ORIGINAL ARTICLE

Genetic diversity and the origin of commercial plantationof Indonesian teak on Java Island

Eko Prasetyo1,2& Widiyatno3

& Sapto Indrioko3& Mohammad Na’iem3

& Tetsuya Matsui4 & Ayumi Matsuo5&

Yoshihisa Suyama5 & Yoshihiko Tsumura6

Received: 29 October 2019 /Revised: 3 February 2020 /Accepted: 26 February 2020 /Published online: 19 March 2020# Springer-Verlag GmbH Germany, part of Springer Nature 2020

AbstractTeak (Tectona grandis) has been widely planted in 70 tropical countries because of the utility and value of its wood. This specieswas introduced to Indonesia more than 100 years ago, and large plantations—covering 1.2 million ha—can be found on JavaIsland. However, little information currently exists about the genetic diversity and origin of these trees. We collected plantmaterials from three regions across Java Island (east, central, and west) and sampled trees spanning three age classes in eachregion, to clarify the genetic diversity and structure of teak plantations on Java Island. We investigated teak plantation and clonalexperiment populations using multiplexed ISSR genotyping by sequencing (MIG-seq) and compared the genetic diversity andstructure with the provenance test populations derived from natural teak forest in India, Myanmar, Thailand, and Laos. Analysesusing 459 single-nucleotide polymorphism (SNP) loci revealed that native provenances had higher genetic diversity than theIndonesian teak plantations. Moreover, old teak plantations demonstrated lower genetic diversity than young plantations. Furtheranalyses showed that most Indonesian teak plantations are genetically related to Laos, Thailand, and Myanmar provenances. Weconclude that there is a weak genetic structure on teak plantations among the regions, which indicates that most plantations wereestablished using plant materials from a specific part of the natural teak distribution. Information regarding the genetic diversityand structure of plantation forests should be taken into account when making future plantation programs.

Keywords Tectona grandis . SNP . Seed origin . Genetic diversity . Genetic constitution

Introduction

Teak (Tectona grandis Linn. f.) (Lamiaceace) has a discontin-uous natural distribution across India, Myanmar, Thailand,and Laos (Kaosa-ard 1989; Behaghel 1999; Verhaegen et al.2010). This species of tree is confined to the moist and dry

mixed tropical deciduous forests of continental Asia (White1991). Teak is a firmly light-demanding species (Kaosa-ard1989; Webb et al. 1984), with relative humidity and annualrainfall the most important climatic factors for growth (Pandeyand Brown 2000). Teak usually grows in areas with between800 and 2500 mm of annual precipitation (Palanisamy et al.

Communicated by F. Isik

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s11295-020-1427-5) contains supplementarymaterial, which is available to authorized users.

* Yoshihiko Tsumuratsumura.yoshihiko.ke@u.tsukuba.ac.jp

1 Graduate School of Life and Environmental Sciences, University ofTsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan

2 Vocational College, Universitas Gadjah Mada, Sekip Unit II,Yogyakarta 55281, Indonesia

3 Faculty of Forestry, Universitas Gadjah Mada, Bulaksumur,Yogyakarta 55281, Indonesia

4 Center for International Partnerships and Research on ClimateChange, Forestry and Forest Products Research Institute, ForestResearch and Management Organization, 1 Matsunosato, Tsukuba,Ibaraki 305-8687, Japan

5 Kawatabi Field Science Center, Graduate School of AgriculturalScience, Tohoku University, 232-3 Yomogida, Naruko-onsen, Osaki,Miyagi 989-67711, Japan

6 Faculty of Life and Environmental Sciences, University of Tsukuba,1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan

Tree Genetics & Genomes (2020) 16: 34https://doi.org/10.1007/s11295-020-1427-5

2009) and a periodic dry period, i.e., 3–6months during whichthere is less than 40 mm rainfall per month (Webb et al. 1984).It has been suggested that a mean annual temperature between22 and 26 °C is suitable for the cultivation of teak (Webb et al.1984). In both mainland Asia and Java, especially the centraland eastern parts, teak is found in the monsoon forests(Kertadikara and Prat 1995). Tanaka et al. (1998) reported thatlimestone areas are usually suitable for teak growth because ofhigh pH, high base content, and adequate soil drainage; theyalso noted that the climate of eastern Java, Indonesia, is similarto that of areas in which teak naturally grows. The averageannual precipitation in Java is 2,235 mm (Yasunari 1981), andthe island has distinct wet and dry seasons (Tanaka et al.1998). Furthermore, Indonesian teak plantations have shownhigher mean annual increment values than plantations in othercountries (Pandey and Brown 2000). Now, teak plantationcovers 1.2 million hectares in Java (Bailey and Harjanto2005) and became the second largest teak plantation in theworld (Midgley et al. 2015).

The origin of teak planted in Indonesia has been reported inseveral previous studies. The first report, published by Altona(1922), stated that teak had been imported to Indonesia fromSouth India between the fourteenth and sixteenth century.When the first European colonizers arrived in the sixteenthcentury, they found large areas of teak forests in central andeastern Java (Smiet 1990). Therefore, it has been postulatedthat teak was imported by Indonesians who traveled to Indiaduring the Hindunization period (between the fourteenth andsixteenth centuries). Teak was introduced to Java for botheconomic and religious reasons (Behaghel 1999), with teakmost probably first planted around temples, after which localrulers asked farmers to plant teak in areas that were unsuitablefor rice fields. Teak later became readily accepted by the localpopulation due to the income associated with teakwood sup-ply (Simatupang 2001). However, genetic data suggest thatteak was most probably introduced to Indonesia from a smallarea with a narrow genetic base in Central Laos or easternThailand (Verhaegen et al. 2010). A more recent genetic anal-ysis by Hansen et al. (2017) also indicated that Indonesianlandraces mainly originate from a specific area of the nativedistribution.

Teak has been widely planted in 70 tropical countries be-cause its wood is very useful and valuable (Kollert andWalotek 2015). In Java, Indonesia, teak is primarily commer-cially cultivated by Perum Perhutani, the State ForestCompany. Most of these plantations are located in centraland eastern Java because these regions have the most favor-able climatic and soil conditions for teak forests (Tanaka et al.1998). Teak plantations usually implement a rotation periodbetween 40 and 80 years, but Perhutani recently selected twoof the best clones to set up plantations with a 20-year rotationperiod (Sadono 2017). The selected teak clones can adapt tovarious environments on Java island and demonstrate superior

growth characteristics (Budiadi et al. 2017). However, theselection of a low number of clones presents several risks,namely, plantation failure, loss of genetic diversity, and sus-ceptibility to disease from genetic gain (Wu 2018). The genet-ic diversity of planting materials become much more impor-tant for avoiding the risks to some unforeseen future bioticthreat such as increasing diseases or harmful insects.

The genetic diversity of teak has been investigatedusing various molecular markers, such as isozyme(Kertadikara and Prat 1995), RAPD (Watanabe andWidyatmoko 2004), AFLP (Vaishnaw et al. 2014), micro-satellite markers (Fofana et al. 2009; Huang et al. 2015;Hansen et al. 2015), and SNP markers (Dunker et al.2019). Natural populations of teak show strong geneticstructure, with genetic diversity decreasing from west toeast along the geographic distribution, i.e., genetic diver-sity is highest in south India and lowest in the Thai andLaos populations (Hansen et al. 2015). Verhaegen et al.(2010) investigated 15 microsatellite markers and re-vealed that three teak provenances from Java (Pati,Blora, and Ngliron) are closely linked to areas inCentral Laos or eastern Thailand. Hansen et al. (2017)reported that two Java provenances (Pati and Ngliron)originated from Thailand and another Java provenance(Temandsang) originated from North India. Based on theresults of these studies, Indonesian teak plantations wereprobably established using trees imported from the Thaiand Laos populations. However, these studies only inves-tigated a total of five teak populations from central andeastern Java. The genetic origin and diversity of teakthroughout Java Island has still not been clearly studied.

The use of single-nucleotide polymorphisms (SNPs) as ge-netic markers has become increasingly popular not only inplant breeding (Mammadov et al. 2012) but also in the mon-itoring of tree genetic resource (Aravanopoulos 2016). Theprevious study have discovered 156 SNP marker on teakand successfully amplified in 172 individuals from 20 popu-lations in Myanmar (Dunker et al. 2019). Suyama andMatsuki (2015) recently presented a new approach for re-search focused on SNP discovery and comparison. They usedmultiplexed inter-simple sequence repeat (ISSR) method thatamplifies thousands of genome-wide regions, after which theyapplied MIG-seq to sequence the genetic markers. UsingMIG-seq to compare genetic variation among populations willrequire the researcher to analyze a far greater number of SNPsthan SSR markers (Morin et al. 2004). Using SNP loci, thepresent study aimed to (1) identify the origin of teak popula-tions planted on Java Island and (2) clarify the genetic consti-tution of plant materials across three regions (western, central,and eastern Java) as well as across age classes. This study willimprove knowledge about the current genetic status of teakplantations across Java. The result may also elucidate theplanting history of teak on Java island.

34 Page 2 of 14 Tree Genetics & Genomes (2020) 16: 34

Material and methods

Plant material

We widely collected plant materials from Java Island and di-vided Java Island into three regions (western, central, and east-ern) according to annual precipitation (Yasunari 1981). In eachregion, we also investigated three age classes of teak planta-tions, more specifically, more than 60 years old (old), 30–60 years old (middle), and less than 30 years old (young). Ineach region, we collected samples between 1 and 4 populationsrepresenting each age class. Thus, samples were collected froma total of 27 teak plantations throughout Java island (Fig. 1;Table 1). In addition, we collected samples from a clonal ex-periment forest in Yogyakarta, Central Java (Table1). At eachsite, leaf samples were collected and stored in a plastic bag withsilica gel to preserve the material until DNA analysis.

We also collected materials from a provenance test forestwhich represents the original habitat of teak. This specificprovenance trial was conducted in 1932 using seed materialsfrom India, Laos, Myanmar, Thailand, and nine Indonesianlandraces, with forests established in two locations:Nglambangan in Bojonegoro and Kesamben in Blitar. Wecollected leaf samples from the Nglambangan of Bojonegoro(S 7° 19′, E 111° 45′) sites using the same procedure describedabove (Table 2).

DNA extraction

Between 27 and 51 individual trees were sampled in eachIndonesian teak plantation, while 11 individual trees fromthe clonal experiment forest were sampled and between 7

and 21 individual trees from each provenance trial forest weresampled. DNA was extracted from a total of 1,172 samples(see Fig. S1) using a modified CTAB (cetyltrimethylam-monium bromide) method (Tsumura et al. 1996). Each 200-mg DNA sample was frozen by liquid nitrogen and groundusing a TAITEC Beads Crusher machine (TAITEC,Nishikata, Japan). The ground powder was mixed with900 μL CTAB buffer (50 mM Tris-HCl, pH 8.0, 0.5 MEDTA, 0.2% ß-mercaptoethanol, 10% PVP-40, 1% bovineserum albumin(BSA)), after which the solution was incubatedat 60 °C for 1 h and then centrifuged for 10 min at 15,000 rpmand 4 °C. A 600 μL portion of the supernatant was then trans-ferred to a micro-centrifuge tube, after which the supernatantwas mixed with isopropanol (2/3 of the recovered volume)and shaken for 5 min at 15,000 rpm and 4 °C. The DNAprecipitated from the aqueous phase and was then washedwith 70% ethanol. The DNA was stored in TE buffer (Tris-HCl 10 mmol/L, pH 8.0, EDTA 1 mmol/L, pH 8.0). DNAquantity was measured using a nanodrop spectrophotometer(Thermo Scientific, Waltham, MA, USA), after which DNAquality was checked using 2% agarose gel electrophoresis.GelRed™ Nucleic Acid Gel Stain (Biotium, Fremont, CA,USA) was added to the gel prior to the pouring step. The gelswere viewed under UV light and were captured using theATTO Printgraph gel documentation system (ATTOCorporation, Tokyo, Japan).

MIG-seq analysis

The DNA samples were then genotyped using MIG-seq anal-ysis based on the protocol of Suyama and Matsuki (2015).According to the protocol, there are two primary steps of

Fig. 1 Locations of the studied teak plantations on Java Island

Tree Genetics & Genomes (2020) 16: 34 Page 3 of 14 34

MIG-seq analysis: construction of a MIG-seq library with twopolymerase chain reactions (PCR) and sequencing. The firstPCR was performed to amplify ISSR regions from genomicDNA using the MIG-seq primer set. The amplificationemployed a specific forward primer – (tail + anchor: CTG)+ SSR + anchor – and a specific reverse primer – (tail +anchor: GAC) + SSR + anchor. This PCR run was conductedin a final volume of 6 μl, containing 1 μl of template DNA,0.96 μl of 1st PCR primers (primer mix with each primer at aconcentration of 20 μM), 3 μl of 2× Multiplex PCR Buffer,0.03 μl of Multiplex PCR Enzyme Mix (Multiplex PCRAssay Kit Ver.2, Takara Bio, Kusatsu, Japan), and 1.01 μl ofwater. The first PCR run employed the following cycling con-ditions: initial activation at 94 °C for 1 min, followed by25 cycles of denaturation at 94 °C for 30 s, annealing at38 °C for 1 min, and extension at 72 °C for 1 min, and endingwith a final incubation at 72 °C for 10 min. The first PCRproducts were diluted 10 times using deionized water. These

products then underwent purification, size selection (to re-move < 250 bp fragments), and normalization with AMPureXP (Beckman Coulter, Brea, CA, USA).

The objective of the second PCR was to add comple-mentary sequences that serve as an index for subsequentanalyses to the primary PCR products. The second PCRrun was performed in a final volume of 6 μl, containing1.6 μl of 1st PCR products, 1.2 μl of 5× PrimeSTARGXL Buffer, 0.48 μl of dNTP mixture, 1.2 μl of the2nd PCR forward primer, 1.2 μl of the second reverseprimer, 0.12 μl of PrimeSTAR GXL polymerase, and0.2 μl of water. . The PCR cycling conditions were 12 cy-cles of denaturation at 98 °C for 10s and annealing at54 °C for 15 s, followed by extension at 68 °C for1 min. Fragments in the 400–800 bp range were isolatedusing AMPure XP (Beckman Coulter) and were quanti-fied using real-time PCR. The DNA library was se-quenced twice using the Illumina MiSeq Sequencer

Table 1 Information about the 28investigated teak populations Region Age Population

abbreviationSamplinglocation

Latitude Longitude Samplesize

Eastern Java 63 EO1 Bondowoso S 7° 47.435′ E 113° 56.951′ 35

87 EO2 Situbondo S 7° 41.309′ E 113° 50.043′ 31

82 EO3 Situbondo S 7° 42.107’ E 113° 49.097′ 33

34 EM1 Bondowoso S 7° 43.493′ E 113° 48.918′ 36

32 EM2 Bondowoso S 7° 53.551′ E 113° 50.502′ 32

52 EM3 Situbondo S 7° 47.238′ E 113° 44.300′ 32

38 EM4 Situbondo S 7° 43.395′ E 114° 07.861′ 33

14 EY1 Bondowoso S 7° 47.092′ E 113° 56.695′ 35

16 EY2 Bondowoso S 7° 53.540′ E 113° 50.463′ 31

23 EY3 Situbondo S 7° 42.155′ E 113° 54.760′ 33

Central Java 69 CO1 Madiun S 7° 29.551′ E 111° 47.243′ 30

88 CO2 Ngawi S 7° 20.240′ E 111° 20.138′ 30

74 CO3 Ngawi S 7° 18.511′ E 111° 21.340′ 29

35 CM1 Madiun S 7° 28.975′ E 111° 47.650′ 30

50 CM2 Ngawi S 7° 20.307′ E 111° 20.097′ 30

58 CM3 Ngawi S 7° 18.574′ E 111° 21.342′ 29

23 CY1 Madiun S 7° 28.971′ E 111° 47.581′ 30

22 CY2 Ngawi S 7° 20.226′ E 111° 20.138′ 30

23 CY3 Ngawi S 7° 20.010′ E 111° 23.552′ 30

9 CY4 Yogyakarta S 7° 54.008′ E 110° 31.001′ 11

Western Java 71 WO1 Purwakarta S 6° 28.402′ E 107° 28.803′ 50

34 WM1 Banten S 6° 38.346′ E 105° 48.001′ 34

35 WM2 Purwakarta S 6° 28.114′ E 107° 28.908′ 51

33 WM3 Cianjur S 7° 29.164′ E 107° 17.843′ 27

31 WM4 Cianjur S 7° 24.465′ E 107° 11.547′ 29

22 WY1 Banten S 6° 38.279′ E 105° 48.122′ 42

15 WY2 Purwakarta S 6° 27.865′ E 107° 28.951′ 46

28 WY3 Cianjur S 7° 29.409′ E 107° 18.164′ 30

E Eastern Java, C Central Java, W Western Java, O old plantation, M middle plantation, Y young plantation

34 Page 4 of 14 Tree Genetics & Genomes (2020) 16: 34

platform and MiSeq Reagent Kit v3 (150 cycles)(Illumina, San Diego, CA, USA).

The sequencing results were filtered using TagDust andFASTX_toolkit to remove adapter and low-quality se-quences. The filtered reads were then input into Stacksv.1.47, which is a software that identifies loci and wasused to detect SNPs (Catchen et al. 2011). Briefly, basedon Catchen et al. (2013), three modul used for buildingloci (“ustacks”), creating the catalog of loci for all sample(“cstacks”) and mapping against the catalog (“sstacks”).We set a minimum minor allele frequency more than twoindividuals and excluded excess of heterozygosity set to60%. Last, “population” module was used to analyze apopulation of individual samples computing a number ofpopulation genetic statistic. The minimum number of apopulation (p) and percentage of individuals within a pop-ulation (r) in which a locus must be present were set as 1and 0.8, respectively. Thus, we retained only those locithat were present in > 80% of individuals.

Genetic analysis

The genetic diversity of each teak plantation was determinedbased on certain variables. For example, the number of alleles(Na), number of effective alleles (Ne), observed heterozygos-ity (Ho), and expected heterozygosity (He) were estimated

using GenAlex 6.5 (Peakall and Smouse 2012). Inbreedingcoefficient (FIS) indices and the significance of FIS were cal-culated using the software FSTAT version 2.9.4 by applyingbootstrapping over loci with a 99% nominal confidence inter-val (Goudet 2003). Allelic richness via refraction (Na(rar))and private allele were calculated in HP-Rare 1.1(Kalinowski 2005).

Intra- and inter-population genetic variation were estimatedby an analysis of molecular variance (AMOVA) usingArlequin 3.5.2.2 (Excoffier and Lischer 2010). For compari-son, AMOVAwas conducted for native provenances and plan-tation populations separately. Pairwise FST values betweenpopulations were calculated by Arlequin. These FST valueswere then imported into MEGA 7 software (Kumar et al.2016) to construct a dendrogram using the Neighbor-Joining(NJ) method (Saitou and Nei 1987). We also conducted PCoA(principal coordinate analysis) in GenAlex to better under-stand the genetic relationships between populations. In addi-tion, we also used Bayesian cluster analyses usingSTRUCTURE software (version 2.3; Pritchard et al. 2000)to clarify the genetic structure of teak plantations in Java.We ran ten replications with each K from 1 to 10 and withburning length 100,000 each including 100,000 MarkovChain Monte Carlo (MCMC) iteration. To determine the mostlikely number of K, we determined by both plotting the logprobability (L(K)) and ΔK the method of Evanno et al. (2005)

Table 2 Information about theeight investigated nativeprovenances in India, Myanmar,Thailand, and Laos provenancesand the nine Indonesian landraces

Country Populationabbreviation

Provenance,landrace

Average annualrainfall (mm)

Soil and topographyat site

Samplesize

India IND1 Malabar 2729 30 m a.s.l, looselaterite, valley

13

IND2 Godavari 1022 400 m a.s.l,sandy loam

12

IND3 Central Prov 1650 500 m a.s.l,sandy loam

14

Myanmar BRM Insein, Burma 2292 Hill 23

Thailand THA1 Phrae 1044 Sandy soil 17

Laos LAO1 Kay Loam hill 19

LAO2 Kouai Sandy loam, plain 13

LAO3 Hinh Sandy loam, plain 13

Indonesia INA1 Ponorogo 1534 Dark clay soil 19

INA2 Margasari 2818 Loamy sandy 12

INA3 Bangilan 1712 Red, old lateric soil 20

INA4 Gundih 2199 Margalitic soil 10

INA5 Pati 1754 Volcanic sandy soil 21

INA6 Muna 1718 Brown loam 18

INA7 Cepu 1964 Dark brown loam 7

INA8 Ngliron 2500 Humous margaliticsoil

10

INA9 Bojonegoro 12

IND India, BRM Burma/Myanmar, THA1 Thailand, LAO Laos, INA Indonesia

From: Suhaendi 1998

Tree Genetics & Genomes (2020) 16: 34 Page 5 of 14 34

using STRUCTURE HARVESTER software (Earl andVonHoldt 2012). Then, the outputs from STRUCTURE andSTRUCTURE HARVESTER were aligned using CLUMPP(Jakobsson and Rosenberg 2007) and illustrated usingDISTRUCT (Rosenberg 2004) . Three rounds ofSTRUCTURE analyses were run. The first analysis was usedto observe the genetic structure of all populations. Then, weexcluded certain populations that showed distinct geneticstructure (more than 40% mixed). The second analysis wasconducted to explore the origin of plantation materials in Javain more detail. The third analysis only included commercialplantation populations so that the genetic structure amongdifferent age groups and regions could be studied. Thus, thethree separate genetic structure analyses included all 45 pop-ulations, 41 populations (excluding three Indian provenancepopulations and CY4), and all 27 plantations (excluding CY4,a breeding program population), respectively.

Results

Genetic diversity

A total of 99,692,395 reads were obtained from 1172 samplesusing MIG-seq analysis. After filtering, trimming, removingshort-reads, and SNP calling, we obtained 459 SNP loci. Theaverage Na and Ne values for all 45 populations were 1.340and 1.113, respectively. Interestingly, only five populationsshowed private alleles. These were not only identified fromprovenance forests such as IND1, IND2, and IND3, but alsothe clonal experiment CY4 and plantation WY2; these loca-tions were characterized by private allelic richness of 0.07,0.01, 0.06, 0.02, and 0.01, respectively (Table 3). The planta-tion populations EM3, EO2, and WO1 showed the lowestallelic richness Ar(r), with a value of 1.05, while the prove-nance populations, specifically IND1 (Ar(r) = 1.30), showedthe highest allelic richness (Table 3). The expected heterozy-gosity ranged from 0.050 to 0.182, while the observed hetero-zygosity ranged from 0.043 to 0.228. The clonal experimentCY4 demonstrated the highest Ho and He values. AmongIndonesian landrace populations, the genetic diversity showedsimilar values as those of plantation populations. A significantFIS value was observed in five of the native provenance pop-ulations (BRM, IND1, IND2, IND3, and LAO1) and three ofthe plantation populations (EY3, WY2 and EO1). The Hovalue was always lower than the He in all of the populationsexcept CY4.

The native provenance populations showed higher geneticdiversity than the plantation populations in the following fourparameters: Na, Ne, Ho, and He. All of the old plantationpopulations (> 60 years old) showed lower genetic diversity(He = 0.053–0.066, Table 4) than the young plantation popu-lations (< 30 years old, He = 0.067–0.100). In all of the

regions, young teak plantations (EY, CY, and WY) showedslightly higher expected heterozygosity (He) than old teakplantations (EO, CO, and WO). The expected heterozygosity(He) for WY was almost double than what was calculated forWO.

Genetic structure

The AMOVA results concerning native provenance popula-tions revealed that most of the variation (44%) resulted fromamong population genetic variation, which was followed bywithin individual (31%) and within population (25%) varia-tion (Table 5A). The FST value among native provenancepopulations was very large, 0.446. Population pairwise FST

values ranged from 0.004 to 0.736 and were statistically sig-nificant (P < 0.05) for all populations; an exception was thatLAO1-BRM, LAO2-BRM and LAO2-LAO3 did not showsubstantial genetic differentiation (Table S1). The PCoA resultshowed that the Indian populations were clearly differentiatedfrom the other native provenance populations (Fig. 2II).

The AMOVA results for commercial plantation popula-tions showed that genetic variance between individualsaccounted for 76.20% of total variance, while among popula-tion genetic variance only contributed to 7.22% of the totalvariance (Table 5B). The FST value among plantation popula-tions was 0.072, while population pairwise FST values rangedfrom 0.000 to 0.237, which represented a narrower range—and a far lower maximum value—than what was measured fornative provenance forest. The highest pairwise FST value wasfound between CO1 and WO1 (Table S2). The PCoA resultshowed that the Central Java population was slightly differen-tiated from the western and eastern populations (Fig. 2III). Weconducted a further AMOVA on samples from the plantationsto detect genetic differentiation between age classes. TheAMOVA results showed that there was no genetic variance(−1.26%) among age classes of teak plantations, but that dif-ferences among the entire population and among populationswithin age classes accounted for 19.18% and 5.87% of thevariation, respectively, while within individual difference ex-plained most of the observed variation (Table S2). TheAMOVA results highlight that regional differences contribut-ed to 3.47% of the genetic variation; furthermore, regionaldifferences were significant for all of the individual trees(P < 0.001).

The PCoA results for all populations showed that the stud-ied teak populations can be divided into two distinct groups,namely, Indian populations (IND1, IND2, and IND3, alongwith CY4) and the other populations (Fig. 2I). The NJ treeagreed with the PCoA results, more specifically, the teak plan-tations in Java were relatively closely linked to the nativeprovenance populations LAO1, LAO2, LAO3, THA1, andBRM (Fig. S2).

34 Page 6 of 14 Tree Genetics & Genomes (2020) 16: 34

Table 3 Various genetic diversityvalues for the teak in Java,calculating using data for 459SNPs

Population Population type Na Ne Ar(r) Ho He FIS Private allele

EO1 Plantation 1.301 1.085 1.06 0.043 0.056 0.236* 0.00

EO2 Plantation 1.266 1.083 1.05 0.046 0.054 0.160 0.00

EO3 Plantation 1.283 1.088 1.06 0.048 0.057 0.188 0.00

EM1 Plantation 1.312 1.090 1.06 0.050 0.059 0.171 0.00

EM2 Plantation 1.394 1.090 1.06 0.050 0.062 0.215 0.00

EM3 Plantation 1.220 1.080 1.05 0.046 0.050 0.105 0.00

EM4 Plantation 1.299 1.088 1.06 0.049 0.058 0.176 0.00

EY1 Plantation 1.305 1.085 1.06 0.047 0.057 0.191 0.00

EY2 Plantation 1.434 1.100 1.07 0.055 0.070 0.228 0.00

EY3 Plantation 1.719 1.153 1.11 0.078 0.110 0.313* 0.00

CO1 Plantation 1.237 1.098 1.06 0.055 0.057 0.049 0.00

CO2 Plantation 1.318 1.098 1.07 0.057 0.064 0.136 0.00

CO3 Plantation 1.314 1.100 1.07 0.061 0.066 0.094 0.00

CM1 Plantation 1.309 1.087 1.06 0.050 0.059 0.160 0.00

CM2 Plantation 1.298 1.095 1.06 0.061 0.062 0.040 0.00

CM3 Plantation 1.331 1.102 1.07 0.064 0.068 0.079 0.00

CY1 Plantation 1.285 1.095 1.06 0.056 0.062 0.118 0.00

CY2 Plantation 1.325 1.098 1.07 0.058 0.065 0.124 0.00

CY3 Plantation 1.314 1.097 1.07 0.056 0.065 0.168 0.00

CY4 Clonal experiment 1.508 1.306 1.19 0.228 0.177 −0.212 0.02

WO1 Plantation 1.277 1.083 1.07 0.047 0.053 0.119 0.00

WM1 Plantation 1.346 1.099 1.07 0.058 0.065 0.129 0.00

WM2 Plantation 1.660 1.118 1.08 0.066 0.084 0.222 0.00

WM3 Plantation 1.325 1.102 1.07 0.062 0.067 0.098 0.00

WM4 Plantation 1.359 1.105 1.05 0.065 0.069 0.078 0.00

WY1 Plantation 1.373 1.097 1.15 0.058 0.065 0.131 0.00

WY2 Plantation 1.773 1.207 1.07 0.087 0.145 0.410* 0.01

WY3 Plantation 1.342 1.098 1.07 0.059 0.065 0.112 0.00

IND1 Native provenance 1.253 1.121 1.30 0.070 0.108 0.400* 0.07

IND2 Native provenance 1.582 1.300 1.19 0.065 0.182 0.675* 0.01

IND3 Native provenance 1.466 1.276 1.18 0.045 0.165 0.754* 0.06

BRM Native provenance 1.641 1.148 1.11 0.061 0.106 0.448* 0.00

THA1 Native provenance 1.227 1.086 1.06 0.048 0.053 0.146 0.00

LAO1 Native provenance 1.285 1.097 1.06 0.049 0.062 0.231* 0.00

LAO2 Native provenance 1.244 1.100 1.07 0.061 0.063 0.078 0.00

LAO3 Native provenance 1.235 1.086 1.06 0.059 0.059 0.051 0.00

INA1 Indonesian landrace 1.279 1.093 1.06 0.057 0.061 0.089 0.00

INA2 Indonesian landrace 1.227 1.091 1.06 0.049 0.058 0.199 0.00

INA3 Indonesian landrace 1.266 1.089 1.06 0.054 0.058 0.102 0.00

INA4 Indonesian landrace 1.251 1.104 1.07 0.061 0.065 0.120 0.00

INA5 Indonesian landrace 1.257 1.094 1.06 0.057 0.060 0.072 0.00

INA6 Indonesian landrace 1.246 1.096 1.06 0.056 0.061 0.108 0.00

INA7 Indonesian landrace 1.185 1.082 1.06 0.050 0.052 0.127 0.00

INA8 Indonesian landrace 1.205 1.094 1.06 0.061 0.058 0.015 0.00

INA9 Indonesian landrace 1.251 1.110 1.07 0.070 0.068 0.029 0.00

Na number of observed alleles, Ne effective number of alleles, Ar(r) allelic richness and private allele calculatedvia rarefraction (two genes), Ho observed heterozygosity, He expected heterozygosity, FIS inbreeding coefficient

*Significance (> confidence Interval 99%)

Tree Genetics & Genomes (2020) 16: 34 Page 7 of 14 34

Based on the STRUCTURE analysis, which applied thedelta K method, the most likely number of clusters was two(K = 2) for all populations, all populations excluding the threeIndian populations and CY4, and all plantations. The logprobability (L(K)) was highest atK = 9, but K = 2 also showeda high value and did not differ much from with K = 9 for allpopulations (Fig. S3.I). The STRUCTURE analysis of allplantations revealed that the most likely number of clusterswas two (K = 2), followed by four (K = 4). Analyses of allpopulations excluding the three Indian populations and CY4and all plantations revealed that there was high likelihood thatthe sites were explained by 10 clusters (K = 10) (Figs. S3.IIand S3.III). In this case, we followed suggestions fromEvanno et al. (2005) that the real number of clusters shouldbe determined based on the value of ΔK. This approach

revealed that the three Indian populations and clonal experi-ment population CY4 belong to one distinct genetic cluster(Fig. 3I). Once these populations were separated, it was ob-served that certain young populations (EY2, EY3,WY2, andWM2) and BRM clustered together. A STRUCTURE analy-sis of the plantation populations in Java showed that the stud-ied teak plantations can be divided into three group roughlydescribed as Eastern, Central, and Western Java (Fig. 3IIIc).

Discussion

MIG-seq analysis can identify SNP markers

The recent application of NGS to SSR genotyping has provid-ed further savings in terms of cost, labor, and time when com-pared to previous methods. However, the analysis of SSRsstill requires several steps such as PCR amplification, electro-phoresis, and genotyping (Davey et al. 2011). The genome-wide SNP markers recently identified for teak can be used toanalyze the genetic diversity among teak plantations and clar-ify the genetic origin of certain landraces (Dunker et al. 2019).The present study demonstrates that SNP genotyping viaMIG-seq can be used to investigate the population structureof commercial teak plantations with significant time and laborsavings. One of the main advantages of MIG-seq is that it canprovide putative neutral loci (Richards et al. 2018), which areuseful for analyses of genetic structure (Gutierrez-Ortega et al.2018). Moreover, MIG-seq analysis is quick, easy to execute,relatively inexpensive, and can be performed with smallamounts of low-quality DNA (Takahashi 2017).

It has been shown that MIG-seq will detect less SNPs thanRAD-seq (Wachi et al. 2018); however, the 459 SNPs identi-fied in this work were sufficient to examine genetic diversityamong teak plantations in Java as well as differentiate theseplantations from other teak populations around the world. Thefirst published study that applied MIG-seq identified between31 and 324 SNP loci from mushrooms, sea cucumbers, sea

Table 5 AMOVA results for thestudied teak populations (bothnative provenance and plantationpopulations) based on data from459 SNPs

Source of variation df Sum of squares Variance components %

(A) Native provenance populations

Among populations 7 3346.878 16.63567 44.56

Within populations 116 2998.196 9.36056 24.78

Within individuals 124 1252.500 11.58472 30.66

Total 247 7597.574 37.78094

(B) Plantation populations

Among populations 26 2388.678 1.23694 7.22

Within populations 881 14,472.029 2.83848 16.58

Within individuals 908 10,493.500 13.04712 76.20

Total 1815 27,354.208 17.12254

Table 4 Average genetic diversity values for each region of teakplantations in Java

Na Ne Ho He

Population

EO 1.359 1.088 0.046 0.058

EM 1.473 1.090 0.048 0.060

EY 1.784 1.112 0.060 0.081

CO 1.370 1.099 0.057 0.066

CM 1.397 1.094 0.058 0.065

CY 1.373 1.098 0.056 0.067

WO 1.277 1.083 0.047 0.053

WM 1.723 1.109 0.063 0.075

WY 1.817 1.137 0.069 0.100

Total 1.508 1.101 0.056 0.069

Plantation 1.900 1.106 0.057 0.074

Indonesian landrace 1.425 1.099 0.057 0.067

Native provenance 1.941 1.233 0.058 0.166

Total 1.755 1.146 0.057 0.102

Na number of observed alleles, Ne effective number of alleles, Ho ob-served heterozygosity, He expected heterozygosity

34 Page 8 of 14 Tree Genetics & Genomes (2020) 16: 34

snails, and orchids (Suyama and Matsuki 2015). Followingthis initial report, MIG-seq was successfully used to detectSNPs from two species of damselfly (270 and 231 SNP loci;Takahashi et al. 2016) and a cycad species (361 SNP loci;Gutierrez-Ortega et al. 2018) in studies of genetic structure.This method has also been successfully leveraged to gain SNPdata for blue coral (Richards et al. 2018), Rhododendronindicum (Yoichi et al. 2018), Saxifraga yuparensis (Tamuraet al. 2018), and Quercus (Binh et al. 2018), providing 166,168, 699, and 19,916 SNP loci, respectively. Binh et al. (2018)used a very low threshold of haplotype frequency in a popu-lation—(r) = 0.001 (in this study, r = 0.8, see “Material andmethods”)—to obtain a vast amount of SNPs. Even the smallnumber of SNP loci in the blue coral study provided a clearerillustration of genetic structure than what was provided bycalculations using microsatellite data (Richards et al. 2018).The 156 SNP loci discovered for teak species by the DoubleDigest Restricted Associated DNA Sequencing method

(ddRAD) is useful for population genetic studies (Dunkeret al. 2019). This provides some context for the present study,as the application of MIG-seq was able to identify a largenumber of SNPs (more specifically, 459 loci) and provide datafor reliable analyses of genetic structure and genetic diversity.

Comparison of genetic diversity between plantationsand native provenances

The results of this study suggest that the Indian population hashigher diversity than other native provenance populations (seeTable 3). This result agrees with what has been reported inprevious studies that relied on microsatellite marker analyses,i.e., Indian populations show the highest heterozygosity of allthe natural teak populations, followed byMyanmar, Laos, andThailand provenances (Huang et al. 2015; Hansen et al. 2015).

Teak native provenances also demonstrated higher hetero-zygosity than the studied plantations in Java (see Table 4).

BRMCY4

IND1

IND2

IND3

LAO1

LAO2LAO3

THA1

PCoA

2 -

10.7

%PCoA 1 - 37.0% variation explained

Native provenance Indonesian plantation & landraceClonal experiment

IND1

IND2

IND3

BRMLAO1

LAO2

LAO3THA1

PCoA

2 -

18.9

%

PCoA 1 - 72.2% variation explained

CM1CM2CM3

CO1CO2

CO3

CY1

CY2CY3

EM1EM2

EM3EM4

EO1EO2EO3EY1

EY2EY3

WM1

WM2

WM3WM4

WO1

WY1

WY2

WY3PCoA

2 -

18.4

%

PCoA 1 - 31.2% variation explained

West JavaCentral JavaEast Java

II

III

I

Fig. 2 PCoA based on the FSTvalue for all populations (I),without India and CY4 (II), andteak plantations without CY4(III). The population names areclarified in Tables 1 and 2

Tree Genetics & Genomes (2020) 16: 34 Page 9 of 14 34

Similar results have been reported for Pinus brutia (Al-Hawijaet al. 2014) and Inga vera subsp. affinis (Neto et al. 2014).However, even artificial populations of the Japanese conifer-ous species Cryptomeria japonica do not show significantdecreases in genetic diversity when compared to natural pop-ulations (Uchiyama et al. 2014). They compared large num-bers of individuals from both the artificial populations of plustrees (i.e., trees showing superior phenotype) and natural pop-ulations; thus, it can be inferred that trees from the artificialpopulation havemaintained comparatively high genetic diver-sity in relation to the natural population. However, in anotherstudy of C. japonica by Iwasaki et al. (2019), the level ofgenetic diversity varied among plantations due to differentseed sources. A previous study that used 15 microsatellitemarkers to study the population structure of teak inMyanmar found that natural and plantation teak populationshave similar levels of genetic diversity (Thwe-Thwe-Winet al. 2015). It is important to state that the situation in Javais most probably different from that in Myanmar, a countrythat includes a wide natural distribution of teak. The resultspresented in this paper agree with the research byHansen et al.2017, in which it is stated that the teak in Java was most likelyimported from a specific area and introduced through a limitednumber of seeds.

Five native provenance populations (BRM, IND1, IND2,IND3, and LAO1) demonstrated significant inbreeding coef-ficient (FIS) values. This result was most probably the result ofestablishing provenance trials with a limited number of

mother trees. The inbreeding coefficient can be expected toincrease further if the limited number of mother trees originatefrom a limited area of the natural distribution range, as thiswill not provide plant materials with high levels of geneticdiversity (Thomas et al. 2014). Even though teak has an ex-tensive natural distribution range in Myanmar and India, theprovenance trials examined in the present study wereestablished using a limited number of populations, which onlyrepresented one and three provenances, respectively. Theprovenance trials were probably set up by planting superiortrees from a specific population. However, this approach is notdesirable, as a small sample of trees from a specific area hasbeen predicted to cause genetic erosion (Rogers andMontalvo2004).

Genetic diversity in teak plantations

It has been suggested that the genetic composition of plantedforests is generally artificially influenced by human activitiesthrough seed collection (Iwaizumi et al. 2018). In this study,the highest Ho and He values were found for CY4, a clonalteak experiment forest in Yogyakarta, Central Java (Fig. 1).CY4 also demonstrates a higher number of private alleles thanthe other plantation populations (Table 3). The origin of thispopulation might come from various location and might befrom Indian provenance. Teak improvement in Indonesiastarted in 1974 (Suhaendi 1998), at a time which teak prove-nance trials has already been established in Indonesia,

Fig. 3 STRUCTURE analysis results using 459 SNPs for all populations(I); Burma, Thailand, and Laos provenances and Java plantation popula-tions without CY4 with (a) K = 2, (b) K = 3 and (c) K = 4 (II); and all

plantations without CY4 with (a) K = 2, (b) K = 3, and (c) K = 4 (III). Thepopulation names are clarified in Tables 1 and 2

34 Page 10 of 14 Tree Genetics & Genomes (2020) 16: 34

including certain trials that used plant materials from Indianprovenances. Plus tree selection was started in the early 1980s(Siswamartana 1998). The provenance trials that wereestablished with Indian plant materials showed good growthperformance (Suhaendi 1998), and it is likely that some of thetrees with superior phenotypes were selected as the startingmaterial for subsequent breeding programs, such as the clonaltests examined in this study. The presence of private allelesand high genetic diversity indicate that CY4 is geneticallydifferent from the other teak plantations on Java island, allof which were heavily influenced by organized breedingprograms.

We observed that young plantations show higher geneticdiversity than old plantations. When CY4 was excluded fromthe analyses, the EY3 and WY2 populations demonstratedconsiderably higher He values than the older populations(Table 3). These two populations also had higher He valuesthan several native provenance populations (LAO andTHA1). This finding agrees with what has been reported byVerhaegen et al. (2010). They stated that an increase in thenumber of teak plantations in Indonesia most probably led tothe genetic mixing of plant materials, which would explainwhy the younger plantations (< 60 years old) demonstratedhigher genetic diversity than the old plantations (> 60 yearsold) (Verhaegen et al. 2010).Midgley et al. (2015) support thisclaim, as they found that teak plantations cover 1.4 million hain 2009, which represents considerable growth from 0.7 mil-lion ha in 1995 (Pandey and Brown 2000). Plant materials forplantation has been used common teak stand and seed produc-tion area (SPAs) for since long ago until 2002. Then, theplantation using seeds from clonal seed orchard has beenstarted since 2002 in Perhutani; Indonesia State OwnedForestry Company (Sadono 2017).

Origin and population structure of teak in Java

This study found high genetic differentiation (FST = 0.446)between the native and provenance populations. This FST val-ue was slightly higher than what has been reported in previousstudies employing microsatellite markers, e.g., Hansen et al.(2015), Fofana et al. (2009), and Huang et al. (2015) reportedFST values of 0.227, 0.220, and 0.146, respectively. This dif-ference can be explained by the use of different geneticmarkers (SNPs and microsatellites), as well as the fact thatthese studies covered different natural populations. The resultalso provides additional evidence that the Indian teak popula-tion is genetically distinct from the populations in Thailandand Laos (Fofana et al. 2009; Huang et al. 2015). Furthermore,the results presented in this paper indicate that the samplesused in the Bojonegoro provenance trial represent variouspopulations from the natural distribution of teak.

Little between-plantation genetic variation was found forthe Java teak populations examined in this study. Moreover,

the three teak plantation age classes could not be geneticallydifferentiated from one another at a significant level. AMOVAresults showed that variance between regions (east, central,and west) only explained 3.47% of the total variation, andSTRUCTURE results also confirmed that the three regionsof teak plantations on Java Island only showed weak geneticstructure (Fig. 3IIIb). This result separated eastern Java plan-tations, with the exception of EY3, as group I (red clustermajor); central Java populations, along with EY3, as groupII (yellow cluster major); and western Java plantations asgroup III (yellow and blue cluster major and complex cluster).This finding probably correlates with the working area ofPerhutani, as the company has divided Java Island into threeforest regions: East Java, Central Java, and West Java andBanten (Perhutani 2015). Seed collections systems often in-fluence the genetic variation between plantations (Iwasakiet al. 2019), and SPAs continue to be the main source of teakseeds for plantations in Indonesia before the emergence ofseed orchard (Sumardi 2011). Suhaendi (1998) reported thatPerhutani has established 463.9 ha of SPA in Central Java,377.7 ha in East Java, and two locations in West Java(41.5 ha in Ciamis and 22.4 in Indramayu). Thus, the geneticstructures of plantations in the three distinct regions can beexplained by different of seed sources. Diverse seed sourcescould influence genetic differentiation in future plantation(Broadhurst et al. 2015).

Ke r t a d i k a r a and P r a t ( 1995 ) r e po r t e d t h a tpaleobiogeographical and climatic data support the naturalmigration of teak from eastern Myanmar and Thailand toIndonesia during the Pleistocene period. The existence ofSundaland during the ice age and the similarities in veg-etation between these areas also support the natural mi-gration hypothesis (Kertadikara and Prat 1995). However,the results of this study do not support the hypothesisbecause private alleles were only observed in WY2, andall of the commercial teak plantations (except for CY4) inJava demonstrated low heterozygosity. According toPCoA and STRUCTURE ana ly se s , t h e na t i v eprovenance populations from Myanmar, Laos, andThailand all showed close genetic relationships to theIndonesian teak plantations. Hansen et al. (2017) alsoreported—based on microsatellite data—that three of thefour investigated Indonesian teak populations are geneti-cally related to the natural populations in Thailand andLaos, while Verhaegen et al. (2010) identified a geneticlink between Indonesian and central Laos teak popula-tions. We investigated 27 plantation populations through-out Java Island and concluded that most of the Indonesianlandraces originate from Myanmar, Laos, and Thailand.

According to the STRUCTURE and PCoA results, CY4 isgenetically related to Indian teak provenances. The CY4 pop-ulation was originally established to act as a source of superiorclones that could be used to increase the productivity of teak

Tree Genetics & Genomes (2020) 16: 34 Page 11 of 14 34

clonal forestry. There is no record of the seed sources for thispopulation, but our results suggest that this population wasestablished using plant materials from various locations, in-cluding Indian provenances, that have shown favorablegrowth in provenance trials. The high genetic diversity ob-served for CY4, as well as the genetic link to Indian nativeprovenance populations, support this hypothesis.

Implication for maintaining genetic variety of teakin Java

Teak plantations in Java, except for the clonal experimentCY4, possess low genetic diversity. However, it is importantto note that the genetic diversity observed in teak plantationson Java is similar to what has been reported for Thai and Laospopulations. Moreover, the Indonesian landraces have shownexcellent growth performance. We conclude that, at the veryleast, the current genetic diversity of Indonesian teak planta-tions must be maintained by seed production at designatedSPAs. When considering the productivity of teak plantations,along with their ability to adapt to global climate change,future breeding projects should include plant materials fromvarious natural populations, such as those in India. This issupported by the results in this paper, e.g., the CY4 popula-tion, which was closely related to Indian natural teak popula-tions, showed the best growth performance of any plantationin Java.

Teak clonal forestry has expanded over the past years be-cause of increasing forest productivity (Monteuuis and Goh2017). Interestingly, our study found the clonal experiments(which included samples from 11 clones) to show the highestheterozygosity among the investigated populations. Perhutanihas recently started to use two superior clones from vegetativepropagation to increase the productivity of teak plantations.However, plantation managers should always consider futurethreats, and global climate change will affect the environmentin which teak grows and increase the risk of pests and disease.As such, Perhutani should consider expanding its set of supe-rior clones by selecting additional clones from the clonal ex-periment forest or introducing new superior phenotype indi-viduals from natural populations (such as Indian prove-nances). In Japan, clonal forestry using C. japonica hasexisted for more than 300 years, with approximately 30 culti-vars applied in the Kyushu district (Ohba 1993). However,each plantation site is established with only one clone, butthe clonal forests become complex when considered on thelarge-scale, and have not experienced any significant prob-lems with disease or pests. The high number of clones mightunderlie the success of clonal forestry in Japan.

Acknowledgements We would like to thank Hideki Mori and SinggihUtomo for assisting with the DNA extraction; Ghani Purnomo, AndyPrakoso, M Jaudin, and Perum Perhutani for helping with sample

collection and Fajar Setiawan for mapping the study site. We would alsolike to thank Gaku Hitsuma for his valuable discussion. The first authorwas supported by a Japanese Government (Monbukagakusho) scholar-ship ID Number 173167. This work was partly supported by the JSPSCore-to-Core Program and A. Advanced Research Networks.

Data archiving statement The SNP data was deposited in Dryad(doi:https://doi.org/10.5061/dryad.r7sqv9s7k).

References

Al-Hawija BN, Wagner V, Hensen I (2014) Genetic comparison betweennatural and planted populations of Pinus brutia and Cupressussempervirens in Syria. Turk J Agric For 38:267–280

Altona T (1922) Teak and Hindoos. Tectona 15:457–506Aravanopoulos FA (2016) Conservation and monitoring of tree genetic

resources in temperate forests. Curr Forestry Rep 2:119–129Bailey JD, Harjanto NA (2005) Teak (Tectona grandis L.) tree growth,

stem quality and health in coppiced plantation in Java, Indonesia.New For 30:55–65

Behaghel I (1999) The state of teak (Tectona grandis L.F.) plantations inthe world. Bois For Trop 262:6–18

Binh HT, Ngoc NV, Bon TN, Tagane S, Suyama Y, Yahara T (2018) Anew species and two new records ofQuercus (Fagaceae) from north-ern Vietnam. PhytoKeys 92:1–15

Broadhurst LM, Fifield G, Vanzella B, PickupM (2015) An evaluation ofgenetic structure of seed sources and the maintenance of geneticdiversity during establishment of two yellow box (Eucalyptusmelliodora) seed-production areas. Aust J Bot 63:455–466

Budiadi, Widiyatno, Ishii H (2017) Response of a clonal teak plantationto thinning and pruning in Java, Indonesia. J Trop For Sci 29:44–53

Catchen JM, Amores A, Hohenleho P, CreskoW, Postlethwait JH (2011)Stacks: building and genotyping loci de novo from short-read se-quences. G3 1:171–182

Catchen JM, Hohenlohe PA, Bassham S, Amores A, Cresko WA (2013)Stacsks: an analysis tool set for population genomics. Mol Ecol 22:3124–3140

Davey JW, Hohenlohe PA, Etter PD, Boone JQ, Catchen JM, Blaxter ML(2011) Genome-wide genetic marker discovery and genotypingusing next-generation sequencing. Nat Rev Genet 12:599–510

Dunker B, Dormontt EE, van Dijk K, Dixon RRM, Jardine DI, Kireta D,Nurtjahjaningsih ILG, Win T-T, Rimbawanto A, Lowe AJ (2019) Aset of 156 SNP markers for teak (Tectona grandis Linn.f.). ConservGenet Resour. https://doi.org/10.1007/s12686-019-01099-7

Earl DA, vonHoldt BM (2012) STRUCTURE HARVESTER: a websiteand program for visualizing STRUCTURE output andimplementing the Evanno method. Conserv Genet Resour 4:359–361

Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clustersof individuals using the software STRUCTURE: a simulation study.Mol Ecol 14:2611–2620

Excoffier L, Lischer HEL (2010) Arlequin suite ver 3.5: a new series ofprograms to perform population genetics analyses under Linux andWindows. Mol Ecol Resour 10:564–567

Fofana IJ, Ofori D, Poitel M, Verhaegen D (2009) Diversity and geneticstructure of teak (Tectona grandis L.f) in its natural range usingDNA microsatellite markers. New For 37:175–195

Goudet J (2003) FSTAT (ver.2.9.4.) a program to estimate and test pop-ulation genetics parameter. http://www.unil.ch/izea/softwares/fstat.html. Accessed 20 September 2018

Gutierrez-Ortega JS, Jimenez-Cedillo K, Perez-Farrera MA, Vovides AP,Martinez JF,Molina-Freaner F, Imai R, TsudaY,Matsuki Y, SuyamaY,Watano Y, Kajita T (2018) Considering evolutionary processes in

34 Page 12 of 14 Tree Genetics & Genomes (2020) 16: 34

cycad conservation: identification of evolutionarily significant unitswithin Dioon sonorense (Zamiaceae) in northwestern Mexico.Conserv Genet 19:1069–1081

Hansen OK, Changtragoon S, Ponoy B, Kjaer ED, Minn Y, Finkeldey R,Nielsen KB, Graudal L (2015) Genetic resources of teak (Tectonagrandis Linn.f.)-strong genetic structure among natural populations.Tree Genet Genomes 11:802

Hansen OK, Changtragoon S, Ponoy B, Lopez J, Richard J, Kjaer ED(2017) Worldwide translocation of teak — origin of landraces andpresent genetic base. Tree Genet Genomes 13:87

Huang GH, Liang KN, Zhou ZZ, Xu JM, Ma HM (2015) Genetic vari-ation and origin of teak (Tectona grandis L.f.) native and introducedprovenances. Silvae Genet 64:33–46

Iwaizumi MG, Miyata S, Hirao T, Tamura M, Watanabe A (2018)Historical seed use and transfer affect geographic specificity in ge-netic diversity and structure of old planted Pinus thunbergii popula-tions. For Ecol Manag 408:211–291

Iwasaki H, Uchiyama K, Kimura M, Saito Y, Hakamata T, Ide Y (2019)Impact of a tree improvement program on the genetic diversity ofsugi (Cryptomeria japonica D Don) plantations. For Ecol Manag448:466–473

Jakobsson M, Rosenberg NA (2007) CLUMPP: a cluster matching andpermutation program for dealing with label switching andmultimodality in analysis of population structure. Bioinformatics23:1801–1806

Kalinowski ST (2005) HP-Rare: a computer program for performingrarefaction on measures of allelic diversity. Mol Ecol Notes 5:187–189

Kaosa-ard A (1989) Teak (Tectona grandis): its natural distribution andrelated factors. Nat Hist Bull Siam Soc 29:55–74

Kertadikara AWS, Prat D (1995) Isozyme variation among teak (Tectonagrandis L.f.) provenances. Theor Appl Genet 90:803–810. https://doi.org/10.1007/BF00222015

Kollert W, Walotek PJ (2015) Global teak trade in the aftermath ofMyanmar’s log export ban. Planted forests and trees working paperFP/49/E. FAO: Rome, Italy. Available at: http://www.fao.org/forestry/plantedforests/67508@170537/en/. Accessed 30 June 2018

Kumar S, Setcher G, Tamura K (2016) MEGA7: molecular evolutionarygenetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

Mammadov J, Aggarwal R, Buyyarapu R, Kumpatla S (2012) SNPmarkers and their impact on plant breeding. Int J Plant Genomics.https://doi.org/10.1155/2012/728398

Midgley S, Somaiya RT, Stevens PR, Brown A, Kien ND, Laity R (2015)Planted teak: global production and markets, with reference tpSolomon Islands. ACIAR Technical Report No.85. AustralianCentre for International Agricultural Research: Canberra,Australia. https://www.aciar.gov.au/node/12356. Accessed 20June 2019

Monteuuis O, Goh D (2017) Origin and global dissemination of clonalmaterial in planted teak forest. In: Kollert W, Kleine M (eds) Theglobal teak study: analysis, evaluation and future potential of teakresources. International Union of Forest Research Organizations,Vienna, pp 30–36

Morin PA, Luikart G, Wayne RK, the SNP Workshop Group (2004)SNPs in ecology, evolution and conservation. Trends Ecol Evol19:208–216

Neto OC, Aguiar AV, Twyford AD, Neaves LE, Pennington RT, LopesAV (2014) Genetic and ecological outcomes of Inga vea subsp.affinis (Leguminosae) tree plantation in a fragmented tropical land-scape. PLoS One. https://doi.org/10.1371/journal.pone.0099903

Ohba K (1993) Clonal forestry with sugi (Cryptomeria japonica). In:Ahuja MR, Libby WJ (eds) Clonal Forestry II. Springer-Verlag,Berlin, pp 66–90

Palanisamy K, Hedge M, Yi JS (2009) Teak (Tectona grandis Linn.F.): arenowned commercial timber species. J For Sci 25:1–24

Pandey D, Brown C (2000) Teak: a global overview. Unasylva 201:3–13Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in excel.

Population genetic software for teaching and research– an update.Bioinformatics 28:2537–2539

Perhutani (2015) Perum Perhutani profile. Perum Perhutani: Jakarta,Indonesia. http://perhutani.co.id/Company%20Profile%20Perhutani.pdf. Accessed 26 July 2019

Pritchard JK, Stephens M, Donnelly P (2000) Inference of populationstructure using multilocus genotype data. Genetics 155:945–959

Richards ZT, Yasuda N, Kikuchi T, Foster T, Mitsuyuki C, Stat M,Suyama Y, Wilson NG (2018) Integrated evidence reveals a newspecies in the ancient blue coral genus Heliopora (Octocorallia).Sci Rep. https://doi.org/10.1038/s41598-018-3269-z

Rogers DL, Montalvo AM (2004) Genetically appropriate choices forplant materials to maintain biological diversity. Report to USDAForest Service. Genetic Resources Conservation Program,University of California: Davis, CA. https://www.fs.usda.gov/internet/FSE_DOCUMENTS/fsbdev3_039080.pdf. Accessed 20December 2018

Rosenberg NA (2004) DISTRUCT: a program for the graphical displayof population structure. Mol Ecol Notes 4:137–138

Sadono R (2017) Temporary site index for two- invented teak clones withgenerative regeneration in the state forestland in East Java,Indonesia. Adv Environ Biol 11:6–12

Saitou N, Nei M (1987) The neighbor-joining method: a new method forreconstructing phylogenetic trees. Mol Biol Evol 4:406–442

Simatupang MH (2001) Chemical properties of teakwood (Tectonagrandis L.f.) as guide for selection of mother trees. In: HardiyantoEB (ed) Potentials and opportunities in marketing and trade of plan-tation teak: challenge for the new millennium. Faculty of ForestryGadjah Mada University, Yogyakarta, pp 255–264

Siswamartana, S (1998) Teak forest management in Indonesia. In KashioM andWhite K (eds.) Teak for the future. Proceedings of the SecondRegional Seminar on Teak: Yangon, Myanmar. http://www.fao.org/3/AC773E/ac773e0d.htm#bm13. Accessed 7 Apr 2019

Smiet AC (1990) Forest ecology on Java: conversion and usage in ahistorical perspective. J Trop For Sci 2:286–302

Suhaendi H (1998) Teak improvement in Indonesia. In Kashio M andWhite K (eds.) Teak for the future. Proceedings of the SecondRegional Seminar on Teak. Yangon, Myanmar. http://www.fao.org/3/AC773E/ac773e0c.htm#bm12. Accessed 8 Jan 2019

Sumardi S (2011) Fertility variation and effective population size in a teakclonal seed orchard. J For Res 8:66–79

Suyama Y, Matsuki Y (2015) MIG-seq: an effective PCR-based methodfor genome-wide single-nucleotide polymorphism genotyping usingthe next-generation sequencing platform. Sci Rep 5:16963

Takahashi Y (2017) Genome-wide population genetic analysis identifiesevolutionary forces establishing continuous population divergence.Ecol Res 32:461–468

Takahashi Y, Suyama Y, Matsuki Y, Funayama R, Nakayama K, KawataM (2016) Lack of genetic variation prevents adaptation at the geo-graphic range margin in a damselfly. Mol Ecol 25:4450–4460

Tamura S, Fukuda T, Pimoneva EA, Petrunenko EA, Krestov PV,Bpndarchuk SN, Chernyagina OA, Suyama Y, Tsunamoto Y,Matsui A, Tsuboi H, Takahashi H, Sato K, Nishikawa Y,Shimamura T, Fujita H, Nakamura K (2018) Molecular and cyto-logical evidences denied the immediate-hybrid hypothesis forSaxifraga yuparensis (sect Bronchiales, Saxifragaceae) endemic toMt. Yubari in Hokkaido, northern Japan. Phytotaxa 373:53–70

TanakaN,Hamazaki T, Vacharangkura T (1998)Distribution, growth andsite requirements of teak. JARQ 32:65–77

Thomas E, Jalonen R, Loo J, Boshier D, Gallo L, Cavers S, Bordács S,Smith P, Bozzano M (2014) Genetic considerations in ecosystemrestoration using native tree species. For Ecol Manag 333:66–75

Tree Genetics & Genomes (2020) 16: 34 Page 13 of 14 34

Tsumura Y, Kawahara T,Wiekneswari R, Yoshimura H (1996)Molecularphylogeny of dipterocarpaceae in Southeast Asia using RFLP ofPCR-amplified chloroplast genes. Theor Appl Genet 93:22–29

Uchiyama K, Miyamoto N, Takahashi M, Watanabe A, Tsumura Y(2014) Population genetic structure and the effect of historical hu-man activity on the genetic variability ofCryptomeria japonica corecollection, in Japan. Tree Genet Genomes 10:1257–1270

Vaishnaw V, Mohammad N, Wali SA, Kumar R, Tripathi SB, Negi MS,Ansari SA (2014) AFLP markers for analysis of genetic diversityand structure of teak (Tectona grandis) in India. Can J For Res 45:297–306

Verhaegen D, Fofana IJ, Logossa ZA, Ofori D (2010) What is the geneticorigin of teak (Tectona grandis L.) introduced in Africa and inIndonesia? Tree Genet Genomes 6:717–733

Wachi N, Matsubayashi KW, Maeto K (2018) Application of next-generation sequencing to the study of non-model insects. EntomolSci 21:3–11

Watanabe A, Widyatmoko A (2004) Discrimination of teak (Tectonagrandis) plus trees using selected random amplified polymorphicDNA (RAPD) markers. J Trop For Sci 16:17–24

Webb DB, Wood PJ, Smith JP, Henman GS (1984) A guide to speciesselection for tropical and sub-tropical plantations. CommontwealthForestry Institute, University of Oxford, Oxford

White KJ (1991) Teak: some aspects of research and development.Publication 1991/17. FAO Regional Office for Asia and thePacific (RAPA): Bangkok, Thailand, p 3

Win T-T, Watanabe A, Hirao T, Isoda K, Ishizuka W, Goto S (2015)Genetic diversity of teak population in native regions and planta-tions in Myanmar detected by microsatellite markers. Bull UnivTokyo For 132:1–15

WuHX (2018) Benefit and risk of using clones in forestry- a review. ScanJ For Res. https://doi.org/10.1080/02827581.2018.1487579

Yasunari T (1981) Temporal and spatial variation of monthly rainfall inJava Indonesia. Southeast Asian Stud 18:170–186

Yoichi W, Kawamata I, Matsuki Y, Suyama Y, Uehara K, Ito M (2018)Phylogeographic analysis suggests two origins for the riparian aza-lea Rhododendron indicum (L.) Sweet. Heredity. https://doi.org/10.1038/s41437-018-0064-3

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