Introduction

Genetic attributes of plant species are impacted by habitat loss and fragmentation, with consequences including genetic erosion, characterised by a reduction in heterozygosity at the individual level and adaptive potential, as well as increased inbreeding, which can decrease long-term survival and population viability (Lowe et al., 2005; Wilson et al. 2016; Niissalo et al. 2018). Fragmentation of natural environments can also alter the genetic structure and connectivity among populations by creating barriers to gene flow, potentially exacerbating genetic isolation due to disrupted migration patterns and dispersal abilities (Young et al. 1996; Leimu et al. 2010; Schlaepfer et al. 2018). Tropical tree species that are isolated, for example by logging and land use change, are more susceptible to genetic erosion than continuous populations (Pither et al. 2003; Bittencourt and Sebbenn 2007; Ang et al. 2016). However, the impact of geographic isolation on genetic variation of tropical trees may be neutralised if effective gene flow can be maintained by pollination vectors (such as wind or insects) that are insensitive to habitat fragmentation (Wang et al. 2012; Minn et al. 2014; Akinnagbe et al. 2019).

By examining genetic changes over time through temporal sampling (where material from individuals that pre-date the impact of interest should be sampled, as well as populations that established after the impact has had the opportunity to affect diversity; Leigh et al. 2021), researchers may gain valuable insights into the adaptive potential of populations, the impact of environmental factors on genetic diversity, and the efficacy of conservation interventions (Díez-del-Molino et al. 2018). While microsatellite markers have traditionally been used to survey genetic variation across age classes (Kettle et al. 2007; Noreen and Webb 2013), the advent of next-generation sequencing (NGS) data offers several advantages to study the conservation genomics of non-model organisms. NGS methods such as genotyping by sequencing (GBS; Hall et al. 2020) or double-digest restriction-site associated DNA (ddRAD) sequencing (Peterson et al. 2012), can provide useful snapshots of genomic variation across the entire genome, allowing for the assessment of genetic variation across a wide range of loci, and fine-scale resolution of genetic diversity and population structure (Hauser et al. 2011; Niissalo et al. 2018; Yang et al. 2022). These methods provide more data than microsatellites, which can only be used for genotyping with a small number of loci that risks underestimating genetic structure (Andrews et al. 2016; Hodel et al. 2017). By facilitating the discovery of novel genetic variants and identification of single nucleotide polymorphisms (SNPs), NGS analyses contribute to a more comprehensive understanding of genetic variation within a species and distinctive characteristics of different age classes.

Very few conservation genetics studies using NGS techniques have been carried out to assess Southeast Asian tropical trees. The assessment of genetic diversity and structure is limited to Dipterocarpaceae species in this region (Ng et al. 2019; Ohtani et al. 2021; Ogasahara et al. 2023) and the only other study of intergenerational genetic characteristics in a native Singapore tree species was carried out on Koompasia malaccensis (Noreen and Webb 2013), all based on characterising between eight and around 20 microsatellite markers. The K. malaccensis study concluded that there is high genetic diversity in all age classes, contrary to expectations of fragmentation-induced genetic erosion, although there were some signs of reduced gene flow in younger cohorts. While microsatellite analysis can detect population divergence, RAD-seq methods have greater power to resolve genetic structure due to the higher number of loci (Hodel et al. 2017; Sunde et al. 2020).

The goal of this study was to employ RAD-seq methods to advance the understanding of how genetic diversity in Palaquium obovatum, a vulnerable tropical tree species in Singapore (Lindsay et al. 2022), has responded to habitat degradation and extreme forest fragmentation in the highly urbanised state of Singapore. Most of the loss of natural ecosystems and species in Singapore had occurred by 1920 (Corlett 1992). Primary lowland rain forest occupies only around 0.2% of its original extent in Singapore, and secondary forest covers around 20% of the land area (Yee et al. 2011, 2019; Ngo et al. 2016). By comparing adults with seedlings from forest fragments affected by disturbance and isolation, we aimed to test the hypothesis that genetic variation and inbreeding statistics would indicate the adverse effects of anthropogenic disturbance and habitat degradation over time (Schlaepfer et al. 2018; Aguilar et al. 2019), and in so doing, also highlight challenges for conservation management of this species.

Materials and methods

Study species

Palaquium obovatum is a species in Sapotaceae, a diverse family of tropical trees that includes species harvested for timber and latex. Representatives of Palaquium, and in fact most of the Sapotaceae family typically register low abundance in Malayan rainforest (LaFrankie 1996; Turner et al. 1997). Palaquium obovatum is native to the lowland rainforests of Southeast Asia, including Indonesia, Thailand, Malaysia and Singapore, and is sometimes found along coasts. In Singapore, its limited range and low density has rendered it vulnerable, with less than 1000 mature individuals in the wild (Lindsay et al. 2022). This distribution makes the species an excellent candidate for investigating genetic responses to extreme habitat fragmentation in an urban landscape, which are relevant to the preservation of remnant tree populations growing in isolated fragments in many other cities, especially against the backdrop of widespread deforestation in Asia making way for man-made habitats. P. obovatum is a medium to tall tree, with a cylindrical, buttressed bole reaching heights of up to 40 m, and diameter at breast height (dbh) of up to 110 cm. The species has perfect flowers exhibiting characteristics of bat pollination (Phang 2023), which also makes it a pertinent study focus in light of anthropogenic disruptions to bat-plant interactions across the world (Aziz et al. 2021). The fruits are fleshy berries, 2–3 cm in diameter, with 1–2 seeds. Small trees (1–2 cm dbh) grew at a mean rate of 0.04 cm in dbh per year from 1993 to 2004 in a 2-ha primary forest plot within Singapore’s Bukit Timah Nature Reserve (LaFrankie et al. 2005). In Peninsular Malaysia, some congeneric species have been observed to have a similarly slow rate of growth (Soerianegara and Lemmens 1993): trees of Palaquium maingayi and P. rostratum were found to take 100 and 70 years respectively to attain a dbh of 55 cm, and P. gutta a dbh of 50 cm in 50 years; however, a tree of P. rostratum in an arboretum attained a dbh of 57 cm in 40 years. A heritage tree of Palaquium obovatum that was planted in 1897 by Henry Ridley in a part of the Singapore Botanic Gardens used to trial plants of economic potential, has a dbh of 131 cm and height of 31.4 m (National Parks Board 2021); however, growth rates in cultivated areas often differ from those in natural forest (Appanah and Weinland 1993). As a chiroptephilous species, Palaquium obovatum is reliant on bats for both pollination and seed dispersal; when fallen, the ripe fruit are quickly consumed by other fauna such as monkeys, squirrels or birds, which curtails seed survivability (Ng 1972; Soerianegara and Lemmens 1993; Chan et al. 2022; Phang 2023). Seedlings that germinate are often outcompeted due to overshadowing vegetation constraining sufficient light (Soerianegara and Lemmens 1993). The genus, in general, does not regularly flower or fruit but P. obovatum has been observed to flower more often than its congeners (c. every 1–2 years), which increases the accessibility to juvenile material for population studies. The smallest fruit-bearing individual recorded had a dbh of 11 cm (pers. obs).

In Singapore, the species has been assessed as Vulnerable (VU; Lindsay et al. 2022) and has some economic importance as a source of gutta percha—a latex used in a myriad of products including the first undersea telegraphic cables. Nothing is currently known about the species’ genetic diversity, levels of inbreeding and levels of gene flow between populations. Polyploidy in the Sapotaceae family is rare and occurs mostly in the tribe Sideroxyleae, with previous studies finding most representatives, including certain Palaquium species, to be diploid (Johnson 1991; Smedmark and Anderberg 2007).

Sample collection

Individuals of Palaquium obovatum were sampled from populations across their natural range in Singapore. Existing literature and past surveys were consulted (Wong et al. 1994; LaFrankie et al. 2005; Yee et al. 2011, 2019; Neo et al. 2013; Hung et al. 2017; Ngo et al. 2016), and forest reserves were searched to locate the largest extant trees of P. obovatum, which likely survived the widespread deforestation that occurred during British colonisation in the 19th century (Corlett 2013). Sampling included individuals in various life stages, from seedling to reproductively capable adults.

In Singapore, the study area encompassed primary and secondary forest areas as well as presumed cultivated populations (i.e. without historical planting records but found in built-up areas) in the Singapore Botanic Gardens (SBG) and St. John’s Island (SJI). Sampling occurred in the remnant primary forest patches within SBG (which contains a 4 ha area of lowland rain forest that formed part of the original establishment in 1959, known as the “Gardens’ Jungle”; Turner et al. 1996), Bukit Timah Nature Reserve (BTNR) and MacRitchie Reservoir area in the Central Catchment Nature Reserve (CCNR). Secondary forest samples were taken from the Upper Seletar area in CCNR, Bukit Batok Nature Park (BBNP), as well as small islands off mainland Singapore including Sentosa, St. John’s Island and Pulau Ubin. From Peninsular Malaysia, one cultivated population was sampled from Kepong Botanic Gardens at the Forest Research Institute Malaysia, and one herbarium sample from a forest in Langkawi (a district in the state of Kedah) was also included (Fig. 1, coordinates plotted using QGIS: QGIS Development Team 2019). In all, 15 populations were sampled, four from primary forest, eight from secondary forest and three from cultivation (Table 1).

Fig. 1
figure 1

Sampling localities. Main, map of Singapore with coloured circles indicating collection localities; inset, map of Peninsular Malaysia with orange stars indicating collection localities, and the island of Singapore at the tip of the peninsula circled in red. BBNP refers to Bukit Batok Nature Park, BTNR: Bukit Timah Nature Reserve, CCNR: Central Catchment Nature Reserve, SBG: Singapore Botanic Gardens, SJI: St. John’s Island, Ubin: Pulau Ubin

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Table 1 Sampling locations, numbers and size characteristics of individuals used for molecular/ddRAD analysis. For more information on sample IDs, see Table S1 in Supplementary Material
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Other than the single herbarium sample from Langkawi, fresh leaf samples from 103 individuals were collected and stored in silica gel. The sampling consisted of 16 adults and of seedlings or saplings collected mostly within a 5 m radius beneath the canopy of these adults (except for the population in BTNR). A total of 18 samples were classified as saplings (> 30 cm in height, dbh < 10 cm) and 70 as seedlings (< 30 cm in height). No seedlings were found in BTNR, and instead nine representative saplings were collected. The dbh of the adult trees was measured and their height was estimated. The seedlings and saplings were grouped into the above size categories, and combined in downstream analyses as juveniles. GPS coordinates and herbarium vouchers were obtained for each adult tree. For adult trees, leaves were obtained either using a pole pruner for branches less than 3 m high, a Big Shot Catapult to launch a weighted line over high branches, or arborist climbers/cranes for sampling in the Singapore Botanic Gardens.

The Singapore samples were collected from eight distinct localities (BTNR, BBNP, CCNR-MacRitchie, CCNR-Seletar, SBG, Sentosa, SJI, Pulau Ubin) and the Malaysia samples from two localities (Langkawi, Kepong). Three populations were from areas of cultivation: one from SBG, one from SJI and the last from Kepong. Trees around or exceeding 100 cm in dbh, i.e. from the Gardens’ Jungle (primary forest), Pulau Ubin, Sentosa and CCNR-Seletar (secondary forest) are likely to be close to or more than 100 years old. Assuming 20–30 years per generation, the oldest trees from wild areas other than BBNP and SJI would have been alive for at least three overlapping generations. The smaller trees from the wild areas of SJI and BBNP, as well as the parent from Kepong Botanic Garden, are unlikely to have produced more than one generation; the larger cultivated instances in SBG and SJI, not more than two generations. There were no seedlings found in the entire 2 ha primary forest plot within BTNR; the sole reproductively capable adult sampled was a young tree only 14 cm in dbh, which is unlikely to have produced the saplings of heights between 1 and 8 m found within the same plot.

DNA extraction, library preparation and sequencing

Genomic DNA was extracted from fresh leaf material using the cetyl-tri-methylammonium bromide (CTAB) method, with 24:1 chloroform: isoamyl alcohol and overnight precipitation in isopropanol at -20 °C (Doyle and Doyle 1987; Cullings 1992). Samples were purified with SeraMag magnetic carboxylate-modified microparticles (Cytiva, USA) diluted to 5%, and the concentration of DNA quantified with a Nanodrop 2000c Spectrophotometer (Thermo Scientific, Singapore). Double-digest Restricted Associated DNA (ddRAD) libraries of 16–99 individuals per library (pools depended on collection and lab access timings) were built using protocols from Peterson et al. (2012) and adapted by Niissalo et al. (2018; 2020), with DNA digested using PstI (rare-cutting) and ApeKI (frequent-cutting) restriction enzymes, and standard Illumina indexing barcodes of 5 bp, adapters and polymerase chain reaction (PCR) primers (Integrated DNA Technologies, Singapore). DNA concentration of 400 ng per sample was used, and SeraMag particles applied to size select 450–550 bp fragments, with quality and size confirmed with a Tapestation 4200 System using High Sensitivity D1000 ScreenTapes (Agilent Technologies, CA, USA). To enrich digested fragments, 12 PCR cycles were conducted for each library, with annealing temperature of 64 °C. Libraries were analysed with a Qubit analyser (Life Technologies Corporation, Carlsbad, CA), pooled to equimolar amounts and sequenced by NovogeneAIT Genomics (Singapore) using an Illumina Novaseq 6000 instrument (San Diego, CA, USA).

Variant calling and quality filtering

Raw reads were quality-checked with FastQC v.0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc). Sequences were then filtered to eliminate poor quality reads, demultiplexed, and reads assigned to individuals by specifying the parameters -q -c -r in the process_radtags program within STACKS 2.53 (Catchen et al. 2013). KMC v. 3.1.0 (Kokot et al. 2017) was used to calculate k-mers, and heterozygous k-mer pairs to assess ploidy levels of demultiplexed reads were estimated using Smudgeplot v. 0.2.3dev (Ranallo-Benavidez et al. 2020). A graph of the percentage of the minor and major common variants was plotted against the kmer count; a peak near 0.5 is expected if the individual is diploid, and 0.167 if hexaploid.

A reference genome for Palaquium obovatum, assembled from short reads, was obtained from the ongoing project to assemble whole genomes of all native species found in BTNR (Niissalo et al., in prep; N50 = 2,381 bp; BUSCO = 64.0%; assembly size = 1.4 Gb: the sizes of other Palaquium species sequenced in the same project were also referenced), and indexed with bwa v.0.17.17 (Burrows-Wheeler Aligner; Li and Durbin 2009; https://github.com/lh3/bwa), Reads for each individual were mapped to the reference genome with the bwa-mem setting in bwa v.0.7.17, then sorted with the sort function in Samtools v.1.14 (Li et al. 2009) to obtain BAM files per sample, which were evaluated by using the samtools stats and depth settings to generate mapping statistics and coverage.

To identify genetic variation, GATK v.4.2.6.1 (https://github.com/broadinstitute/gatk) was used, along with Picard v.2.19 (https://github.com/broadinstitute/picard) and Samtools v.1.14 (https://github.com/samtools/) to sort, mark duplicates, and index the data, followed by HaplotypeCaller with ploidy set at the estimated level (according to Smudgeplot results) to call SNPs on individual samples to create GVCF files with alignment to the most likely haplotypes. These GVCF files were consolidated using GenomicDBimport and jointly genotyped with GenotypeGVCF. Indels and SNPs of low base quality and depth of coverage were removed using the following hard-filtering parameters in GATK: “QD < 2.0 || QUAL < 30.0 || SOR > 3.0 || FS > 60.0 || MQ < 40.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0”. To minimise the influence of singletons, further filtering was then done with bcftools v.1.14 (https://github.com/samtools/bcftools) to remove multiallelic SNPs, those below a set frequency (maf 0.01) and all with missing data.

Genetic diversity and population structure

Genetic deterioration through the accumulation of deleterious genes is normally detected in measures of excess homozygosity and inbreeding, which eventually manifests in reduced fitness of organisms (Bosse and van Loon 2022). Heterozygosity was assessed by generating related metrics (i.e., distribution and frequency of heterozygous and homozygous genotypes in the dataset) with the stats -s parameter in bcftools. A custom script was then applied to only retain common alleles present as one or two copies out of a total of six in the whole sample set, effectively converting the vcf into one with diploid calls allowing direct comparison of the same genetic parameters across all samples (Supplementary File 1). To address linkage disequilibrium between SNP pairs, the dataset for unlinked SNPs was generated using the pruning parameters --indep-pairwise 50 5 0.5 (i.e. window size of 50 variants, window shift of 5 variants and r2 of 0.5) in plink v.1.90 (Chang et al. 2015). This pruned vcf file was examined for genetic variation by generating heterozygotic and inbreeding sample-specific statistics using the --het parameter in plink v.1.90 (Chang et al. 2015), as well as nucleotide diversity (π) and Tajima’s D averaged across loci using parameters --window-pi and --TajimaD in vcftools v.0.1.16, with reference to Tajima (1989) for critical values indicating deviation from mutation-shift balance. Paired t-tests were run to determine whether differences in heterozygotic and inbreeding measures were significant between cohorts of age classes and type of sampling locality (i.e. between wild and cultivated populations, as well as between primary and secondary forest populations).

To assess genetic similarity, a pairwise comparison of heterozygous single nucleotide polymorphisms (SNPs) was performed using the script from https://github.com/mattiniissalo/Genepop_clonality_script but adapted by manually converting the STRUCTURE file to genepop format (typically used by the Stacks pipeline). Specifically, each heterozygous SNP present in a sample was compared to its corresponding locus in another sample. A heatmap was also generated for these pairwise comparisons using the R package ggplot2 v.3.4.0 (Wickham 2011) in Rstudio v.4.1.3. After converting the pruned vcf file to STRUCTURE format with PGDSpider2 v1.1.5 (Lischer and Excoffier 2012), STRUCTURE (Pritchard et al. 2000) and Structure_threader v1.3.10 (Pina-Martins et al. 2017) were used to infer population structure, with K values (i.e. number of genetic clusters) ranging from one to 15, using 10 replicates for each value of K, with a burn-in of 20,000 and Markov Chain Monte Carlo (MCMC) repetitions at 100,000. The optimal value of K was estimated with the software Kfinder2 v.1.0.0.0 (Wang, 2019), which generates estimators of the best K using the ΔK (Evanno et al. 2005) and Pr[X|K] (Pritchard et al. 2000) methods, which take into consideration the rate of change in log probability between consecutive values of K (ΔK). The R package pophelper v.2.3.1 (Francis 2017) was applied to produce barplots for each value of K.

Plink was used to convert the consolidated VCF file to eigenvector/eigenvalue format for principal components analysis (PCA), with the first eight dimensions visualised using the tidyverse v.1.3.2 package (Wickham et al. 2019) in Rstudio v.4.1.3. The association between genetic and geographical distance was assessed by a Mantel test with 9999 permutations performed with the ade4 v.1.7–20 package (Dray and Dufour 2007) in Rstudio v.4.1.3. To further assess population differentiation, weighted FST (Bhatia et al. 2013) was calculated using vcftools v 0.1.14 (Danecek et al. 2011). Gene flow amongst populations was investigated using the ABBA-BABA test in D-suite (Malinsky et al. 2021); the sample from Langkawi was fixed as the outgroup (it had emerged as one of the earliest diverging samples in the Palaquium obovatum clade from a previous phylogenetic study, Phang et al. 2023), and the dataset was split into 20 jackknife blocks.

Results

The final ddRADseq dataset consisted of 104 samples of Palaquium obovatum: 98 from Singapore and six from Malaysia (Supporting Information, Table S1). Around 780 million paired-end reads were obtained from the Illumina sequencing, and about 750 million were retained after quality-control filtering and demultiplexing. ∼660 million reads were mapped and paired with the reference genome, with the average percentage of paired reads being 82.46% (Supplementary Fig. 3). Average sequencing depth was 44X with a minimum of 19X and a maximum of 75X. Smudgeplot analyses indicated that the ploidy level was six for every individual, as k-mer peaks were all around 16.7% (Supplementary Fig. 1). The reference genome of the hexaploid Palaquium obovatum had an assembly size (1.4 Gb/1 C) of about twice that of other presumed diploid Palaquium species found in BTNR (P. microphyllum = 0.8 Gb, P. impressinervium = 0.7 Gb, P. oxleyanum = 0.77 Gb). The vcf file obtained after joint genotyping in the GATK pipeline contained 680,585 variants, reduced to 603,679 after hard-filtering. Filtering out SNPs below a set frequency (maf 0.01) or with missing loci reduced the number of SNPs to 32,984. Reducing the SNPs to those with only a single copy of a variant and five copies of the dominant SNP produced a dataset with 3,066 SNPs. The final subset of independent SNPs after filtering for linkage-disequilibrium was 2,436.

Measures of genetic diversity were similar for the polyploid and “diploid” datasets (average Ho = 0.12 and 0.11, respectively; He = 0.09 and 0.14, respectively), hence the diploid set was deemed appropriate for downstream analyses. Significant differences (p < 0.05) in genetic diversity and inbreeding (FIS) were found between adults and juveniles from all populations, except for the wild samples from CCNR—MacRitchie and SJI (Table 2). When considered against similar statistics from other Palaquium species in Singapore (Phang et al. 2023), observed heterozygosity (Ho) indicated fairly low to moderate levels of diversity in wild (adults Ho=0.113 to 0.136, juveniles Ho=0.09 to 0.144) and cultivated populations (adults Ho=0.096 to 0.112, juveniles Ho=0.077 to 0.121). Most populations showed that juveniles had consistently lower genetic diversity and higher inbreeding than adults, except for the cultivated populations in SBG and SJI, and the wild population in SJI, where juveniles had higher diversity than adults. This wild population in SJI was also the only one where juveniles did not show significant levels of inbreeding (FIS = -0.019). Pairwise nucleotide diversity (π) showed limited levels of genetic variation (0.000079) and the small negative value for Tajima’s D (-0.326) indicated no significant deviation from a mutation-drift balance at the 95% level (Tajima 1989). No significant differences in Ho and FIS were found between wild populations in similar age classes (i.e., SBG, CCNR, Sentosa and Ubin), or between populations from primary and secondary forest. Adults in cultivated populations however, had significantly lower observed heterozygosity than adult wild populations.

Table 2 Comparisons of genetic diversity across populations and age classes. Ho, mean observed heterozygosity; FIS, mean inbreeding coefficient. Asterisks denote significant difference between adult and juvenile classes at *p < 0.05; **p < 0.01; ***p < 0.001
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Investigation into the clonality of individuals indicated higher percentage pairwise similarity for parent-offspring pairs, however the highest similarity value of 75% indicated that no pairs were clones of each other (Fig. 2). Using the ΔK and Pr[X|K] estimators, the STRUCTURE analysis suggested that the most likely number of distinct genetic groups in the data set was K = 12 (Supplementary Fig. 2), with clustering corresponding to localities, including separate clusters for cultivated and wild populations as well as further subdivision of two clusters within the Singapore Botanic Gardens rainforest (Fig. 3). This was corroborated by the heatmap generated from percentage pairwise similarity between individuals, showing 12 distinct clusters based on the same localities as the STRUCTURE analysis (Fig. 4). Principal components analysis showed that most populations clustered in their own genetic space (Fig. 5). A Mantel test using genetic and geographic distance between sampling sites found no support for an isolation by distance mechanism of population differentiation, which proposes that populations closer to each other are also more closely related (r = -0.07, p-value 0.835). Pairwise FST values showed substantial amounts of differentiation between populations (Table 3). No significant gene flow between populations was detected (Table S2 in Supplementary Material).

Fig. 2
figure 2

Dot plot of sample clonality analyses. The x-axis represents increasing sequence similarity for each pair of loci, and the y-axis represents pairwise similarity of heterozygous SNPs

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Fig. 3
figure 3

Population structure inferred from 2,436 unlinked SNPs with the STRUCTURE software; colours represent genetically distinct clusters. BBNP refers to Bukit Batok Nature Park, BTNR: Bukit Timah Nature Reserve, CCNR_M: Central Catchment Nature Reserve_MacRitchie, CCNR_S: Central Catchment Nature Reserve_Seletar, Kepong: Kepong Botanic Gardens, SBG_c: Singapore Botanic Gardens_cultivated, SBG_r: Singapore Botanic Gardens_rainforest, SJI: St. John’s Island, SJI_c: St. John’s Island_cultivated, Ubin: Pulau Ubin

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Fig. 4
figure 4

Heatmap of pairwise similarity values of heterozygous SNPs across individuals. BTNR refers to Bukit Timah Nature Reserve, BBNP: Bukit Batok Nature Park, CCNR_M: Central Catchment Nature Reserve_MacRitchie, CCNR_S: Central Catchment Nature Reserve_Seletar, Kepong: Kepong Botanic Gardens, SBG_c: Singapore Botanic Gardens_cultivated, SBG_r: Singapore Botanic Gardens_rainforest, SJI: St. John’s Island, SJI_c: St. John’s Island_cultivated, Ubin: Pulau Ubin

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Fig. 5
figure 5

PCA scatter plots of genetic variation in Palaquium obovatum populations using 2,436 independent SNPs presented through two bivariate plots of the first three principal components. A, PCA1 vs. PCA2. B, PCA2 vs. PCA3. BBNP refers to Bukit Batok Nature Park, BTNR: Bukit Timah Nature Reserve, CCNR_MacRitchie: Central Catchment Nature Reserve_MacRitchie, CCNR_Seletar: Central Catchment Nature Reserve_Seletar, Malaysia/Kedah: Langkawi in the Malaysian state of Kedah, Malaysia/Kepong: Kepong Botanic Gardens, SBG_c: Singapore Botanic Gardens_cultivated, SBG_r: Singapore Botanic Gardens_rainforest, SJI: St. John’s Island, SJI_c: St. John’s Island_cultivated, Ubin: Pulau Ubin

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Table 3 Population pairwise weighted FST (Wright’s index of fixation)
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Discussion

The overriding objective of this investigation was to assess the response of a vulnerable tree species, Palaquium obovatum, to extreme habitat fragmentation within a highly urban landscape. Using genome-wide SNPs, we found (i) significant differences in genetic diversity between adults and juveniles across almost all wild populations and (ii) strong population differentiation and genetic structure with little mixing between areas sampled, even for proximate populations. Our results provide evidence for genetic erosion due to anthropogenic disturbances. We also discuss potential pollen and seed-mediated dynamics that could shape conservation measures for managing this tree species, which have potential to be generalised to other species with related traits.

Previous chromosome counts indicated that most representatives of Sapotaceae are diploid (Johnson 1991; Smedmark and Anderberg 2007), and this study provides the first observation of polyploidy in the Isonandreae tribe of Sapotaceae. Palaquium obovatum is confirmed as hexaploid and the fact that the assembly size of the reference genome of this species was only about twice that of other diploid Palaquium was likely due to a collapsed assembly (where some chromosome copies were not fully distinguished), which also resulted in six copies being present along the majority of the assembly. The reference genome is incomplete and a higher-quality assembly would be preferred, also the lack of available data prevented the possibility of using pangenomes across multiple close-related individuals. However, using even a lower-quality reference genome gives advantages against de novo clustering, as reference-based methods allow for all compatible reads to map to a single part of the contig (Fitz-Gibbon et al. 2017). In a previous phylogenetic study (Phang et al. 2023), no gene tree conflict was detected for placement of Palaquium obovatum, which suggests that the species is likely an autopolyploid. While collapsed, our reference genome has the advantage of being a conspecific assembly from within the study area. This close relatedness, bolstered by high sequencing depth (Deng et al. 2022) increases the number and accuracy of variant calls, and we achieved above 80% mapping of paired reads and more than 2000 quality-filtered SNPs.

As Palaquium obovatum has a wider distribution (extending northwards up to India) and has more frequent reproductive cycles (i.e., flowering and fruiting) than other Palaquium species in Singapore, the genetic variability afforded by polyploidisation may have had a positive impact on higher growth and survival. Genome multiplications are known to be beneficial and therefore more likely retained in novel or changing environments, where the surplus gene copies can be used as material for new gene functions (Van de Peer et al. 2021; Edgeloe et al. 2022).

Despite the potential advantages of polyploidy, there are signals of genetic erosion in response to habitat degradation. The genetic structure indicates high population differentiation and decreasing genetic diversity in juveniles compared to adults indicating that anthropogenic change of the natural environment is exerting detrimental genetic consequences. This is consistent with previous studies where significant reductions in heterozygosity have been found between populations encountering at least a century of fragmentation (Aguilar et al. 2008). In addition to lower observed heterozygosity, average inbreeding coefficients across juveniles were nearly double the values observed in adults. The only wild population where observed heterozygosity was greater in juveniles than adults was found around a young tree in secondary coastal forest on St. John’s Island, which provides some indication that genetic diversity can be maintained in degraded landscapes and may have benefited from increased coastal protection in recent years (National Parks Board 2010).

The most likely reason for higher levels of inbreeding in juveniles is that urbanisation has reduced the abundance of reproductively mature trees in remnant populations, increasing self-fertilisation or biparental inbreeding (Kettle et al. 2007). It is possible that more inbreeding and genetic erosion is contributing to low seedling survival and the exceptionally low seed set within the primary forests in the Bukit Timah Nature Reserve and Singapore Botanic Gardens (pers. obs.). Increased inbreeding is not evident between adults and juveniles in cultivated populations in Singapore, even though observed heterozygosity is discernibly lower than in wild populations.

Strong differentiation between adult populations, even over short spatial scales of less than 2 km, indicates that gene flow is likely to have been limited over longer periods rather than just recently. Previous studies have indicated higher genetic differentiation among populations of tropical forest tree species than in temperate forests.(Finkeldey and Hattemer 2007; Yang et al. 2020). Genetic pools can be unique to localities even within small areas, such as the wild populations within 1 km of each other in the Singapore Botanic Gardens primary rainforest that reinforces conclusions derived from studies of other Southeast Asian trees (Tito de Morais et al. 2015, Smith et al. 2018). This suggests that non-random distribution of genetic variation may have been influenced by pre-existing natural barriers to gene flow, which could be exacerbated by fragmentation, shaping the genetic patterns observed in degraded habitats (Hamrick, 2010; Browne et al. 2015).

Limited pollen- and seed-mediated gene flow are likely contributors to increased genetic erosion in fragmented habitats (Nistelberger et al. 2015). Loss of genetic diversity among recruits found directly beneath the adult canopy might occur if pollen dispersal is disrupted by fragmentation (Collevatti and Hay 2011), leading to higher self- or sibling-breeding and reduced survival of inbred progeny (Nutt et al. 2016). Genetic differentiation among more distantly dispersed recruits is more likely due to inhibited seed dispersal (Browne et al. 2015). Taking into account previous findings attributing pollination and seed dispersal failure to landscape degradation (Carvalheiro et al. 2010; Wee et al. 2015; Bovo et al. 2018), our study also suggests that both pollen and seed dispersal processes have been affected negatively by habitat fragmentation, given the high levels of relatedness between recruits directly beneath the adult, as well as between dispersed recruits found in BTNR and BBNP despite the proximity of these sites (within 2 km), which may reflect the negative effects of limited pollen- and seed-dispersal on genetic diversity.

Known vectors of seed dispersal include fruit bats, squirrels and monkeys, which consume the fleshy fruits of Palaquium (Ng 1972; Soerianegara and Lemmens 1993). Changes in frugivore communities as a result of habitat loss would thus affect the dispersal dynamics of plants with sizable seeds (Mittelman et al., 2020; Escobar et al. 2023), such as in Palaquium. While the full range of pollinators is not known, P. obovatum was found to exhibit chiropterophilous characteristics with very brief periods of flowering (around 24 h; Phang 2023) coinciding with extensive bat visitation. Aguilar et al. (2019) observed that habitat fragmentation had fewer negative effects in terms of genetic erosion on the progeny of vertebrate-pollinated species than wind and insect-pollinated species, as the greater flight range of pollinators such as birds and bats allowed more movement across an urban landscape. In Singapore, pollination efficiency is likely to have been eroded by a dramatic decline in the abundance and species richness of bats, resulting in estimated local extinctions of 33–72% of species, with forest-dependent bats particularly affected (Lane et al. 2006). Although Cynopterus brachyotis, the bat species observed visiting P. obovatum flowers (Phang 2023) persists, foraging is preferred near their roosts, and they readily visit the flowers of non-native species in urbanised sites (Chan et al. 2021), which may reduce the likelihood that they exploit food sources arising from ephemeral Palaquium flowering. The effectiveness of seed dispersal and seedling establishment may also decline where bat roosts occur over non-natural areas sealed by concrete and human traffic (Vendan and Kaleeswaran 2011; Chan et al. 2021). Furthermore, Cynopterus brachyotis may have divergent lineages specific to forests or disturbed habitats (Campbell et al., 2004), and only the latter has so far been confirmed for Singapore (Lane et al. 2006). Other bat species dependent on intact mature forests (Balionycteris maculata, Chironax melanocephalus, Megaerops ecaudatus, Megaerops wetmorei, Cynopterus horsfieldii) have been extinct in Singapore for more than a hundred years.

In the only other study investigating possible genetic erosion of a forest tree species in Singapore, Noreen and Webb (2013) found high genetic diversity in Koompassia malaccensis, with connectivity between BTNR, CCNR and the Singapore Botanic Gardens facilitated by a mobile pollinator, the giant honeybee Apis dorsata (Noreen et al. 2016). However, there were already signs of increased relatedness between individuals at short distances. While reviews of key forest fragments, such as the well-studied primary forest plot in BTNR, have indicated relative resilience in tree species diversity and biomass (LaFrankie et al., 2005; Lum and Ngo 2021), the observed lack of old mature trees and seedlings, as well as evidence of genetic degradation in P. obovatum, might suggest that cryptic loss of genetic diversity may be accumulating through time, resulting in lagged effects contributing to a potential extinction debt. The species composition of secondary forests in Singapore displays evidence of strong dispersal limitations and poor convergence to the original primary forest species, including significantly lower seedling density (Goldsmith et al. 2011; Chua et al. 2013; Corlett 2022). These observations suggest that natural regeneration mechanisms may have been disrupted, especially in rarer species within genera like Palaquium (Wong et al. 1994; LaFrankie 1996). Other native representatives of the Sapotaceae family across Southeast Asia, as well as species in numerous other families, are likely to be even more prone to the effects of fragmentation, as most flower and fruit less often than P. obovatum.

Conservation implications

To improve the long-term survival of Palaquium obovatum in isolated forest patches, we suggest the following conservation measures: (1) As P. obovatum is a dispersal-limited species in need of restoration efforts (Martínez-Garza and Howe 2003) to maintain gene flow across patchy distributions (Finger et al. 2011), ex situ populations should be established to ensure long-term persistence of the species, and the material to establish such populations should be collected from a wide range in order to adequately capture most of the existing genetic diversity in the wild (Wei and Jiang 2021). Seedlings can then be transplanted into botanical gardens or suitable sites as our results suggested that cultivated populations can have high levels of diversity, preserving the genetic diversity of native wild populations across Singapore. (2) Plant-pollinator and seed dispersal mutualisms should also be restored, including a continuation of efforts to build green corridors to facilitate fauna movements (Chan and Davison 2019), and the possible establishment of ex situ populations near known bat roosts.

Conclusions

In this paper we report the first population genomics study in the Isonandreae tribe of the Sapotaceae family, and also one of the first to use NGS methods to estimate gene flow and genetic diversity between different age classes of a forest tree species in Southeast Asia. Contrary to Noreen and Webb (2013), we present novel evidence that contemporary disturbances and habitat fragmentation have led to reduced genetic diversity and increased inbreeding in wild populations across primary and secondary forests, an outcome likely supported by the decline of bat species functioning as key pollinators for P. obovatum. The strong differentiation and genetic structure between localities indicate limited directional gene flow within a small geographical location, which raises concerns for overall population health.

Singapore is expected to face a period of ‘extinction debt’ with delayed but likely extinctions (Niissalo et al., 2018). If small, isolated populations like Palaquium obovatum and similar species are not regenerating in situ or dispersed effectively, even if they persist in the short-term, they are in fact already ‘living dead’ (Guimarães et al. 2008; McConkey et al. 2012; Aguilar et al. 2019), unless conservation measures are proactively undertaken to preserve valuable germplasm across the landscape, as well as to increase the connectivity of natural habitats.