately 100,000 to 300,000, indicating that genetic contributions substantially influenced pollen number, as indicated by the pSEM (Figure3b). Figure4b presents the model of pollen number using all samples and two predictors (male strobilus weight and pollen size). Male strobilus weight was strongly and positively correlated with pollen number, as suggested by the model (Figure3b). Every 10-mg increase in male strobilus weight was associated with an increase in pollen number of approximately 100,000 (Figure4b ). Moreover, for male strobili of equal weight, samples with a smaller pollen size tended to have a higher pollen number (Figure4b, light blue vs. orange symbols). This finding suggests that pollen size also affects the pollen number. Kondo et al. (1992) counted pollen grains in 10 clones and they also found a correlation between male strobilus weight and Plants 2021, 10, 856 8 of 15

Plants 2021, 10, x FOR PEER REVIEW 8 of 16 pollen number [23]. We propose that pollen number can be estimated more accurately using male strobilus weight and pollen size.

Figure 3. Piecewise Structural Equation Modeling (pSEM) of factors affecting pollen number. Hy- pothetical model (a) with six factors (pollen number, pollen size, male strobilus weight, direction, height, and clone) and the best model (b) with five factors (pollen number, pollen size, male strobilus weight, direction, and clone). Solid black arrows indicate a significant effect (p < 0.001), and dashed arrows indicate no significant effect. Double-headed arrows indicate that two factors are related but Figure the3. piecewise direction Structural of the effect Equation is unclear. Modeling Black numbers(pSEM) of indicate factors standardizedaffecting pollen effect number. size, andHy- positive potheticaland model negative (a) with numbers six factors represent (pollen positive number, and pollen negative size, effects, male respectively.strobilus weight, Red direction, numbers indicate height, theand contribution clone) and the ratio best (0–1) model of each(b) with factor five in factors the model. (pollen Note number, that numberspollen size, of male the same stro- color are bilus weight,comparable, direction, whereas and clone). those ofSolid differing black colorsarrows are indicate not (see a significant also Section effect 2.4). (p < 0.001), and dashed arrows indicate no significant effect. Double-headed arrows indicate that two factors are related but the direction of the effect is unclear. Black numbers indicate standardized effect size, and positive and negative numbers represent positive and negative effects, respectively. Red numbers indicate the contribution ratio (0–1) of each factor in the model. Note that numbers of the same color are comparable, whereas those of differing colors are not (see also Section 2.4).

3.3. Factors Affecting Pollen Number The best model indicated that pollen number was significantly affected by male strobilus weight and pollen size, and was influenced by genetic contributions (Figure 3b). Figure 4a presents a bar plot of pollen number by clone; pollen number varied from approximately 100,000 to 300,000, indicating that genetic contributions substantially influenced pollen number, as indicated by the pSEM (Figure 3b). Figure 4b presents the model

Plants 2021, 10, x FOR PEER REVIEW 9 of 16

of pollen number using all samples and two predictors (male strobilus weight and pollen size). Male strobilus weight was strongly and positively correlated with pollen number, as suggested by the model (Figure 3b). Every 10-mg increase in male strobilus weight was associated with an increase in pollen number of approximately 100,000 (Figure 4b). More- over, for male strobili of equal weight, samples with a smaller pollen size tended to have a higher pollen number (Figure 4b, light blue vs. orange symbols). This finding suggests that pollen size also affects the pollen number. Kondo et al. (1992) counted pollen grains Plants 2021, 10, 856 in 10 clones and they also found a correlation between male strobilus weight and pollen 9 of 15 number [23]. We propose that pollen number can be estimated more accurately using male strobilus weight and pollen size.

Figure 4. Factors affecting pollen number. Distribution of pollen number among clones (a). This

plot was made using raw data, and thus reﬂects different environmental conditions (see Section 3.6). FigureScatterplot 4. Factors of affecting pollen number pollen number. and male Distribution strobilus weightof pollen from number all samples among clones (b). Samples (a). This with smaller plot was made using raw data, and thus reflects different environmental conditions (see Section pollen grains (< 34.1 µm) are indicated by light blue circles and those with larger grains (> 34.1 µm) 3.6). Scatterplot of pollen number and male strobilus weight from all samples (b). Samples with smallerare indicated pollen grains by orange (< 34.1 μ diamonds.m) are indicated Trend by lines light indicateblue circles predicted and those pollen with larger number grains as a(> function of 34.1grain μm)size: are indicated small (29.5 by orangeµm; dashed diamonds. line), Trend middle lines (34.1 indicateµm; solidpredicted line), pollen and largenumber (36.8 as µa m; bold line). 3.4. Factors Affecting Male Strobilus Weight pSEM and pollen number modeling suggested that male strobilus weight is among

the most important factors inﬂuencing pollen number. pSEM predicted that male strobilus weight is signiﬁcantly affected by direction and genetic contributions (Figures3b and5a ). A lower pollen number was associated with lower male strobilus weight, and vice versa (Figures4a and5a); however, several clones, including Higashikubiki-5, Iwafune-17, Nishikanbara-1, and Iwafune-15, exhibited only weak relationships between pollen number and male strobilus weight. Higashikubiki-5 and Nishikanbara-1 generally had low male strobilus weights but high pollen numbers, while the inverse was true for Iwafune-17 and Iwafune-15 (Figures4a and5a). This apparent contradiction was resolved by accounting for pollen size: Higashikubiki-5 and Nishikanbara-1 had smaller pollen grains whereas Iwafune-17 and Iwafune-15 had larger grains (Figure6a). These results support the hypoth- esis that pollen number is affected by not only male strobilus weight but also by pollen size. Plants 2021, 10, 856 10 of 15

Plants 2021, 10, x FOR PEER REVIEW 11 of 16

Figure 5

o W

Clone

b c All data Wogon-sugi only

)

(

30 t h

h 20

20 s

t t

s 10 10

a M 0 M

N NW/NE W/E SW/SE S N W/E S Direction Direction Figure 5. FigureFactors affecting 5. Factors male strobil affectingus weight male. The strobilus distribution weight. of male strobilus The distribution weight among of clone males (a strobilus). (b,c) Violin weight among plots of male strobilus weight and direction with the full dataset (n = 523; (b)) and Wogon-sugi (n = 101; (c)). Plots and the trendline clonesshow male (a). strobilus (b,c) Violin weight, plotswhich ofwas male greater strobilus on the south weight side. The and trendline direction was withobtained the from full the dataset linear (n = 523; (b)) mixed effectand model Wogon-sugi (LMM) included (n = in 101; the (bestc)). model Plots (Figure and the 3b). trendline show male strobilus weight, which was greater on the south side. The trendline was obtained from the linear mixed effect model (LMM) included in the best model (Figure3b).

To explore the relationship between male strobilus weight and direction, we generated violin plots based either on all 523 samples, or the 101 samples collected from Wogon-sugi, which were collected from all directions (Figure5b,c). Both datasets suggest that male strobili collected from the south side of trees tended to have approximately 1.5-fold greater male strobilus weight compared to those collected from the north side (1.475-fold for all data [Figure5b], 1.458-fold for the Wogon-sugi subset [Figure5c]). This is likely due to the amount of sunlight that male strobili are exposed to, as the south sides of trees in Japan receive more sunlight than the north sides [39]. It is known that the amount of solar radiation positively affects the male strobilus number [33]. Our data also indicate that the amount of sunlight also positively affects male strobilus weight (pollen number indirectly).

3.5. Factors Affecting Pollen Size Few studies have examined the pollen size variation among Japanese cedar clones. The pSEM predicted that pollen size is signiﬁcantly correlated with male strobilus weight and inﬂuenced by genetic variation (Figure3b). Plot data showed that pollen size varied Plants 2021, 10, 856 11 of 15

from 32 to 36 µm among clones (Figure6a). Interestingly, the mean rank of pollen size was not signiﬁcantly correlated with the mean rank of pollen number (Spearman’s rank correlation = 0.212, p = 0.298; Figure4a) or mean male strobilus weight (Spearman’s rank correlation = 0.305, p = 0.129; Figure5a). This suggests that pollen size follows a pathway that is independent from pollen number. Figure6b shows the relationship between male strobilus weight, pollen size, and clone; samples from different clones are represented by different symbols Table1. We draw two conclusions from this plot; ﬁrst, samples from the same clone were clustered, and second, within the same clone, heavier male strobili tended to be associated with large pollen. These results suggest that pollen size is primarily controlled by genetic variation, but is also affected by male strobilus weight. Our results Plants 2021, 10, x FOR PEER REVIEW suggest that male strobilus weight is also positively related to sunlight,12 of 16 and, as such, that

sunlight indirectly inﬂuences pollen size. Figure 6

)

(

o W

Clone b

35 )

g 30

(

t h

g 25

e w

i b

o 15

l 10

a M 5

30 32 34 36 Pollen size (µm)

FigureFigure 6. Factors 6. Factors affecting affecting pollen size pollen. Distribution size. of Distribution pollen size among of pollen clones size: scatter among plot of clones: pol- scatter plot of pollen len number and and pollen pollen size (a size). The (a relationship). The relationship between pollen between size, male pollen strobilus size, weight, male and strobilus weight, and clone clone (b). Each symbol indicates a clone (see Table 1). (b). Each symbol indicates a clone (see Table1).

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3.6. Direction-Corrected Pollen Number Allows for More Accurate Comparisons of Pollen Number among Clones It is often difﬁcult in ﬁeld studies to sample in a way that is not confounded by variations in environmental conditions. We collected male strobilus samples from branches facing different directions because the open-air side of the tree differed among clones (Table1, Figure1a,c). Ideally, comparisons of pollen numbers among clones should control for the effects of variations in environmental conditions, in our case direction, as we have demonstrated that pollen number is indirectly affected by direction via variations in male strobilus weight (Figures3b and5b). We corrected the pollen number for each clone by constructing LMMs that included direction as a predictor, and extracted the mean pollen number per clone for samples representing the same direction. Raw and corrected pollen numbers are shown in Figure7. In some cases, the corrected pollen numbers differed from Plants 2021, 10, x FOR PEER REVIEW 14 of 16 the raw data (e.g., Minamikanbara-3, Iwafune-15, Iwafune-1, and Iwafune-12). Corrected pollen numbers enable us to make more precise comparisons among clones.

Figure 7. Comparison of corrected pollenpollen numbers.numbers. PollenPollen numbersnumbers per per clone clone based based on on raw raw (orange) (or- andange) predicted and predicted (blue) (blue) data. Predicteddata. Predicted data were data correctedwere corrected for the for effects the effects of sampling of sampling direction direction using theusing LMMs the LMMs included included in the bestin the model best model (Figure (Figure3b). 3b).

4. ConclusionsOur data indicate that the mean pollen number varied approximately 3-fold among clones,We from quantified 100,000 the to number 300,000 pollen of pollen grains grains per per male male strobilus. strobilus Three using studies an efficient have calculatedcounting method pollen, graincomplemented number per by male modeling strobilus: and statistical Ikuse (1965) methods reported. Modeling that single revealed male strobiluswhich factors produces affect 396,000pollen number pollen grainss in Japanese [22]; Saito cedar, and and Takeoka correct (1987)ed pollen found number 237,000s en- to 442,000abled us pollen to make grains accurate from comparisons six clones [24 among]; and clones Kondo. Because et al. (1992) we fo reportedund more from than 182,019 3-fold tovariation 656,289 among pollen clones, grains incounting 10 clones pollen [23]. grain We found numbers that will clones help produced to select fromclones 100,000 with less to 300,000pollen. Modeled pollen grains predictions each; fewer will thanalso thebe useful other reports.for examining There are other several traits possible in the field reasons re- forsearch this,. including genetic variation. We found that pollen number has a large genetic variation among the clones and we did not examine the same clones studied in the other reports.Supplementary In C. japonica Materials:, DNA The markers following indicate are available that there online is at genetic www.mdpi.com/ differentiationxxx/s1 between. Supple- clonesmentary growing Figure S1. mostly Scatterplot on the of Paciﬁcmale strobilus Ocean weight side of and Japan area and. All those samples growing are shown mostly in this on thefigure. Japan Weight Sea and side area [40 are]. We strongly measured correlated clones (r = from0.892). Niigata Different Prefecture, clones are represented on the Japan by Seadif- ferent symbols (see Table 1). Supplementary Table S1. Data for all samples. Author Contributions: Conceptualization, H.K., R.S. and Y.M.; methodology, H.K. and E.T; data collection, H.K. and E.T.; statistical analyses, H.K., E.T. and R.S; writing—original draft preparation, H.K.; writing—review and editing, H.K., E.T., R.S, and Y.M.; visualization, H.K. and R.S.; supervision, Y.M.; project administration, H.K. and Y.M.; funding acquisition, H.K. and Y.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Japan Society for the Promotion of Science KAKENHI Grant Number 19K05976 to H.K., Project of the NARO Bio-oriented Technology Research Advancement Institution (Research program on development of innovative technology) Grant Number 28013BC to Y.M. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request.

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side, while the previous studies measured many clones from the Pacific Ocean side [22–24]. Additionally, because it is known that male strobilus number varies markedly by year, annual differences in the pollen number per male strobilus are possible [33,41]. Counting pollen grains from a larger area over multiple years will provide more comprehensive data on pollen numbers. To counter pollinosis, pollen number per male strobilus can possibly contribute some aspects. For example, we can further select a clone with a low pollen number from the clones that have selected by few numbers of male strobilus. It is important to consider the amount of allergen to assess its effects on pollinosis patients. Cry j 1 is a major Japanese cedar allergen and the amount of Cry j 1 (µg g−1) differs up to 80 times difference among the clones [42]. One interesting approach would be to analyze the correlation between the amount of allergen and pollen number (or pollen size). Introducing the pollen number to a Japanese cedar pollinosis breeding strategy may help counter pollinosis. Pollen grain number varies in several plant species and the locus or gene controlling pollen number was discovered recently. Nguyen et al. investigated rye chromosome addition lines in wheat and found that the 4R addition line had 33% more pollen, which might have applications in breeding hybrid wheat [43]. Recently, the gene controlling pollen numbers was identified in Arabidopsis thaliana [44]; the pollen number varied around 4-fold among 144 Arabidopsis thaliana accessions (approximately 2000–8000 pollen grains per flower). Functional analysis revealed REDUCED POLLEN NUMBER 1 (RDP1) as the gene controlling pollen number, and an evolutional analysis revealed that RDP1 is related to the recent pollen reduction in selfing A. thaliana. Although Japanese cedar has much larger genome (10.8 GB) than A. thaliana, a recent genetic study identified the male sterility gene in Japanese cedar [45,46]. Combination with DNA marker analysis and efficient pollen counting method may contribute to revealing the pollen number controlling locus (or gene) in the future.

4. Conclusions We quantified the number of pollen grains per male strobilus using an efficient counting method, complemented by modeling and statistical methods. Modeling revealed which factors affect pollen numbers in Japanese cedar, and corrected pollen numbers enabled us to make accurate comparisons among clones. Because we found more than 3-fold variation among clones, counting pollen grain numbers will help to select clones with less pollen. Modeled predictions will also be useful for examining other traits in the field research.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/plants10050856/s1. Supplementary Figure S1. Scatterplot of male strobilus weight and area. All samples are shown in this ﬁgure. Weight and area are strongly correlated (r = 0.892). Different clones are represented by different symbols (see Table1). Supplementary Table S1. Data for all samples. Author Contributions: Conceptualization, H.K., R.S. and Y.M.; methodology, H.K. and E.T; data collection, H.K. and E.T.; statistical analyses, H.K., E.T. and R.S; writing—original draft preparation, H.K.; writing—review and editing, H.K., E.T., R.S, and Y.M.; visualization, H.K. and R.S.; supervision, Y.M.; project administration, H.K. and Y.M.; funding acquisition, H.K. and Y.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Japan Society for the Promotion of Science KAKENHI Grant Number 19K05976 to H.K., Project of the NARO Bio-oriented Technology Research Advancement Institution (Research program on development of innovative technology) Grant Number 28013BC to Y.M. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request. Plants 2021, 10, 856 14 of 15

Acknowledgments: We thank Yukiko Ito (Niigata Prefectural Forest Research Institute) for providing breeding materials, and Naoto-Benjamin Hamaya (University of Zurich) for valuable suggestions. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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