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Characterization of the complete chloroplast genome sequence of Dalbergia species and its phylogenetic implications

The pantropical plant genus Dalbergia comprises approximately 250 species, most of which have a high economic and ecological value. However, these species are among the most threatened due to illegal logging and the timber trade. To enforce protective legislation and ensure effective conservation of Dalbergia species, the identity of wood being traded must be accurately validated. For the rapid and accurate identification of Dalbergia species and assessment of phylogenetic relationships, it would be highly desirable to develop more effective DNA barcodes for these species. In this study, we sequenced and compared the chloroplast genomes of nine species of Dalbergia. We found that these chloroplast genomes were conserved with respect to genome size, structure, and gene content and showed low sequence divergence. We identified eight mutation hotspots, namely, six intergenic spacer regions (trnL-trnT, atpA-trnG, rps16-accD, petG-psaJ, ndhF-trnL, and ndhG-ndhI) and two coding regions (ycf1a and ycf1b), as candidate DNA barcodes for Dalbergia. Phylogenetic analyses based on whole chloroplast genome data provided the best resolution of Dalbergia, and phylogenetic analysis of the Fabaceae showed that Dalbergia was sister to Arachis. Based on comparison of chloroplast genomes, we identified a set of highly variable markers that can be developed as specific DNA barcodes.

their potential value as effective barcodes for Dalbergia. Accordingly, there exists a need to develop more effective genetic markers for Dalbergia in order to assess phylogenetic relationships and facilitate rapid and accurate species identification.
Chloroplast genome sequences have been demonstrated to be effective molecular resources that can be applied in species identification and phylogenetic studies 11 . In most angiosperms, the chloroplast genomes have a circular structure and are composed of four regions, namely, two inverted repeat regions that separate the remainder of the genome into a large single-copy (LSC) and a small single-copy (SSC) region 12 . Angiosperm chloroplast genomes typically range in size from 115 to 165 kb and contain approximately 130 genes, among which there are 80 protein-coding genes, four rRNA genes, and 30 tRNA genes. Owing to their slower evolution than that of nuclear genomes, lack of recombination, and general uniparental inheritance, chloroplast genome sequences are a primary source of data for species identification and inferring plant phylogenies 13,14 . Most relevant studies have revealed that chloroplast genomes are characterized by distinct clusters of mutations, known as "hotspots, " or highly variable regions, which can serve as DNA markers for the accurate identification of plant species. We thus reasoned that a comparative study of the chloroplast genomes of Dalbergia species might provide potentially useful insights for developing DNA barcodes that could be applied to facilitate the reliable identification of these species.
In this study, we accordingly sequenced the chloroplast genomes of nine Dalbergia species using the Illumina HiSeq X platform. Our main goals were to (1) evaluate the interspecific variation among chloroplast genomes within the genus Dalbergia, (2) provide information regarding the most suitable chloroplast molecular markers for species identification, and (3) infer the chloroplast phylogenomic relationships of Dalbergia. We believe that the findings of this study will provide valuable information for determining the phylogenomics of Dalbergia species as well as facilitating the identification and phylogeographic characterization of these species, thereby making an important contribution toward the development of conservation strategies for endangered Dalbergia species.

Materials and Methods
Plant materials and DNA extraction. According to the phylogeny of the Dalbergia 1,2,15 , we selected nine Dalbergia species at different divergence levels. We collected fresh healthy leaves (those lacking any apparent disease symptoms) from nine Dalbergia species growing in the Jianfengling Nature Reserve, Hainan Province, South China. These leaves were immediately dried using silica gel prior to DNA extraction. Total genomic DNA was isolated from all samples following the method described by Li et al. 16 .
Illumina sequencing, assembly, and annotation. Using an ultrasonicator, approximately 5-10 μg of total DNA was sheared into 350-bp fragments. Paired-end (2 × 150 bp) sequencing was performed by Novogene Bioinformatics Technology Co. Ltd (Beijing, China), using the Illumina Hiseq X-Ten platform, and generated approximately 4.0 Gb of raw data for each sample.
For raw data processing, we used Trimmomatic v 0.32, and the resulting clean data were used for assembly and post analysis 17 . The clean reads were assembled using SPAdes 3.6.1 18 with different K-mer parameters. Chloroplast genome contigs were selected based on BLAST searches, using the published Dalbergia odorifera chloroplast genome sequence (GenBank accession number: MF668133) as a reference. The selected contigs were secondarily assembled using Sequencher 5.4.5 (Gene Codes, Ann Arbor, MI).
The IR-SC boundaries and the gaps between the contigs were amplified and sequenced using specific primers. Chloroplast genome annotation was performed with Plann 19 using the Dalbergia odorifera reference sequence and then manually corrected. The complete assembled chloroplast genome sequences of the nine Dalbergia species were submitted to GenBank with the accession numbers MN251241 to MN251249. A chloroplast genome map was drawn using OGDRAW software 20 .
Analysis of tandem and single sequence repeats. Simple sequence repeats in the nine Dalbergia chloroplast genomes were detected using GMAT 21 with the minimal repeat number set to 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexanucleotide sequences, respectively. Five types of repeat sequences, namely, forward, reverse, complementary, palindromic, and tandem repeats, were identified in the Dalbergia chloroplast genome. Forward, reverse, palindrome, and complementary sequences were determined by running the REPuter program 22 with a minimum repeat size of 30 bp and similarities of 90%. Tandem repeats were identified using Tandem Repeats Finder (https://tandem.bu.edu/trf/trf.html), with alignment parameters being set to 2, 7, and 7 for matches, mismatches, and indels, respectively.

Sequence divergence analysis and divergent hotspot identification. The sequences of all nine
Dalbergia chloroplast genomes were aligned using MAFFT v7 23 , and then adjusted manually using Se-Al 2.0 24 . MEGA 7.0 software 25 was used to calculate the variable and parsimony-informative base sites and the k2p-distances among the chloroplast genomes.
To identify rapidly evolving molecular markers that can be used in further phylogenetic studies, we conducted a sliding window analysis using DnaSP v5.10 software 26 , with the step size and window length set to 200 and 800 bp, respectively.
We evaluated the hypervariable barcodes and compared the chloroplast genes rbcL, matK, and trnH-psbA using tree-based methods. Neighbor joining (NJ) trees were constructed for each hypervariable marker and different marker combinations using MEGA 7.0 based on a k2p-distance model. The relative support for branches of the NJ tree was assessed via 1000 bootstrap replicates.
Phylogenetic reconstruction. We inferred phylogenetic relationships within the family Fabaceae by constructing a maximum likelihood tree based on the sequences of 81 protein-coding genes. For phylogenetic reconstruction, we used 71 species from the family Fabaceae and one species from the family Moraceae as an outgroup (Table S1). The protein-coding genes were extracted from the GenBank formatted file containing all chloroplast genomes using Geneious v11, and gene alignment was performed using MAFFT v7 23 . The data for whole chloroplast genome sequences were used to infer the phylogenetic relationship among Dalbergia species.
The concatenated data were analyzed using maximum likelihood and Bayesian inference methodologies. Prior to maximum likelihood and Bayesian analyses, a general time reversible and gamma distribution (GTR + G) model was selected using the ModelFinder v.1.6 27 under the Akaike Information Criterion. Maximum likelihood analyses were performed using RAxML v.8.1 28 . Nodes supports was calculated via rapid bootstrap analyses with 1000 replicates.For Bayesian analysis, we used MrBayes v.3.2 29 in the CIPRES Science Gateway. The Markov chain Monte Carlo algorithm was run for ten million generations, with trees sampled every 1000 generations and the first 25% of generations discarded as burn-in. The remaining trees were used to construct a 50% majority-rule consensus tree and estimate posterior probabilities. Posterior probabilities (PP) > 0.95 were considered significant support for a clade.

Results
Genome sequencing and assembly. Illumina paired-end sequencing produced between 32,198,750 and 43,895,392 paired-end clean reads per species. On the basis of BLAST searches, the contigs mapped to D. odorifera chloroplast genome sequence were then used for reconstructing the chloroplast genomes of the nine examined Dalbergia species. After screening these paired-end reads through alignment with D. odorifera chloroplast genome using Geneious V9, 198,763 to 1,744,392 chloroplast genome reads were extracted with 191 × (D. hainanensis) to 1,677 × (D. tonkinensis) coverage (Table S2).  Table 1). Furthermore, the nine genomes were found to have similar GC contents, ranging from 35.9% to 36.1%.
Considering only single copies of the duplicated genes in the IR regions, we detected a total of 110 different genes, comprising 76 protein-coding genes, 30 tRNAs, and 4 rRNAs in each of the Dalbergia chloroplast genomes. Gene number, order, and type were found to be very similar among the nine Dalbergia species. Furthermore, we identified 15 duplicated genes in the IR regions, among which there are seven tRNA gene, four rRNA genes, and four protein-coding genes. Eighteen genes were found to harbor introns (one class I intron in the trnL-UAA region and 17 class II introns), among which, 14 genes have only a single intron, whereas ycf3 and clpP each contain two introns. The trnK-UUU region containing the matK gene has the largest intron (2,618-2,648 bp).

Analysis of repeat elements.
A total of 148-178 SSR loci were detected in the nine Dalbergia chloroplast genomes ( Fig. 2A). These SSR loci are located primarily in the LSC region (76.3-86.9%), followed by the SSC region (19.1-23.7%, Fig. 2B). Mono-, di-, tri-, tetra-, penta-, and hexanucleotide SSRs were detected in each of the nine species, with the average percentages of mono-, di-, tri-, and tetranucleotide SSRs being 71.83%, 21.69%, 2.52%, and 3.81%, respectively. In all the sequenced genomes, we found pentanucleotide SSRs to be very rare (Fig. 2C), and were unable to detect any hexanucleotide SSR in these genomes. SSRs in the Dalbergia chloroplast genomes were found to be particularly rich in AT sequences and rarely contain CG (Fig. 2D). A majority of the SSRs (68.08%) are mononucleotide A/T repeats, with only two C/G mononucleotide SSRs being detected per genome, and the majority of the dinucleotides are composed of AT and TA. An AATAA pentanucleotide SSR was found only in D. odorifera, whereas TTTTA repeat units were only detected in D. oliveri.
Sequence divergence. The chloroplast genomes of the nine Dalbergia were fully aligned with an alignment matrix of 165,085 bp. The alignment revealed a high degree of sequence similarity across the Dalbergia species, suggesting that the sequences are highly conserved. We searched for nucleotide substitutions and determined k2p-distances in each of the chloroplast genomes, and accordingly detected 4,071 variable sites (0.41%), including 2,663 parsimony-informative sites (0.27%), across the nine chloroplast genomes. We found that the average value for nucleotide diversity (pi) was 0.00858, and a comparison nucleotide diversity in the LSC, SSC, and IR regions of the Dalbergia chloroplast genomes, revealed that the SSC region exhibits the highest nucleotide diversity (0.01718), whereas the IR regions show the least divergence (0.00146).
In addition, we detected variation in the number of nucleotide substitutions and k2p-distances among the nine Dalbergia species (Table S3). In pairwise comparisons of the nine species, the k2p-distances were observed to range from 24 to 1,978, and the number of nucleotide substitutions ranged from 0.0002 to 0.0129. The D. hupeana and D. cochinchinensis pair showed the lowest sequences divergence, whereas the D. sissoo and D. tonkinensis pair showed the largest divergence.
Phylogenomic analysis. In this study, we used 81 protein-coding genes to calibrate the phylogenetic position of Dalbergia in the Fabaceae (Table S1), and used the complete chloroplast genome sequences to examine the feasibility of reconstructing the phylogeny of Dalbergia. We found that the phylogenetic relationships determined based on the 81 protein-coding genes using the maximum likelihood approach were identical to those obtained  www.nature.com/scientificreports www.nature.com/scientificreports/ using Bayesian inference analysis, the results of which are presented in Figs. 5 and S2. Most nodes were validated with maximum support (1.00 posterior probability, 100% bootstrap support). We found that Cercidoideae and Caesalpinioideae + Papilionoideae + Detarioideae formed sister groups, whereas Dalbergia and Arachis formed a clade, although the position of this clade was uncertain owing to lower bootstrap support and posterior probability values (77% bootstrap support, 0.98 posterior probability). As shown in previous studies [30][31][32] , we that found terminal branches were well supported, whereas in contrast, the internal nodes tended to have poorer bootstrap support, an indication of rapid radiation.
Overall, we found that phylogenetic analyses based on both 81 protein-coding genes (Fig. 5) and complete chloroplast genomes (Fig. S3)

Discussion
Chloroplast genome evolution in Dalbergia. Next-generation sequencing methods have enabled the rapid and cost-efficient sequencing of plant genomes. In this study, we used these methods to sequence the chloroplast genomes of nine Dalbergia species. These genomes were found to have the typical stable quadripartite structure, namely a pair of IRs separating the LSC and SSC regions 33 . The Dalbergia chloroplast genomes range in size from 155,726 to 156,698 bp, which is within the range of the previously sequenced angiosperm chloroplast genomes 14,34 . Among the nine selected Dalbergia species, we found that all the sequenced chloroplast genomes encode the same set of 110 unique genes in a uniform gene order, thereby indicating the highly conserved nature of these genomes.
Indeed, the divergence among the Dalbergia chloroplast genome was found to be lower than that reported for other plant species, such as Paris 35,36 , Lilium 37 , and Oryza 38,39 , with an average k2p-distance of 0.0092 (range: 0.0002 to 0.0129) (Table S3). In this regard, previous studies have demonstrated that species with short generation times tend to evolve more rapidly and exhibit fast evolutionary rates 40,41 . The relatively long life cycles of Dalbergia species can thus probably explain the slower evolution of the chloroplast genomes of these species. As expected, the IR and coding regions are more highly conserved than the LSC and SSC regions and non-coding regions, as has been observed in other flowering plants 42 .   6 , and since that time an increasing number of researchers have focused on the selection of one or a few standard markers as DNA barcodes. For example, the two chloroplast-encoded genes rbcL and matK are considered core barcodes for land plants 43 , along with two supplementary non-coding regions, namely, the plastid trnH-psbA intergenic spacer and the internal transcribed spacer (ITS) from the nuclear ribosomal DNA 44 . However, despite their broad utility, these markers have been demonstrated to have extremely low discriminatory power in certain plant groups [45][46][47] . Bhagwat, et al. 7 and Hartvig, et al. 8 have previously examined the success rate of Dalbergia species identification using standard DNA barcodes, and found that these barcodes have good discriminatory ability. However, these authors examined only a relatively few Dalbergia species (31/250 or 10/250) in those studies. When a larger number of species has been sampled, the number of successful identifications has been found to decrease significantly. For example, a success rate of only 10-33% was obtained at the species level when using 121 Dalbergia samples collected in Madagascar 2 . Therefore, the screening and validation of more highly variable markers are considered to be priority prerequisites with respect to Dalbergia DNA barcoding.
It is known that there are certain mutation hotspot regions within chloroplast genomes that are associated with high numbers of SNPs, and are accordingly defined as highly variable markers. On the basis of a comparison the nine Dalbergia chloroplast genomes, we identified eight such highly variable regions, namely, six intergenic markers (trnL-trnT, atpA-trnG, rps16-accD, petG-psaJ, ndhF-trnL, and ndhG-ndhI) and two genic makers (ycf1b www.nature.com/scientificreports www.nature.com/scientificreports/ and ycf1a) (Fig. 4). Among these, the ycf1 gene, which encodes the Tic214 protein that is essential for plant viability, is the second largest in the chloroplast genome, and has recently been assessed for its DNA barcoding potential. Dong et al. 11,48 have proposed that the two highly variable regions ycf1a and ycf1b are the most variable loci within the chloroplast genome, showing greater variability than the existing chloroplast candidate barcodes (such as rbcL, matK and trnH-psbA), and thus might have potential utility as DNA barcodes for land plants.
The intergenic spacer regions trnL-trnT, in conjunction with universal primers, have a long history of use in plant phylogenetic studies 49,50 , and it has been reported that the trnL-trnT spacer has greater variation than either the trnL-F spacer and trnL intron 51 . However, these spacers often contain large A/T-rich regions that may lead a low sequence quality 52 . In the present study, however, we detected poly C and poly T structures within these regions in the Dalbergia chloroplast genome.
The atpA-trnG region consists of two intergenic spacers, atpA-trnR and trnR-trnG. ndhG-ndhI, located within the SSC, with an average length of 1,308 bp (range:1,281-1,377 bp) is the most highly variable marker in the Dalbergia chloroplast genome (Fig. 4). Four large indels were observed in Dalbergia. The atpA-trnG, petG-psaJ, and ndhG-ndhI markers have previously been little used in plant phylogenetic studies and DNA barcoding. The rps16-accD intergenic spacer, which contains a 50-kb inversion between rps16-trnQ and rbcL-accD regions, is specific to Papilionoid chloroplast genomes 53 .
The ndhF-trnL region includes two intergenic spacers (ndhF-rpl32 and rpl32-trnL) in the SSC region of the chloroplast genome. This region has previously been shown to have a high level of positional variability by Shaw et al. 52,54 and Dong et al. 11 , and is probably the best marker for molecular studies at low taxonomic studies. This region is approximately 2 kb in size and harbors a number of variable and informative sites ( Table 2), which may represent the best molecular markers for investigations in Dalbergia. Therefore, although we have identified a number of candidate barcoding regions, further research is still necessary to determine whether these highly divergent markers could be used in the identification of Dalbergia species.

conclusion
In this study, we sequenced and compared the chloroplast genomes of nine Dalbergia species. The structure, size, and gene contents of the Dalbergia chloroplast genomes were found to be well conserved, and comparative analyses revealed low levels of sequence variability. Mononucleotide SSR and tandem repeats were observed abundantly in the Dalbergia chloroplast genomes. In addition, the SSRs identified herein should be useful in characterizing the population genetic structure of Dalbergia species. Moreover, we identified eight mutation hotspot regions with potential utility as DNA barcodes for Dalbergia species identification. These highly variable markers and the whole chloroplast genome sequences provided sufficient genetic information for species identification and phylogenetic reconstruction of the genus Dalbergia.

Data availability
The datasets generated for this study can be found in GenBank with the accession numbers MN251241-MN251249.