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Article

Insights into Phylogeny, Taxonomy, Origins and Evolution of Crataegus and Mespilus, Based on Comparative Chloroplast Genome Analysis

by
Jiaxin Meng
1,*,†,
Yan Wang
1,†,
Han Song
2,
Wenxuan Dong
3 and
Ningguang Dong
1,*
1
Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100093, China
2
College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China
3
College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2025, 16(2), 204; https://doi.org/10.3390/genes16020204
Submission received: 29 December 2024 / Revised: 24 January 2025 / Accepted: 6 February 2025 / Published: 7 February 2025
(This article belongs to the Section Plant Genetics and Genomics)
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Abstract

:
Hawthorns (Crataegus L.) are widely distributed and well known for their medicinal properties and health benefits. Nevertheless, the phylogenetic relationships among Crataegus native to China remain unclear. Additionally, no consensus exists on the origin and evolution of Crataegus, and the relationship between Crataegus and Mespilus is is unclear. Here, we sequenced 20 chloroplast (cp) genomes (19 from Crataegus and 1 from Mespilus) and combined them with 2 existing cp genomes to investigate the phylogenetic relationships, divergence times and biogeographic history of Crataegus. Four hypervariable loci emerged from the newly sequenced genomes. The phylogenetic results indicated that the 14 Chinese Crataegus species analyzed clustered into two clades. One clade and the North American Crataegus species grouped together, while the other clade grouped with the European Crataegus species. Our results favor recognizing Mespilus and Crataegus as one genus. Molecular dating and biogeographic analyses showed that Crataegus originated in Southwest China during the early Oligocene, approximately 30.23 Ma ago. Transoceanic migration of East Asian Crataegus species across the Bering land bridge led to the development of North American species, whereas westward migration of the ancestors of C. songarica drove the formation of European species. C. cuneata may represent the earliest lineage of Chinese Crataegus. The uplift of the Qinghai–Tibet Plateau (QTP) and the Asian monsoon system may have led the ancestors of C. cuneata in south-western China to migrate toward the northeast, giving rise to other Chinese Crataegus species. This study offers crucial insights into the origins of Crataegus and proposes an evolutionary model for the genus.

1. Introduction

Hawthorn (Crataegus L.), widely distributed throughout temperate regions of the Northern Hemisphere, belongs to the genus Crataegus in the family Rosaceae [1]. The hawthorn fruit is widely used for its sweet and sour flavor, as well as its nutritional properties; it is favored by consumers because of its freshness, ease of processing, and medicinal properties [2,3]. Hawthorn has garnered significant global interest as an exceptional medicinal tonic due to its abundance of biologically active compounds [4,5]. In addition, hawthorns have significant ecological and ornamental value; their fruits serve as a food source for birds and mammals, while their dense branches offer ideal nesting locations for birds [6,7].
Phipps [8] suggested that C. scabrifolia of Southwest China and C. mexicana of Mexico are among the most primitive species and are similar to each other based on cladistic analysis. The evolution of the extant species began with C. scabrifolia, which spread into western Eurasia (one major line) and eastern Asia through Beringia to initiate a North American diversification (a minor line), as well as a minor North American diversification from C. mexicana. Owing to molecular and morphological characters, other researchers consider Crataegus as part of the subtribe Pyrinae and suggested that it originated in North America [9]. The relationships among species from eastern Asia, North America, and Europe were examined using sequences of chloroplast (cp) DNA regions and internal transcribed spacers (ITSs). The results indicated that North America and Europe might be the most recent areas of origin for Crataegus [10]. Generally, the origin of Crataegus remains unclear, and information about its evolutionary process is incomplete; thus, further research is required. In addition, a consensus is lacking on the relationship between Mespilus (medlar) and Crataegus (hawthorn), which are sister genera in the tribe Pyreae of the Rosaceae [11].
China is one of the places of origin of wild and cultivated Crataegus species, with a total of 18 species and six varieties [12]; however, others recognize 20 species of Chinese Crataegus and seven varieties across China [13]. Previous studies provide insight into the phylogenetic history of cultivated Crataegus in China. To date, there seem to be a lack of consensus on the phylogeny, taxonomy, origins and evolution of Crataegus. This hypothesis is in agreement with the study of Du et al. [14]. Based on specific-locus amplified fragment sequencing (SLAF-seq), Du et al. [14] suggested that the south-western Chinese species C. scabrifolia was the earliest Chinese species to separate and that this species shares a gene pool with European Crataegus species. However, others hold different views. Lo et al. [10] also proposed that North America and Europe are likely to be the most recent areas of origin for Crataegus based on sequencing of chloroplast (cp) DNA regions and internal transcribed spacers (ITSs); their findings indicated that Crataegus migrated from Europe to eastern Asia and then gave rise to C. hupehensis, C. songorica and C. pinnatifida. The ITS regions encompass ITS1, 5.8S rDNA and ITS2 spacer. ITS1 is located between 18S rDNA and 5.8S rDNA, whereas ITS2 is located between 5.8S rDNA and 28S rDNA. The intragenomic variability of the ITS2 region in Crataegus has negative consequences for DNA barcoding [15,16]. The length of this ITS in Crataegus is 675 bp. Its divergence among Crataegus species in east Asia, eastern North America and western North America is 0–0.85%, 0–2.3% and 0.41–1.72%, respectively [10].
A consensus is lacking on the relationship between Mespilus (medlar) and Crataegus (hawthorn), which are sister genera in the tribe Pyreae of the Rosaceae [11]. The genus Mespilus was considered to be a new, monotypic section in Crataegus based on two nuclear (ribosomal ITS and LEAFY intron 2) and four intergenic chloroplast DNA regions (trnS-trnG, psbA-trnH, trnH-rpl2 and rpl20-rps12) by Lo et al. [11]. However, Phipps [17] supports the existence of two genera according to morphological and cladistic evidence. Chloroplast genome sequences have been widely used to study plant evolutionary relationships at almost all taxonomic levels due to their maternal inheritance characteristic, lack of recombination, low nucleotide substitution rates and haploidy [18,19].
Genomic information for Crataegus is currently scarce, which complicates research pertaining to the taxonomy, genetics, identification and conservation of Crataegus species. Valuable information is contained in cp genome sequences, which permits the taxonomic classification and phylogenetic reconstruction of plant species and individuals [16,20]. Compared with nuclear genomes, cp genomes are much smaller in size and have high copy numbers per cell, which enables their complete sequencing [21]. The typical cp genomes in angiosperms usually have a circular structure containing a large single-copy (LSC) and a small single-copy (SSC) region separated by two inverted repeats (IRs) [22,23]. The rapid development of next-generation sequencing technologies has permitted a faster and more frugal cp genome sequencing. Complete cp genome analysis has been effectively employed in taxonomic and phylogenetic analyses, thus improving our understanding of evolutionary relationships, especially at lower taxonomic levels at which rapid radiation and recent speciation hamper the examination of sequence variations using conventional methods [24,25].
Here, the overall cp genomes of 19 Crataegus species (including 14 Crataegus species native to China, 2 Crataegus species from Europe, and 3 Crataegus species from North America) and 1 Mespilus species from Europe were sequenced using next-generation sequencing. For phylogenomic analysis, molecular dating and biogeographic analysis, the entire cp genomes of two published Crataegus species from the GenBank database, i.e., C. marshallii (MK920293.1) and Crataegus sp. BBL (MK920293.1), were obtained. The main objectives of this study were to analyze common structures and patterns of the Crataegus cp genomes; detect regional hotspots of sequences divergence; reconstruct the phylogenetic relationships among Crataegus species native to China; elucidate the relationship between Crataegus (hawthorn) and Mespilus (medlar); and determine the origin and evolution of the genus Crataegus. Our results will enable species identification in Crataegus, as well as support research in phylogenetic, phylogeographic and population genetic, which will in turn give a hand in the conservation and use of Crataegus resources.

2. Materials and Methods

2.1. Plant Material and DNA Extraction

Our sampling aimed to maximize the taxonomic and geographical coverage of Crataegus. Based on a preliminary field investigation and literature review, we finally chose 14 species native to China. Five exotic Crataegus species were chosen with respect to four major geographical areas (Eastern and Central North America, Eastern and Southern North America, Southeastern United States, and Europe). One Mespilus species was included, as only a single species is comprised in Mespilus.
Leaf material from the 14 Chinese Crataegus species was sampled from the National Germplasm Repository for Crataegus in Shenyang, Liaoning Province, China, while the leaves of the 5 exotic Crataegus species and 1 Mespilus species were sampled from Shanghai Chenshan Botanical Garden in Shanghai, China. The specific biogeographic regions and sampling locations of the 19 Crataegus species and 1 Mespilus species are displayed in Table 1. The CTAB method was used to extract DNA [26].

2.2. Chloroplast Genome Sequencing, Assembly and Annotation

Genomic DNA was fragmented to 400 bp fragments by ultrasound. Then, total DNA was purified and end-repaired. A 400 bp (insert size) paired-end library was constructed using a NEBNext Ultra DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA), and PE150 sequencing (Illumina, Inc., San Diego, CA, USA) was performed on an Illumina high-throughput sequencing platform (HiSeq 4000) (Illumina, Inc., San Diego, CA, USA). After sequencing, the raw reads were assembled into whole chloroplast genomes in a four-step approach. Firstly, adaptors were removed, and low-quality sequences were trimmed using Trimmomatic 0.39 [27] with the following parameters: LEADING = 20, TRAILING = 20, SLIDINGWINDOW = 4:15, MINLEN = 36, and AVGQUAL = 20. Secondly, the paired-end reads were evaluated for quality and assembled using SPAdes 3.6.1 (Kmer = 95) [28]. Thirdly, the assembled plastome sequences were selected from the initial assembly by a Basic Local Alignment Search Tool (BLAST)-based algorithm [29] using the chloroplast genome of C. hupehensis (MW201730) as a reference. Sequencher 4.10 (https://www.genecodes.com/) software was used to assemble selected contigs. At last, the command line Perl script Plann was used to annotate the Crataegus cp genomes [30]. Intron positions, putative starts and stops and initial annotation were determined with the C. hupehensis (MW201730) cp genome as a reference. Subsequently, Genome Vx was used to generate a physical map of the cp genome [31].

2.3. Molecular Marker Identification and Sequence Divergence Analysis

MAFFT v7 was employed to align the 20 cp genome sequences with the default parameters [32]. The nucleotide variability of the chloroplast genome was determined via sliding window analysis in DnaSP version 6.0 [33]. The analysis was conducted with a window size of 600 bp, and the step size was 100 bp. MEGA 7.0 software was employed to assess the variable and parsimony-informative base sites across the whole cp genomes and in the LSC, SSC and IR regions of the 20 cp genomes [34]. The p-distance among Crataegus plastomes was determined to assess the divergence pattern of the Crataegus species with MEGA software.

2.4. Phylogenetic Analysis

We subjected the 20 cp genome sequences generated, along with 2 whole cp genome sequences of Crataegus and the sequences of another 10 plastomes from the species of Maloideae downloaded from Genbank, to phylogenetic analysis. The maximum-likelihood (ML) and Bayesian inference (BI) methods were used to construct phylogenetic trees. Modelfinder was used to identify the optimal model GTR + F + I + G4 on the basis of the Bayesian information criterion [35]. ML analysis was performed with IQ-TREE 2.2.0 software [36], and sampling was repeated 1000 times. BI analysis was performed in the MrBayes program. The BI model was constructed, with ngen set to 1,000,000 burnin = ngen × 0.25/sample freq., Lset nst = 6 rates = invgamma, Prset statefreqpr = dirichlet (1, 1, 1, 1); graphical visualization of the STRUCTURE results was performed using Clumpak. Markov chain Monte Carlo (MCMC) analyses were performed for 10,000,000 generations. The trees were sampled every 1000 generations, and the first 25% sampled generations were discarded as burn-in samples. Finally, the average and standard deviation of split frequencies >0.01 were verified.

2.5. Divergence Time Estimation

Divergence time estimation was implemented using a relaxed log-normal clock model run on the BEAST v2.6.7 platform and a general time-reversible (GTR) substitution model and speciation tree prior based on the Yule speciation process [37]. We used one fossil constraint and one secondary calibration to estimate the divergence time: the stem of Amelanchier was dated to 33.9 Ma ago based on fossils of A. peritula and A. scudderi [38], and the estimated divergence date (50.06 Ma ago) of clade Maleae was used as the secondary calibration point [39].
BEAST analysis was executed for 100 million generations, sampling every 1000 generations. Using Tracer v1.6, the stationary phase was examined in terms of convergence and to secure an adequate sample size (>200) for all parameters. The initial 10% of trees were discarded (burn-in period). Subsequently, we used TreeAnnotatorv2.4.8 to summarize the time-calibrated species as a maximum clade credibility (MCC) tree [40]. For constructing the MCC tree, we set the credible set to 95%. This means that the top 95% of trees with the highest posterior probabilities were selected.

2.6. Ancestral Area Reconstruction

To rebuild the broad-scale biogeographic history of Crataegus, the distribution of each extant Crataegus species was encoded as a character with seven states based on the floristic regions reported by Zhao et al. [12] and Dong et al. [13] (Table 1) as follows: A, South-western China; B, Central Plains and Qinling Mountains of China; C, North-eastern China; D, Mongolia–Siberian region; E, Central and Western Asia; F, Europe; G, North America. We estimated the ancestral areas based on the collapsed MCC tree from the entire plastome datasets using the dispersal–extinction–cladogenesis (DEC) model, which was performed in RASP 4.0 [41]. Regarding the DEC analysis, the initial dispersal probabilities between areas were set as equal, with all entries in the dispersal constraint matrix assigned a value of 1. Only the most likely status (MLS) at the center of the pie diagram for each node is displayed. For detailed setting parameters, refer to Wang et al. [42]. The global temperature variation over the past 30 Ma was gained from Zachos et al. [43].

3. Results

3.1. Basic Characteristics of the Chloroplast Genome

We submitted overall 20 complete cp genome sequences to the GenBank database with accession numbers MW201730 and OP963999~OP964017. The lengths of the nucleotide sequences of the 19 Crataegus cp genomes and 1 Mespilus cp genome ranged from 159,654 bp in C. viridis to 159,977 bp in C. crussgalli (Figure 1 and Table 2). All 20 cp genomes exhibited the typical quadripartite structure characteristic of angiosperms, including a pair of IRs (26,331–26,396 bp) separated by LSC (87,682–88,061 bp) and SSC (19,139–19,312 bp) regions. The overall GC content of the 20 cp genomes was 36.6–36.7%, with similar GC contents among all 20 genomes.

3.2. Sequence Divergence and Hotspots

We aligned the complete cp genome sequences of the 19 Crataegus species and 1 Mespilus species to determine sequence variations (Table 4). The alignment matrix spanned 163,856 bp in size, with 1115 variable sites (0.68%) including 654 parsimony-informative sites (0.4%) and 461 singleton sites (0.28%). The overall nucleotide diversity among the 20 cp genome sequences was 0.0018. The IR regions exhibited the lowest nucleotide sequence diversity (0.00032); that of the SSC regions was the highest (0.00355).
To pinpoint sequence divergence hotspots, the nucleotide diversity (π) value was assessed within a sliding window of 600 bp (Figure 2). The π values ranged from 0 to 0.012. Four highly variable regions (π > 0.01), including trnR(UCU)-atpA, ndhC-trnV(UAC), ndhF-rpl32 and ndhA, were found in the Crataegus and Mespilus cp genomes (Figure 2). The trnR(UCU)-atpA region had the highest π values (π = 0.012), followed by the ndhC-trnV(UAC), ndhA and ndhF-rpl32 regions. Among these sequences, trnR(UCU)-atpA and ndhC-trnV(UAC) are located in the LSC region, while ndhF-rpl32 and ndhA are located in the SSC region. Meanwhile, the four hypervariable markers and universal DNA barcodes (rbcL, matK and trnH-psbA) were compared in greater detail (Table 5). The count of variable sites across the four marker regions ranged from 22 (ndhC-trnV(UAC)) to 36 (trnR(UCU)-atpA), and the number of universal DNA barcode regions ranged from 8 (rbcL and trnH-psbA) to 16 (matK). Nucleotide diversity was much higher in the two major mutation hotspots (trnR(UCU)-atpA and ndhC-trnV(UAC)) than in the typical universal cp DNA barcode regions (matK, rbcL and trnH-psbA).

3.3. Phylogenetic Analysis

We conducted a phylogenetic analysis using 22 entire cp genomes obtained from 21 Crataegus species and 1 Mespilus species, with cp genomes of 10 other Maloideae species as the outgroup. As shown in Figure 3, the topologies of the ML and BI phylogenetic trees were generally consistent, showing two major clusters. Crataegus was closely related to the genus Amelanchier. Additionally, Crataegus and Mespilus formed a strongly supported (100%) monophyletic group.
Four well-supported clades (I–IV) were revealed within the ingroup based on a complete cp genome analysis, which successfully elucidated the species relationships within the clades (Figure 3). Apparently, these four clades clustered into two large lineages, in which Clades I and II were sister clades, as were Clades III and IV. Clade I consists of six sympatric or parapatric Chinese species (C. dahurica, C. sanguinea, C. kansuensis, C. maximowiczii, C. bretschneideri and C. altaica) found in the Mongolia–Siberian region, north-eastern China, and Central Plains and Qinling Mountains of China, along with one species from south-western China (C. chungtienensis). Clade II contained five North American species (C. marshallii, C. viridis, C. sp. BBL 2019, C. crussgalli, C. phaenopyrum). Clade III consisted of six Chinese species, i.e., C. pinnatifida, C. pinnatifida var. major, C. hupehensis, C. cuneata, C. scabrifolia and C. wilsonii, with distribution ranges including south-western, central, northern, eastern, and north-eastern China, as well as the Mongolia–Siberian region. In Clade IV, two European species (C. laevigata and C. monogyna) fell into a common subclade, with one Chinese species (C. songarica) found in Central and Western Asia forming a sister subclade.

3.4. Divergence Time Estimation

On the basis of the complete cp genome datasets and fossil calibration, divergence time analysis showed that Crataegus originated and the split between Clades I + II and Clades III + IV occurred during the early Oligocene, approximately 30.23 Ma ago (Figure 4). The differentiation between Clades I and II was estimated to have occurred at approximately 22.39 Ma ago, while the split between Clade III and IV was estimated at 18.8 Ma ago. Early predecessors of the four clades began to emerge during the Miocene, with rapid divergence happening from the late Miocene to the Pliocene. In Clade I, the first Chinese Crataegus species to split were C. altaica and C. chungtienensis, which diverged around 9.47 Ma ago, followed by C. bretschneideri and C. maximowiczii, which split around 7.35 and 4.74 Ma ago, respectively, while C. kansuensis, C. sanguinea and C. dahurica successively split approximately 0.71 Ma and 0.45 Ma ago. Species diversification in Clade II was gradual, with C. phaenopyrum and C. cruss-galli splitting approximately 14.55 and 8.76 Ma ago, respectively, whereas divergence among C. sp. BBL, C. viridis and C. marshallii happened from 4.13 to 0.75 Ma ago. In Clade III, C. cuneata, which diverged around 9.22 Ma ago, was the first Crataegus to diverge, followed by C. scabrifolia and C. wilsonii, which split approximately 3.53 Ma ago. Divergence among C. hupehensis, C. pinnatifida and C. pinnatifida var. major emerged recently (4.14–0.75 Ma ago). In Clade IV, the intercontinental split between Asian and European species happened around 4.67 Ma ago, and divergence between M. germanica and European Crataegus occurred approximately 1.62 Ma ago. The split between C. monogyna and C. laevigata was estimated to have occurred 0.78 Ma ago.

3.5. Biogeographic History

Southwest China, the Central Plains and the Qinling Mountains of China are the most likely ancestral areas of Crataegus, according to the results of the DEC analysis (Figure 4). The current distribution of Crataegus appears to be shaped by a combination of dispersal and vicariance events. Integrating the results of the DEC analysis and molecular dating, we identified a long-distance dispersal route across the Bering land bridge from East Asia to North America (AB–G), which led to the divergence of the North American species C. phaenopyrum in the middle Miocene (approximately 14.55 Ma ago). Another long-distance spreading route across the Bering land bridge in the opposite direction (G–D–C) facilitated the occurrence of C. altaica and other species in north-eastern China (Clade I). The third long-distance spreading from East Asia to the Europe (AB–E–F) gave rise to speciation of the Chinese species C. songarica and the European species Crataegus in the early Pliocene (approximately 4.67 Ma ago) and Pleistocene (1.62–0.78 Ma ago), respectively. Within China, two important dispersals (A–B; B–C) and subsequent vicariance resulted in the colonization of Southwest China, the Central Plains, Qinling Mountains and Northeast China by Crataegus.

4. Discussion

4.1. Features of the Crataegus and Mespilus Chloroplast Genomes

In the present study, we sequenced the cp genomes of 19 Crataegus species and 1 Mespilus species using next-generation sequencing methods. The sizes of the 20 newly sequenced cp genome ranged in length from 159,654 bp (C. viridis) to 159,977 bp (C. crussgalli). All species contained a total of 113 genes, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The genes ycf15 and ycf68 are not annotated due to their status as pseudogenes, which contain several internal stop codons [18]. Additionally, the genes ycf2, rpl23 and accD are missing from the plastomes in some species [22,44,45]; on the contrary, these genes generally appear in Crataegus. The structures of the 20 newly sequenced cp genome gained in present study are generally analogous to those found in most plants, and no recombination events were found. The lowest sequence divergence was observed in IR regions, while all high-variation regions are located in the LSC region (Table 4); this finding is consistent with recent studies [18,23]. The most possible reason for this difference is that, in the cp genome, which is present in multiple copies within individual plant cells, gene conversion, which exhibits a minor bias against new mutations, is more effective at reducing the mutation load in the two IR regions than in the single-copy regions, primarily due to the duplicative inherent quality of the IRs [46,47,48,49]. The high-variation regions could be utilized to develop DNA barcoding methods for the estimation of plant phylogeny at low taxonomic levels, such as among species of Crataegus.

4.2. Potential Highly Variable Chloroplast Barcodes

A growing body of research shows that universal DNA barcodes (rbcL, matK and trnH-psbA) have low sequence divergence and do not offer a clear resolution of the phylogenetic relationship [28,50]. A few nuclear and cp DNA barcodes have been applied for the genetic characterization of Crataegus and to determine its phylogeny, taxonomy, origins and evolution [10,51,52]. Due to the formation of apomictic complexes, active introgression events and relatively rare segregation events related to older hybridizations, DNA barcoding and the taxonomic assessment of the genus Crataegus are challenging [8]. Hence, it is essential to utilize additional markers and expand the taxonomic sampling to achieve better resolution at low taxonomic levels. The hawthorn fruit is widely used for its sweet and sour flavor, as well as its nutritional properties [3]; however, genomic resources for Crataegus are scarce, which is a serious barrier to the progress of its taxonomy, genetics, conservation and utilization. Further study can be conducted to determine its genomic evolution and produce valuable genetic resources with the help of cp genome sequences. Mutation events in the cp genome may not have occurred randomly throughout the genome sequence; instead, they are often focused in hotspot regions [53]. An efficient means for detecting mutation hotspots is comparing cp genome sequences; these identified highly variable regions can be used as specific DNA markers. In the present study, four hypervariable regions, including trnR(UCU)-atpA, ndhC-trnV(UAC), ndhF-rpl32 and ndhA (Figure 2), were identified.
The trnR(UCU)-atpA region is the most variable region in the 20 cp genomes (Figure 2). It is part of the trnG-atpA intergenic marker, which is split between two intergenic regions: trnG-trnR and trnR-atpA. The trnG-atpA region could serve as a high-variability marker in Corylus [54]. ndhC-trnV(UAC) is part of the ndhC-trnV(UAC)-trnM(CAU) region and has been reported as a high-variability marker that can be used for DNA barcoding and molecular phylogenetic studies [55]. The ndhF and ndhF-rpl32 regions are considered divergence hotspots by Wu et al. [56], who compared nine Crataegus cp genomes and identified five highly variable markers. These two regions, ndhF and ndhF-rpl32, have been widely used in plant phylogenetic studies [50,57].

4.3. Phylogenetic Relationships

As China is one of the places of origin of wild and cultivated Crataegus species, the collection and cultivation of hawthorns has been documented in China for >1000 years [12]. Although a total of 18 species and six varieties have been identified and confirmed [58], the phylogenetic relationships among Crataegus species native to China remain undetermined.
Crataegus is a genus of the Maloideae subfamily in the Rosaceae [59]. The taxonomy of the genus Crataegus is complicated by a mix of biological and historical factors. Frequent interspecific hybridization, widespread polyploidy and apomixis can all obscure the distinctions between Crataegus species [60,61]. The genetic relationships among some species of Crataegus have been identified using DNA markers such as simple sequence repeats (SSRs), cpSSRs (non-coding cp regions), restriction fragment length polymorphism (RFLP) and ITS [6,7,10,62]. However, these molecular markers suggest that considerable information is missing. In recent years, the cp genome has become an effective strategy to elucidate phylogenetic relationships at almost all taxonomic levels [17,63]. Wu et al. [64] published a phylogenetic study of Chinese Crataegus based on cp genome sequences; however, in that study, only 4 of 18 sequences were derived from whole-genome assemblies. In our study, the newly sequenced cp genomes of 20 species (including 14 Crataegus species native to China, 2 Crataegus species from Europe, 3 Crataegus species from North America and 1 Mespilus species from Europe), and two whole cp genome sequences of Crataegus obtained from GenBank were analyzed comparatively; the higher coverage in this study may shed more light on the interspecific relationships among Crataegus species native to China.
A well-supported phylogenetic tree for 14 Chinese Crataegus species was constructed, according to the complete cp genome sequences (Figure 3). The ML phylogenetic tree showed that 14 Chinese Crataegus species clustered into two clades (Clade I and Clade III, Figure 3). Based on morphological characters, Chinese Crataegus species can be divided into four groups [12], which form two clades derived from the cp genomes. Clade I consists of C. sanguinea, C. kansuensis, C. dahurica, C. maximowiczii, C. bretschneideri, C. altaica and C. chungtienensis, while Clade III comprises C. pinnatifida, C. pinnatifida var. major, C. hupehensis, C. cuneata, C. scabrifolia, C. wilsonii and C. songarica. From a geographic perspective, Clade I is a northern group except for C. chungtienensis, whereas Clade III is widely distributed, with species ranges in south-western, central, northern, eastern, and north-eastern China. Clade I and the North American Crataegus species grouped together, as did Clade III and the European Crataegus species. These results provide evidence for a genetic exchange between Chinese and North American or European species of Crataegus, consistent with previous studies [8,10,14]. In Clade I, C. sanguinea, C. kansuensis and C. dahurica clustered together, with very little difference seen among the three species. In accordance with our results, Wu et al. [65] and Zhang et al. [62] implied that C. sanguinea, C. kansuensis and C. dahurica are closely related based on RFLP and SSR analyses.
As noted above, Clade I is a northern group except for C. chungtienensis. C. chungtienensis is predominantly found in alpine areas of Southwest China, at elevations of around 2500–3300 m [12], whereas C. altaica is endemic to north-western China. However, C. chungtienensis and C. altaica grouped together. The variation among the cp sequences aligned with the geographic distribution of each species, indicating that repeated cp DNA introgression resulted in the present distribution [66]. Hybridization and introgression are effective means to generate new species in plant evolution, or ecotypes, and drive reticulate evolution [15,67,68]. The cytoplasm of one species might be replaced by that of another via gene flow infiltration through hybridization and repeated backcrossing. Consequently, the genetic components of one species includes not only the nuclear genome inherited from its parents but also newly acquired cp gene sequences. Introgression-induced cp capture events have been commonly reported in sympatric and parapatric plant taxa [54,68,69,70]. Multiple cp capture events within Crataegus may have been enabled by frequent colonization events associated with the long-distance dispersal of fruits by birds. The spread of C. chungtienensis and C. altaica has likely relied on birds. The pomes of Crataegus, as well as those of other berry-like fruits approximately 1 cm in size, are commonly considered as bird-dispersed fruits [8,64,71].
Research on the relationship between Crataegus (hawthorn) and Mespilus (medlar) has been ongoing for a long time, but controversies persist, and researchers have not yet reached a consensus regarding these two genera. The longstanding consensus among academic taxonomists is that Mespilus and Crataegus are distinct genera [9,59]. Two nuclear and four intergenic cp DNA regions were used to assess the relationship between Mespilus and Crataegus; the results did not confirm a distinction between the genera Mespilus and Crataegus [11]. Talent et al. [60] proposed conserving the name Crataegus rather than Mespilus. However, Phipps [17] contested that morphological and cladistic evidence favors the recognition of two distinct genera. Mespilus and Crataegus are treated as two distinct genera by some researchers [38,72]. In the present study, Mespilus germanica fell within the genus Crataegus in the phylogenetic tree based on complete cp genome sequences (Figure 3). Furthermore, Mespilus germanica and the two European Crataegus species grouped together, and C. songarica was the sister group. Consequently, our results favor the recognition of Mespilus and Crataegus as one genus.

4.4. Origin and Spread of Crataegus

The current biogeographic patterns and species speciation processes in the northern temperate zone are the result of both geological events and long-term climate changes emerging since the Tertiary [69,73]. Molecular dating and biogeographic analysis (Figure 4) showed that Crataegus emerged around 30.23 Ma ago, which is adjacent to the era of the Eocene–Oligocene climate transition (approximately 34 Ma ago) [74]. Therefore, the early development of Crataegus is related to the temperature. As the temperature in high-latitude regions (45–70 degrees in both hemispheres) decreased during the Eocene–Oligocene climate transition [74], Crataegus became distributed in Southwest China, as well as in the Central Plains and Qinling Mountains of China. Southwest China, ranging from the eastern Himalayas and Yarlung Zangbo Canyon to the entire Hengduan Mountains, is recognized as one of thirty-four biodiversity hotspots by Conservation International [70]. This region is considered the place of origin of the genus Crataegus, consistent with previous biogeographic studies [8].
On the origin and spread of Crataegus, there are many hypotheses. Phipps [8] postulated that Crataegus migrated eastward from East Asia to North America via the Bering land bridge in the early Tertiary or Miocene, as well as westward from Southwest China to Europe. Nevertheless, other researchers have divergent views. Evans and Campbell [9] proposed that Crataegus originated in North America. Lo et al. [10] posited that Crataegus originated in Europe and eastern North America and that the Chinese Crataegus species C. songarica, C. pinnatifida and C. hupehensis derived from the European Crataegus that spread into eastern Asia. Our results showed three intercontinental migrations based on ancestral area reconstruction (Figure 4). One migratory pathway involved East Asian Crataegus species migrating to North America across the Bering land bridge 30.23–22.39 Ma ago. The opposite dispersal route, along which Crataegus migrated westward from North America to East Asia across the Bering land bridge, was active 22.39–14.9 Ma ago. The Bering land bridge made a significant contribution to the interchange between Asia and North American biotas and is therefore globally regarded as a key area for paleogeographic and biogeographic research [75]. A possible explanation regarding the disjunct distributions of related extant flora in eastern Asia and western North America is spreading across the Bering land bridge [76]. Dispersal from Asia into North America reached a peak during the late Oligocene warming period (26–24 Ma ago), while influx from North America into Asia emerged at a lower rate than migration in the opposite direction throughout the Cenozoic, with peak rates of dispersal occurring in the early to middle Miocene (16–14 Ma ago) [76]. Along the third intercontinental migratory pathway, Chinese Crataegus in East Asia migrated to Central Asia 18.8–4.67 Ma ago, resulting in the speciation of C. songarica, which migrated further into Europe 4.67–0.78 Ma ago and led to the formation of the M. germanica and European Crataegus species. The uplift of the Qinghai–Tibet Plateau (QTP) was a pivotal geological event in the Cenozoic era. The analysis of magnetic susceptibility and soil particle size in the north-eastern area of the QTP indicated a significant uplift 7–8 and 2.6 Ma ago [74,77]. The geography and climate of Asia significantly changed due to the uplift of the QTP, which drives the formation of the East Asian winter monsoon system [78]. The Asian monsoon system leads to precipitation occurring mainly at low latitudes, leaving the mid-latitude areas of Asia in arid conditions [64]. Due to these events, the ancestors of C. songarica in north-western China migrated westward to Europe.
In addition to these three intercontinental migrations, the results of ancestral area reconstruction also suggested two other important dispersals (A–B; B–C) (Figure 4). Du et al. [14] suggested that a divergence event for Crataegus species native to China happened in south-western China and led to their migration northward and eastward to north-eastern China. C. scabrifolia in south-western China, which was determined as the earliest-diverging Chinese species, progressed toward the northeast and split to form C. pinnatifida [14]. However, according to the results of our molecular dating analysis (Figure 4), C. cuneata may represent the earliest lineage of Crataegus. C. cuneata, which is widespread in the warm temperate and south-eastern regions of China, split approximately 9.22 Ma ago. Based on the results of ancestral area reconstruction (Figure 4), the ancestor of C. cuneata is likely to have migrated northward and eastward, splitting to form C. scabrifolia, C. hupehensis, C. pinnatifida and C. pinnatifida var. major. This migratory route is consistent with a migration pathway from the Hengduan Mountains north-eastward through the Qinling Range, i.e., the eastern fringe of the Loess Plateau including the Taihang Range, Yinshan Range and Changbai Mountains, as suggested by Wang [79] based on the distribution patterns and migration routes of some East Asian flora. An intense uplift of the QTP occurred three times during the Cenozoic era [80]. The uplift of the QTP significantly altered the climate in Asia and resulted in the formation of the Asian monsoon system [81,82]. As a result, precipitation in south-western China is not well distributed, which leads to the migration of plants [83,84]. Together, these climatic events may have prompted the ancestors of C. cuneata in south-western China to migrate toward the northeast.
The present study provides the complete cp genomes of 19 Crataegus species and 1 Mespilus species and sheds light on the phylogeny, taxonomy, origins and evolution of Crataegus. However, the limited number of species analyzed in the present study may have resulted in possible errors in the phylogenetic analysis. In future studies, a higher number of species and more extensive nuclear gene information should be included.

5. Conclusions

In this study, the complete cp genomes of 19 Crataegus species and 1 Mespilus species were sequenced. A comparative analysis of the plastomes led to the identification of four hypervariable hotspots, trnR(UCU)-atpA, ndhC-trnV(UAC), ndhF-rpl32 and ndhA, which could serve as molecular markers for the genus Crataegus. In subsequent studies, species of hawthorn can be identified by designing primers to further determine whether the hypervariable hotspots can be used for identification.
A phylogenetic analysis based on the complete cp genome revealed that the 14 Chinese Crataegus species examined could be divided into two clades to help elucidate the phylogenetic relationships among Crataegus species in China. One clade and the North American Crataegus species grouped together, while the other clade grouped with European Crataegus species. Additionally, our findings support the recognition of Mespilus and Crataegus as one genus.
Molecular dating and biogeographic analyses indicated that Crataegus originated in Southwest China during the early Oligocene, approximately 30.23 Ma ago. The transoceanic migration of east Asian Crataegus species across the Bering land bridge led to the development of North American species, whereas westward migration of the ancestors of C. songarica drove the formation of European species. The uplift of the Qinghai–Tibet Plateau (QTP) and the Asian monsoon system may have led the ancestors of C. cuneata in south-western China to migrate toward the northeast, giving rise to other Chinese Crataegus species. Overall, the present study provides valuable information about the phylogeny, taxonomy, origins and evolution of Crataegus.

Author Contributions

Conceptualization, N.D. and J.M.; methodology, Y.W.; software, Y.W.; validation, H.S.; formal analysis, W.D.; investigation, H.S.; resources, W.D.; data curation, N.D.; writing—original draft preparation, J.M. and Y.W.; writing—review and editing, N.D.; visualization, N.D.; supervision, W.D. and N.D.; project administration, N.D.; funding acquisition, N.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Special Fund for the Construction of Scientific and Technological Innovation Capability (KJCX20251401) and the Youth Research Foundation of the Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Science (LGSJJ202302).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The complete chloroplast genomes generated during the current study were submitted to the NCBI database and are available with the GenBank accession numbers MW201730 and OP963999~OP964017.

Acknowledgments

We sincerely thank Haiyang Shou (Shanghai Chenshan Botanical Garden) for providing some samples.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. General chloroplast genome map of 19 Crataegus and 1 Mespilus species. The species sizes for the chloroplast genomes are shown in Table 2. Genes located inside the circle are transcribed in a clockwise direction, whereas those outside are transcribed counterclockwise. Genes are color-coded according to their function categories. The dashed area in the inner circle represents the GC content, while the lighter gray area indicates the AT content.
Figure 1. General chloroplast genome map of 19 Crataegus and 1 Mespilus species. The species sizes for the chloroplast genomes are shown in Table 2. Genes located inside the circle are transcribed in a clockwise direction, whereas those outside are transcribed counterclockwise. Genes are color-coded according to their function categories. The dashed area in the inner circle represents the GC content, while the lighter gray area indicates the AT content.
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Figure 2. Sliding-window analysis of nucleotide variability across the 20 newly sequenced entire chloroplast genomes (window length: 600 bp; step size: 100 bp). x-Axis: position of the midpoint of each window; y-axis: nucleotide diversity of each window. The red dashed line means nucleotide diversity value is 0.01. The regions over the red dashed line mean they were characterized by high variability and high polymorphism. LSC, large single-copy; IRa, inverted repeat a; SSC, small single-copy; IRb, inverted repeat b.
Figure 2. Sliding-window analysis of nucleotide variability across the 20 newly sequenced entire chloroplast genomes (window length: 600 bp; step size: 100 bp). x-Axis: position of the midpoint of each window; y-axis: nucleotide diversity of each window. The red dashed line means nucleotide diversity value is 0.01. The regions over the red dashed line mean they were characterized by high variability and high polymorphism. LSC, large single-copy; IRa, inverted repeat a; SSC, small single-copy; IRb, inverted repeat b.
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Figure 3. Phylogenetic analysis of Crataegus from maximum-likelihood analyses using complete chloroplast genomes. The four major Clades (I–IV) in the ingroup are represented by vertical bars, with the blocks on the right corresponding to the distribution of each species. The map indicates the species distribution of Crataegus used in the analysis. Seven regions were defined: (A) South-western China; (B) Central Plains and Qinling Mountains of China; (C) North-eastern China; (D) Mongolia–Siberian region; (E) Central and Western Asia; (F) Europe; (G) North America. The first and second numbers on the branches correspond to ML bootstrap support (BS) and Bayesian posterior probability (PP) values, respectively. The asterisks (*) indicate BS = 100% or PP = 1.0.
Figure 3. Phylogenetic analysis of Crataegus from maximum-likelihood analyses using complete chloroplast genomes. The four major Clades (I–IV) in the ingroup are represented by vertical bars, with the blocks on the right corresponding to the distribution of each species. The map indicates the species distribution of Crataegus used in the analysis. Seven regions were defined: (A) South-western China; (B) Central Plains and Qinling Mountains of China; (C) North-eastern China; (D) Mongolia–Siberian region; (E) Central and Western Asia; (F) Europe; (G) North America. The first and second numbers on the branches correspond to ML bootstrap support (BS) and Bayesian posterior probability (PP) values, respectively. The asterisks (*) indicate BS = 100% or PP = 1.0.
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Figure 4. ML phylogenetic tree reconstruction of Crataegus species obtained from BEAST 2 analysis, with ancestral area reconstruction performed using the DEC model in RASP. The divergence times were estimated using a single fossil calibration (see text for more details). The branches are annotated with the most recent common ancestor (MRCA) times and their corresponding 95% highest posterior density (HPD) intervals (in Ma); cyan bars represent the 95% HPD intervals for node ages. Seven regions were defined: (A) South-western China; (B) Central Plains and Qinling Mountains of China; (C) North-eastern China; (D) Mongolia–Siberian region; (E) Central and Western Asia; (F) Europe; (G) North America. The letters and colors in the legend denote extant ancestral areas, as well as their combinations. The pie charts at each node illustrate the most probable ancestral areas.
Figure 4. ML phylogenetic tree reconstruction of Crataegus species obtained from BEAST 2 analysis, with ancestral area reconstruction performed using the DEC model in RASP. The divergence times were estimated using a single fossil calibration (see text for more details). The branches are annotated with the most recent common ancestor (MRCA) times and their corresponding 95% highest posterior density (HPD) intervals (in Ma); cyan bars represent the 95% HPD intervals for node ages. Seven regions were defined: (A) South-western China; (B) Central Plains and Qinling Mountains of China; (C) North-eastern China; (D) Mongolia–Siberian region; (E) Central and Western Asia; (F) Europe; (G) North America. The letters and colors in the legend denote extant ancestral areas, as well as their combinations. The pie charts at each node illustrate the most probable ancestral areas.
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Table 1. Biogeographic regions, sample collection sites and related information of 19 Crataegus species and 1 Mespilus species.
Table 1. Biogeographic regions, sample collection sites and related information of 19 Crataegus species and 1 Mespilus species.
TaxonIdentification CodeBiogeographic RegionCollection SiteUsageMain Characteristics
C. bretschneideriFSZNorth-eastern China, Mongolia–SiberiaShenyangFresh eating, processing, medicine, ornamental plant, afforestationFruit, small, prismatic and oblate, peel, bright red
C. pinnatifidaSLHNorth-eastern China, Mongolia–SiberiaShenyangMedicine, ornamental plant, afforestationFruit, subglobose or pear-shaped, peel, dark red
C. pinnatifida var. majorSDDJXCentral Plains, Qinling Mountains of China and North-eastern ChinaShenyangFresh eating, processing, medicine, afforestationFruit, large, broad obovate, peel, dark red or purple red
C. maximowicziiMSZNorth-eastern China, Mongolia–SiberiaShenyangFresh eating, ornamental plant, afforestation, timberFruit, spherical, pilose when young, glabrous after, peel, red
C. hupehensisHBSZ1HCentral Plains and Qinling Mountains of ChinaShenyangFresh eating, processing, medicine, ornamental plant, afforestationFruit, small, subglobose, peel, bright red
C. sanguineaLNSZNorth-eastern China, Mongolia–SiberiaShenyangFresh eating, medicine, ornamental plant, afforestationFruit, extremely small, oblate, peel, blood red and smooth
C. kansuensisGSSZCentral Plains and Qinling Mountains of ChinaShenyangFresh eating, processing, medicine, afforestationFruit, subspheroidal, peel, red or orange
C. scabrifoliaYNSZSouth-western ChinaShenyangFresh eating, processing, medicineFruit, medium size, oblate, peel, stone yellow
C. songaricaZGESZCentral and Western AsiaShenyangMedicine, ornamental plantFruit, spherical or oblate, peel, reddish black
C. cuneataXYSZSouthwest, Central Plains and Qinling Mountains of ChinaShenyangProcessing, medicine, rootstockFruit, spherical or oblate, peel, red or yellow
C. altaicaAETSZCentral and Western Asia, Mongolia–SiberiaShenyangMedicine, ornamental plantFruit, small and spherical, peel, golden yellow
C. dahuricaGYSZNorth-eastern China, Mongolia–SiberiaShenyangFresh eating, medicine, afforestation, rootstockFruit, spherical or
oblong, peel, orange-red or orange
C. chungtienensisZDSASouth-western ChinaShenyangOrnamental plant, afforestationFruit, small, spherical or oblong, peel, red
C. wilsoniiHZSZSouthwest, Central Plains and Qinling Mountains of ChinaShenyangProcessing, afforestationFruit, small, oblong, peel, red
C. crussgalliJJSZNorth AmericaShanghaiOrnamental plant, afforestationThorn, strong, fruit, small, peel, red
C. viridisLSZNorth AmericaShanghaiOrnamental plant, afforestationFruit, small, peel, bright red
C. phaenopyrumHSDSZNorth AmericaShanghaiOrnamental plant, afforestationFruit, small, peel, bright red
C. laevigataHHSZEuropeShanghaiOrnamental plantFruit, scarce, peel, dark red
C. monogynaDZSZ1HEuropeShanghaiOrnamental plant, afforestationdunga-runga, peel, dark red
M. germanicaOZEuropeShanghaiFresh eating, processing, afforestationDunga-runga, fruit, apple-shaped, peel, brown
Table 2. General characteristics of the 20 newly sequenced chloroplast genomes.
Table 2. General characteristics of the 20 newly sequenced chloroplast genomes.
SpeciesPlastome Size (bp)LSC
Length (bp)		IR
Length (bp)		SSC
Length (bp)		Gene NumberProtein-Coding GenestRNA GenesrRNA GenesOverall GC (%)GenBank Accession Number
C. pinnatifida159,67687,74426,38419,1641137943036.60%OP963999
C. pinnatifida var. major159,65687,74926,38419,1391137943036.70%OP964000
C. maximowiczii159,80087,77826,38419,2541137943036.60%OP964001
C. cuneata159,71887,76626,38419,1841137943036.60%OP964002
C. sanguinea159,85187,84426,38419,2391137943036.60%OP964003
C. scabrifolia159,74487,75926,38319,2191137943036.60%OP964004
C. songarica159,87187,88026,38919,2131137943036.60%OP964005
C. kansuensis159,82787,80026,38419,2591137943036.60%OP964006
C. altaica159,70487,68226,37719,2681137943036.60%OP964007
C. bretschneideri159,71787,71126,34719,3121137943036.60%OP964008
C. dahurica159,85287,84726,38419,2371137943036.60%OP964009
C. chungtienensis159,83587,80326,38419,2641137943036.60%OP964010
C. laevigata159,70787,69826,38419,2411137943036.60%OP964011
C. monogyna159,76387,77926,38419,2161137943036.60%OP964012
C. hupehensis159,76687,85226,38519,1441137943036.60%MW201730
C. wilsonii159,77987,77326,38319,2401137943036.60%OP964013
C. crussgalli159,97788,01426,36319,2371137943036.60%OP964014
C. phaenopyrum159,95388,06126,33119,2301137943036.60%OP964015
C. viridis159,65487,70426,36519,2201137943036.60%OP964016
M. germanica159,81187,80426,39619,2151137943036.60%OP964017
Note: LSC, large single-copy; SSC, small single-copy; IR, inverted repeat in the cp genomes of Crataegus and Mespilus; a total of 113 genes were identified, containing 79 protein-coding genes, 4 ribosomal RNA (rRNA) genes, and 30 transfer RNA (tRNA) genes (Figure 1 and Table 3). Eight protein-coding genes (ndhB, rpl2, rpl23, rps7, rps12, rps19, ycf1 and ycf2), seven tRNA genes (trnI-CAU, trnL-CAA, trnV-GAC, trnI-GAU, trnA-UGC, trnR-ACG and trnN-GUU), and all four rRNA genes are duplicated in IR regions (Figure 1 and Table 3). Sixteen genes include introns, with two genes (clpP and ycf3) having two introns each, while the remaining fourteen genes (atpF, petB, petD, ndhA, ndhB, rps12, rpl2, rpl16, trnA-UGC, trnG-GCC, trnI-GAU, trnK-UUU, trnL-UAA and trnV-UAC) each contain one intron (Figure 1 and Table 3). Notably, rps12 is trans-spliced, with its 5′ exon located in the LSC region, and its 3′ exon and intron duplicated and located in the IR regions (Figure 1). The trnK-UUU gene has the longest intron, which contains the matK gene.
Table 3. Gene composition of the 20 newly sequenced chloroplast genomes.
Table 3. Gene composition of the 20 newly sequenced chloroplast genomes.
Gene CategoryGene GroupGene Names
Photosynthesis-related genesRubiscorbcL
Photosystem IpsaA, psaB, psaC, psaI, psaJ
Assembly/stability of photosystem I* ycf3, ycf4
Photosystem IIpsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
ATP synthaseatpA, atpB, atpE, * atpF, atpH, atpI
Cytochrome complexpetA, * petB, * petD, petG, petL, petN
Cytochrome synthesisccsA
NADPH dehydrogenase* ndhA, * ndhB, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Transcription- and translation-related genesTranscriptionrpoA, rpoB, rpoC1, rpoC2
Ribosomal proteinsrps2, rps3, rps4, rps7, rps8, rps11, * rps12, rps14, rps15, rps16, rps18, rps19, * rpl2, rpl14, * rpl16, rpl20, rpl22, rpl23, rpl32, rpl33, rpl36
Translation initiation factorinfA
RNA genesRibosomal RNArrn5, rrn4.5, rrn16, rrn23
Transfer RNA* trnA-UGC, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-UCC, * trnG-GCC, trnH-GUG, trnI-CAU, * trnI-GAU, * trnK-UUU, trnL-CAA, * trnL-UAA, trnL-UAG, trnfM-CAUI, trnM-CAU, trnN-GUU, trnP-UGG, trnQ-UUG, trnR-ACG, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC, * trnV-UAC, trnW-CCA, trnY-GUA
Other genesRNA processingmatK
Carbon metabolismcemA
Proteolysis* clpP
Fatty acid synthesisaccD
Genes of unknown functionConserved reading frames 2ycf1, ycf2
Intron-containing genes are marked with asterisks (*).
Table 4. Results of variable-site analysis of the 20 newly sequenced chloroplast genomes.
Table 4. Results of variable-site analysis of the 20 newly sequenced chloroplast genomes.
RegionLength
(bp)		Variable SitesParsimony-
Informative Sites		Singleton SitesNucleotide Diversity
Number%Number%Number%
LSC91,1718030.884610.513420.380.00232
SSC19,8812541.281590.80950.480.00355
IR26,402290.11170.06120.050.00032
Total163,85611150.686540.404610.280.00180
Table 5. The variability of four highly variable markers and universal chloroplast DNA barcodes (rbcL, matK and trnH-psbA) was investigated in Crataegus and Mespilus.
Table 5. The variability of four highly variable markers and universal chloroplast DNA barcodes (rbcL, matK and trnH-psbA) was investigated in Crataegus and Mespilus.
MarkerLength
(bp)		Variable SitesParsimony-
Informative Sites		Nucleotide Diversity
Number%Number%
trnR(UCU)-atpA847364.25172.010.01381
ndhC-trnV(UAC)758222.90121.580.01227
ndhF-rpl321221310.56180.490.00732
ndhA2310261.05170.660.00352
trnH-psbA29784.2552.010.00781
rbcL142882.9071.580.00188
matK1521162.54101.470.00293
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Meng, J.; Wang, Y.; Song, H.; Dong, W.; Dong, N. Insights into Phylogeny, Taxonomy, Origins and Evolution of Crataegus and Mespilus, Based on Comparative Chloroplast Genome Analysis. Genes 2025, 16, 204. https://doi.org/10.3390/genes16020204

AMA Style

Meng J, Wang Y, Song H, Dong W, Dong N. Insights into Phylogeny, Taxonomy, Origins and Evolution of Crataegus and Mespilus, Based on Comparative Chloroplast Genome Analysis. Genes. 2025; 16(2):204. https://doi.org/10.3390/genes16020204

Chicago/Turabian Style

Meng, Jiaxin, Yan Wang, Han Song, Wenxuan Dong, and Ningguang Dong. 2025. "Insights into Phylogeny, Taxonomy, Origins and Evolution of Crataegus and Mespilus, Based on Comparative Chloroplast Genome Analysis" Genes 16, no. 2: 204. https://doi.org/10.3390/genes16020204

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Meng, J., Wang, Y., Song, H., Dong, W., & Dong, N. (2025). Insights into Phylogeny, Taxonomy, Origins and Evolution of Crataegus and Mespilus, Based on Comparative Chloroplast Genome Analysis. Genes, 16(2), 204. https://doi.org/10.3390/genes16020204

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