Comparative Plastome Analyses and Phylogenetic Applications of the Acer Section Platanoidea

: The Acer L. (Sapindaceae) is one of the most diverse and widespread genera in the Northern Hemisphere. Section Platanoidea harbours high genetic and morphological diversity and shows the phylogenetic conﬂict between A. catalpifolium and A. amplum . Chloroplast (cp) genome sequencing is e ﬃ cient for the enhancement of the understanding of phylogenetic relationships and taxonomic revision. Here, we report complete cp genomes of ﬁve species of Acer sect. Platanoidea . The length of Acer sect. Platanoidea cp genomes ranged from 156,262 bp to 157,349 bp and detected the structural variation in the inverted repeats (IRs) boundaries. By conducting a sliding window analysis, we found that ﬁve relatively high variable regions ( trnH - psbA , psbN - trnD , psaA - ycf3 , petA - psbJ and ndhA intron) had a high potential for developing e ﬀ ective genetic markers. Moreover, with an addition of eight plastomes collected from GenBank, we displayed a robust phylogenetic tree of the Acer sect. Platanoidea, with high resolutions for nearly all identiﬁed nodes, suggests a promising opportunity to resolve infrasectional relationships of the most species-rich section Platanoidea of Acer .


Introduction
Chloroplasts (cp) are essential organelles in plant cells for the processes of photosynthesis and carbon fixation [1]. They possess uniparental inheritance and their genome has a high conservation structure in most land plants [2]. Generally, cp genomes in most angiosperms are circular DNA molecules composed of four parts, namely two inverted repeats (IRs) at approximately 20-28 kb, a vast single-copy region (LSC) at 80-90 kb and a small single-copy region (SSC) at 16-27 kb [3]. The composition of angiosperm cp genomes is relatively conserved and encodes four ribosomal RNAs (rRNAs), roughly 30 transfer RNAs (tRNAs) and approximately 80 single-copy genes [4]. With the rapid development of next-generation sequencing (NGS) and other methods for obtaining the cp genome sequences, the availability of cp genome sequences has increased dramatically for land plants, offering opportunities for the comprehensive structure comparison, improvement of horticultural plant breeding [5,6] and reconstruction of evolutionary relationships [7,8]. In most angiosperms, the cp genome is inherited from the patrilineal lineage and exhibits little or no recombination [9]. Due to its relatively conserved features, cp sequences are commonly used as DNA barcodes for genetic identification, plant systematic studies, and research into plant biodiversity, biogeography, adaptation, etc. [10,11].
Acer L. (Maple), a diverse genus of family Sapindaceae L., contains more than 124 species [12]. Most extant species of the genus are native to Asia, whereas others occur in North America, Europe and North Africa [12][13][14]. Most Acer species are famous ornamental plants [13], and also another usage for pharmaceutical and chemical products [15]. To date, 17 species belonging to the Acer section Platanoidea have been recognised in China, in which the section shows high genetics and morphology diversity. [12]. Among them, four species are widespread in various vegetation regions (A. amplum, A. longipes, A. mono and A. truncatum) [16], and some are endangered, such as A. catalpifolium, A. miaotaiense, and A. yangjuechi.
However, some species with diversified leaf morphology have taxonomic controversy due to unresolved phylogenetic relationships (for example, A. longipes and A. amplum) and require further studies and clarification [17]. The comparative plastome analysis allows detailed insights to affirm the phylogenetic placement of these plants and will be useful for species identification, to verify taxonomic levels and identify phylogenetic relationships [8,18]. Recently, cp genomes of A. miaotaiense and A. truncatum have been reported, but merely the sequence information was provided without further analyses [19,20]. Thus, a comparative study among these two published cp genomes and five newly generated plastomes of sect. Platanoidea is conducted and applied to address phylogenetic and taxonomic validity.
Firstly, we reported newly completed cp genomes of five species of the Acer sect. Platanoidea (A. catalpifolium, A. amplum, A. longipes, A. yanjuechi and A. mono). Then, we compared the gene contents and the plastomic organisation with two published cp genomes in the sect. Platanoidea to identify variable loci that can apply to the species or population-level studies on Acer. The aims of this study are: (i) to deepen our understanding of the genetic and structural diversity within the sect. Platanoidea, (ii) to increase our understanding of phylogenetic relationships of species within the sect. Platanoidea, and (iii) to reconstruct a phylogenetic tree based on these plastomes. Our study also provides genetic resources for future research in this genus.

Sampling and DNA Extraction
Young leaves of five Acer species (A. catalpifolium, A. amplum, A. longipes, A. yanjuechi and A. mono) were collected and dried immediately with silica gel for preservation, for each species we collected the leaves from one healthy plant. Collection information of the plant materials is showed in Table S1. Vouchers taxonomical determination was by the Beijing Forestry University herbarium and deposited at the College of Forestry, Beijing Forestry University, China. We isolated total genomic DNA from silica gel-dried leaves according to the modified CTAB method [21].

Divergence Hotspot Identification
In order to determine the divergence level, a MAFFT: multiple sequence alignment program [27] was used to align cp sequences of seven Acer sect. Platanoidea species, and then sliding windows of the nucleotide variability (pi) was conducted using DnaSP 5.0 with 600-bp window length and 200-bp step size [28].

Phylogenomic Reconstruction
To determine the phylogenetic relationship of the Acer sect. Platanoidea, we performed phylogenetic analyses using 13 cp genome sequences, which comprised five plastome sequences generated in this study, six plastomes of the Acer species collected from GenBank, and two of the Dipteronia species as an outgroup (Table S2). Consequently, a total length of 160,886 bp was aligned using MAFFT [27]. The best-fitting substitution model (GTR + I + G) was inferred using Modeltest 3.7 [29]. Finally, phylogenomic relationships were reconstructed with Bayesian Inference (BI) and Maximum Likelihood (ML) using MrBayes 3.2 [30] and phyML v3.0 [31], respectively. For the BI tree, 10 million generations were simulated using two parallel Markov Chain Monte Carlo (MCMC) simulations, sampled every 1000 generations. The first 25% of the simulations were discarded (burn-in) to generate a consensus tree. For the ML tree, 1000 bootstrap replicates were conducted to evaluate the supporting values of each node.

Comparative Analysis of the Genomic Structure
Contraction and expansion of the IRs, LSC and SSC are important to the evolution of cp genomes [33,34], which is the leading cause of gene order and the size changes of the cp genome [35]. Detailed structure comparisons among the 13 cp genomes of the Acer species were presented in Figure 2.  rpl22 and rps19 gene order changes in the IRA/LSC borders were also observed and has become the most varied rearrangement in this section [36]. Similarly, the rearrangement has been reported in the Apiales species, which also has two structure types of rpl23 and rps19 in the LSC/IR boundary [37]. Length variations of the Acer cp genomes were found by contraction and expansion of the LSC and IRs, the length of the LSC varies from 85,379 bp to 86,327 bp, and the length of the IR varies from 26,085 bp to 26,769 bp ( Figure 2). Moreover, the type of unique structural borders of the cp genome (A. truncatum, A. miaotaiense, and A. buergerianum) also show a contraction of IRs, which indicates variations in boundary genes may be caused by variations in length.

Comparative Analysis of the Genomic Structure
Contraction and expansion of the IRs, LSC and SSC are important to the evolution of cp genomes [33,34], which is the leading cause of gene order and the size changes of the cp genome [35]. Detailed structure comparisons among the 13 cp genomes of the Acer species were presented in Figure 2. rpl22 in the LSC/IR boundary and rps19 was the last gene in most Acer sect. Platanoidea cp genomes included A. mono, A. amplum, A. catalpifolium, A. yangjuechi, A. longipes, A. morrisonense, A. davidii and A. griseum. However, the different structures of rps19 in the LSC/IR boundary and the last gene rpl2 were found in A. truncatum, A. miaotaiense and A. buergerianum. Among the Arecoideae species, rpl22 and rps19 gene order changes in the IRA/LSC borders were also observed and has become the most varied rearrangement in this section [36]. Similarly, the rearrangement has been reported in the Apiales species, which also has two structure types of rpl23 and rps19 in the LSC/IR boundary [37]. Length variations of the Acer cp genomes were found by contraction and expansion of the LSC and IRs, the length of the LSC varies from 85,379 bp to 86,327 bp, and the length of the IR varies from 26,085 bp to 26,769 bp ( Figure 2). Moreover, the type of unique structural borders of the cp genome (A. truncatum, A. miaotaiense, and A. buergerianum) also show a contraction of IRs, which indicates variations in boundary genes may be caused by variations in length.

Divergence Hotspot of the Acer sect. Platanoidea Species
Sliding window analysis of the whole cp genome was performed to identify hotspots of the Acer sect. Platanoidea species. In Figure 3, it is apparent that the trnH-psbA, psbN-trnD, psaA-ycf3, petA-psbJ and ndhA intron nucleotide variability was higher than other regions. Most divergent hotspot loci are located in the LSC region, which allows for the proper design of the genetic markers. Only one hotspot ndhA intron was located in the SSC region. The IR regions were much more conserved. This result Forests 2020, 11, 462 6 of 10 was similar to other cp genomes [37,38]. The general barcode trnH-psbA has demonstrated extreme variation in plant groups [39,40]. Thus, the highest variation trnH-psbA has the potential to be used in DNA barcoding in the Acer sect. Platanoidea. Additionally, the evolutionary history of A. mono has been inferred by using psbA-trnH, trnL-trnF and an intron of rpl16 [16]. The regions of the psaA-ycf3, petA-psbJ and ndhA intron have been indicated as high variations in previous studies. In witch-hazel (genus Hamamelis L., Hamamelidaceae), appropriate variations of psaA-ycf3 were used to reconstruct phylogenetic relationships [41]. In Scutellaria, petA-psbJ was one of six fast-evolving DNA sequences in the cp genome [42], while a systematic study shows that Muhlenbergiinae has high variation at the ndhA intron [43].
are located in the LSC region, which allows for the proper design of the genetic markers. Only one hotspot ndhA intron was located in the SSC region. The IR regions were much more conserved. This result was similar to other cp genomes [37,38]. The general barcode trnH-psbA has demonstrated extreme variation in plant groups [39,40]. Thus, the highest variation trnH-psbA has the potential to be used in DNA barcoding in the Acer sect. Platanoidea. Additionally, the evolutionary history of A. mono has been inferred by using psbA-trnH, trnL-trnF and an intron of rpl16 [16]. The regions of the psaA-ycf3, petA-psbJ and ndhA intron have been indicated as high variations in previous studies. In witch-hazel (genus Hamamelis L., Hamamelidaceae), appropriate variations of psaA-ycf3 were used to reconstruct phylogenetic relationships [41]. In Scutellaria, petA-psbJ was one of six fast-evolving DNA sequences in the cp genome [42], while a systematic study shows that Muhlenbergiinae has high variation at the ndhA intron [43].
The endangered plants, A. catalpifolium, A. yangjuechi and A. miaotaiense in the Acer sect. Platanoidea, have small population sizes [44]. Population genetics studies of these species are relatively weak, and with limited conservation goals [45,46]. These high variability regions can provide alternative sites for subsequent studies and will contribute to the conservation of endangered plants.

Phylogenetic Analysis
The phylogenomic inference based on cp genomes shows high bootstrap supports in most nodes that provided robust evolutionary placement and relationship of the Acer species (Figure 4). The results showed that seven sampled species of the Acer sect. Platanoidea formed a single clade, which is consistent with previous studies of the Acer phylogeny [14,47]. In this clade, A. catalpifolium, A. mono, A. miaotaiense and A. yangjuechi had the closest phylogenetic relationship and formed a subclade, while A. truncatum, A. longipes, A. amplum formed another one. Previous phylogenetic inference of the Acer did not contain species with a small population size in the sect. Platanoidea (such as A. catalpifolium and A. yangjuechi) [14,47]. Phylogenetic analysis exhibited in this study for the issue of A. longipes A. amplum complex in the sect. Platanoidea raised earlier [17], and the phylogenetic position of A. catalpifolium was also redefined.
It is somewhat surprising that A. mono is not a sister with A. truncatum but with A. yangjuechi, which differs from some published studies [13]. Since A. mono is widespread in Asia and comprised The endangered plants, A. catalpifolium, A. yangjuechi and A. miaotaiense in the Acer sect. Platanoidea, have small population sizes [44]. Population genetics studies of these species are relatively weak, and with limited conservation goals [45,46]. These high variability regions can provide alternative sites for subsequent studies and will contribute to the conservation of endangered plants.

Phylogenetic Analysis
The phylogenomic inference based on cp genomes shows high bootstrap supports in most nodes that provided robust evolutionary placement and relationship of the Acer species (Figure 4). The results showed that seven sampled species of the Acer sect. Platanoidea formed a single clade, which is consistent with previous studies of the Acer phylogeny [14,47]. In this clade, A. catalpifolium, A. mono, A. miaotaiense and A. yangjuechi had the closest phylogenetic relationship and formed a subclade, while A. truncatum, A. longipes, A. amplum formed another one. Previous phylogenetic inference of the Acer did not contain species with a small population size in the sect. Platanoidea (such as A. catalpifolium and A. yangjuechi) [14,47]. Phylogenetic analysis exhibited in this study for the issue of A. longipes A. amplum complex in the sect. Platanoidea raised earlier [17], and the phylogenetic position of A. catalpifolium was also redefined.
It is somewhat surprising that A. mono is not a sister with A. truncatum but with A. yangjuechi, which differs from some published studies [13]. Since A. mono is widespread in Asia and comprised of multiple local varieties, we cannot rule out that the possibility of the grouping of A. mono and A. yangjuechi due to adjacent sampling localities of A. mono and A. yangjuechi in Lin'an, Zhejiang province of China, the only extant habitat of A. yangjuechi [48]. Liu et al. [49] showed that the population composition of the Lin'an population is significantly different from the neighbouring populations of A. mono. The clustering of A. mono and A. yangjuechi inferred in this study may not only reflect the truth of geographic divergence in the genetics of A. mono but also implies the chloroplast capture by ancient hybridisation events between the two species in Lin'an.
yangjuechi due to adjacent sampling localities of A. mono and A. yangjuechi in Lin'an, Zhejiang province of China, the only extant habitat of A. yangjuechi [48]. Liu et al. [49] showed that the population composition of the Lin'an population is significantly different from the neighbouring populations of A. mono. The clustering of A. mono and A. yangjuechi inferred in this study may not only reflect the truth of geographic divergence in the genetics of A. mono but also implies the chloroplast capture by ancient hybridisation events between the two species in Lin'an.

Conclusions
In this study, we firstly reported complete cp genomes of five Acer sect. Platanoidea species (A. catalpifolium, A. amplum, A. longipes, A. yanjuechi and A. mono) using the NGS technology. In comparison with other published Acer species from NCBI, we found that the Acer species have similar cp genome structure and gene content. The divergence hotspots identified in the cp genome of the Acer sect. Platanoidea could be applied to develop molecular markers for further population genetics studies. The high variation at the IR/LSC and IR/SSC boundaries were also reported. The phylogenetic analysis strongly supported that A. catalpifolium has the closest relationship with A. miaotaiense, followed by A. mono, and A. yanjuechi, which confirms the species-complex relationship of A. longipes and A. amplum. The available genomic data presented in this paper provides a basis for further research on the evolutionary history and conservation genetics of endangered species of genus Acer.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: General features of the Acer sect. Platanoidea chloroplast genomes compared in this study. Table S2: Acer taxa sampled in this study.

Conclusions
In this study, we firstly reported complete cp genomes of five Acer sect. Platanoidea species (A. catalpifolium, A. amplum, A. longipes, A. yanjuechi and A. mono) using the NGS technology. In comparison with other published Acer species from NCBI, we found that the Acer species have similar cp genome structure and gene content. The divergence hotspots identified in the cp genome of the Acer sect. Platanoidea could be applied to develop molecular markers for further population genetics studies. The high variation at the IR/LSC and IR/SSC boundaries were also reported. The phylogenetic analysis strongly supported that A. catalpifolium has the closest relationship with A. miaotaiense, followed by A. mono, and A. yanjuechi, which confirms the species-complex relationship of A. longipes and A. amplum. The available genomic data presented in this paper provides a basis for further research on the evolutionary history and conservation genetics of endangered species of genus Acer.