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The ML tree inferred from the nrITS dataset. The name of each clade of Ilex is shown on the right by bars. The Bayesian posterior probabilities greater than 0.5 are shown below the branches and the maximum likelihood (ML) and the maximum parsimony (MP) bootstrap percentages greater than 50 above.
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A new species, Ilex venusta, is described and illustrated from the Luoxiao Mountains, Jiangxi Province, China. It is included in a phylogenetic analysis using nuclear ribosomal nrITS and two plastid DNA markers (the atpB-rbcL and trnL-F spacers). This species is a member of Ilex sect. Paltoria., which can be distinguished from other sections in Ile...
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... the great discrepancy between nuclear and plastid trees found by Manen (2010), data for nrITS and combined plastid DNA datasets were analyzed separately. Parsimony analyses and data properties for the datasets used in this study are presented in Table 3. The ML tree was chosen as the basis for both nrITS and the combined plastid DNA trees. The nrITS tree ( Fig. 1) is generally congruent with morphological classification except for four species, I. venulosa (Hooker 1875: 602), I. fragilis (Hooker 1785: 602), I. serrata (Thunburg 1784: 78), I. rotunda (Thunburg 1784: 77). However, since introgression is well documented in Ilex (Manen et al. 2010), the plastid DNA tree (Fig. 2) is incongruent with the morphological classification. We mainly illustrate the nrITS tree here. Ilex sect. Paltoria is monophyletic with strong support (Fig. 1, 89/100/1; Fig. 2 This new species is a member of Ilex sect. Paltoria and can be distinguished from other sections in Ilex by solitary pistillate inflorescence. Ilex venusta is similar to I. viridis, I. triflora, but I. venusta differs in its narrow leaves, slender trunk and drooping branches (Table 3). Shrubs, 1.0-2.5 m tall, 1-2cm in diameter. Bark grey to brownish. Branchlets of first and second year slender, pendulous, green, terete, longitudinally ridged slightly apically, puberulent. Leaves persist one-three years on branchlets. Stipules deciduous, brown, lanceolate, ca. 0.7 mm; petiole 1.9-2.7 mm, puberulent. Leaf blade abaxially ...
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... the great discrepancy between nuclear and plastid trees found by Manen (2010), data for nrITS and combined plastid DNA datasets were analyzed separately. Parsimony analyses and data properties for the datasets used in this study are presented in Table 3. The ML tree was chosen as the basis for both nrITS and the combined plastid DNA trees. The nrITS tree ( Fig. 1) is generally congruent with morphological classification except for four species, I. venulosa (Hooker 1875: 602), I. fragilis (Hooker 1785: 602), I. serrata (Thunburg 1784: 78), I. rotunda (Thunburg 1784: 77). However, since introgression is well documented in Ilex (Manen et al. 2010), the plastid DNA tree (Fig. 2) is incongruent with the morphological classification. We mainly illustrate the nrITS tree here. Ilex sect. Paltoria is monophyletic with strong support (Fig. 1, 89/100/1; Fig. 2 This new species is a member of Ilex sect. Paltoria and can be distinguished from other sections in Ilex by solitary pistillate inflorescence. Ilex venusta is similar to I. viridis, I. triflora, but I. venusta differs in its narrow leaves, slender trunk and drooping branches (Table 3). Shrubs, 1.0-2.5 m tall, 1-2cm in diameter. Bark grey to brownish. Branchlets of first and second year slender, pendulous, green, terete, longitudinally ridged slightly apically, puberulent. Leaves persist one-three years on branchlets. Stipules deciduous, brown, lanceolate, ca. 0.7 mm; petiole 1.9-2.7 mm, puberulent. Leaf blade abaxially ...
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... I. venusta bears all the typical characters of I. sect. Paltoria, such as being a shrub with solitary pistillate inflorescences, four pyrenes, and conspicuous dark glands on abaxial side of the leaves, which are distinguishable from other sections in this genus. This section is distinctive not only in morphology but also in its phylogenetic position (Fig. 1). However, species within this section are difficult to distinguish from each other. Considering about this, we added molecular data into our ...
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... the great discrepancy between nuclear and plastid trees found by Manen (2010), data for nrITS and combined plastid DNA datasets were analyzed separately. Parsimony analyses and data properties for the datasets used in this study are presented in Table 3. The ML tree was chosen as the basis for both nrITS and the combined plastid DNA trees. The nrITS tree ( Fig. 1) is generally congruent with morphological classification except for four species, I. venulosa (Hooker 1875: 602), I. fragilis (Hooker 1785: 602), I. serrata (Thunburg 1784: 78), I. rotunda (Thunburg 1784: 77). However, since introgression is well documented in Ilex (Manen et al. 2010), the plastid DNA tree (Fig. 2) is incongruent with the morphological classification. We mainly illustrate the nrITS tree here. Ilex sect. Paltoria is monophyletic with strong support (Fig. 1, 89/100/1; Fig. 2 This new species is a member of Ilex sect. Paltoria and can be distinguished from other sections in Ilex by solitary pistillate inflorescence. Ilex venusta is similar to I. viridis, I. triflora, but I. venusta differs in its narrow leaves, slender trunk and drooping branches (Table 3). Shrubs, 1.0-2.5 m tall, 1-2cm in diameter. Bark grey to brownish. Branchlets of first and second year slender, pendulous, green, terete, longitudinally ridged slightly apically, puberulent. Leaves persist one-three years on branchlets. Stipules deciduous, brown, lanceolate, ca. 0.7 mm; petiole 1.9-2.7 mm, puberulent. Leaf blade abaxially ...
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... the great discrepancy between nuclear and plastid trees found by Manen (2010), data for nrITS and combined plastid DNA datasets were analyzed separately. Parsimony analyses and data properties for the datasets used in this study are presented in Table 3. The ML tree was chosen as the basis for both nrITS and the combined plastid DNA trees. The nrITS tree ( Fig. 1) is generally congruent with morphological classification except for four species, I. venulosa (Hooker 1875: 602), I. fragilis (Hooker 1785: 602), I. serrata (Thunburg 1784: 78), I. rotunda (Thunburg 1784: 77). However, since introgression is well documented in Ilex (Manen et al. 2010), the plastid DNA tree (Fig. 2) is incongruent with the morphological classification. We mainly illustrate the nrITS tree here. Ilex sect. Paltoria is monophyletic with strong support (Fig. 1, 89/100/1; Fig. 2 This new species is a member of Ilex sect. Paltoria and can be distinguished from other sections in Ilex by solitary pistillate inflorescence. Ilex venusta is similar to I. viridis, I. triflora, but I. venusta differs in its narrow leaves, slender trunk and drooping branches (Table 3). Shrubs, 1.0-2.5 m tall, 1-2cm in diameter. Bark grey to brownish. Branchlets of first and second year slender, pendulous, green, terete, longitudinally ridged slightly apically, puberulent. Leaves persist one-three years on branchlets. Stipules deciduous, brown, lanceolate, ca. 0.7 mm; petiole 1.9-2.7 mm, puberulent. Leaf blade abaxially ...
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... I. venusta bears all the typical characters of I. sect. Paltoria, such as being a shrub with solitary pistillate inflorescences, four pyrenes, and conspicuous dark glands on abaxial side of the leaves, which are distinguishable from other sections in this genus. This section is distinctive not only in morphology but also in its phylogenetic position (Fig. 1). However, species within this section are difficult to distinguish from each other. Considering about this, we added molecular data into our ...
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A new species, Ilex jiangmenensis, is described from Gudoushan Provincial Nature Reserve of Jiangmen City, a coastal city in southern Guangdong, China. It was observed to grow in rock crevices or beside the evergreen broad-leaved forests in the mountains of Gudoushan and Nanfengshan at an elevation of 50–550 m. The new species differs from its morp...
Citations
... Three DNA loci, the internal transcribed spacer (ITS) and the chloroplast psbA-trnH and petA-psbJ regions, were sequenced for phylogeny reconstruction, with the primers 17SE and 26SE (Sun et al. 1994), psbAF and trnHR (Sang et al. 1997a), and petA and psbJ (Shaw et al. 2007) employed, respectively. The detailed protocols of DNA extraction, PCR amplification, and DNA sequencing followed that of Jiang et al. (2017) and Yang et al. (2017). For all the individuals of I. sanqingshanensis, direct sequencing produced the superimposed chromatograms and unreadable peaks on most sites at the ITS maker, hence we implemented cloning sequencing to separate the PCR products. ...
Identification of natural hybrids considered as endangered species is of vital importance in taxonomy and biodiversity conservation, as natural hybrids will usually waste the conservation resource and efforts. During field surveys in the Sanqing Mountain, we found the endangered endemic species, Ilex sanqingshanensis, strictly co-occurred with I. ficoidea and I. pernyi and then hypothesized a hybrid origin for this taxon. Combing molecular analyzes of the nrDNA ITS and cpDNA (petA-psbJ + psbA-trnH) with morphological analyzes of eight leaf characters, we confirmed that this taxon is truly a hybrid between I. ficoidea and I. pernyi and we therefore to rename it as I. × sanqingshanensis. Despite the presence of intermediate morphological characteristics, this hybrid is sharply distinct from the two parents in almost all tested traits, misleading botanists who treated it as a species. Considering the inadequacies of morphological distinctions in distinguishing holly hybrids, we have emphasized the necessity of molecular evidence for erecting Ilex species.
... The total species number in the genus is still ill-defined due to insufficient taxonomic revision of the genus worldwide [3]. A total of 204 species have been recorded, and additional new species have been discovered and added to the checklist of Ilex in China [4][5][6][7][8][9][10][11]. The majority of these Ilex species are distributed in montane forests in southern areas of the Yangtze River [4]. ...
Many holly species are dominant species in evergreen broad-leaved forests, and they play an important role in the formation and maintenance of forest ecosystems in tropical and subtropical regions. Here, a new holly species (Ilex danxiaensis K. W. Xu & Q. Fan) of Aquifoliaceae was discovered from Guangdong, China, when conducting biodiversity investigations in the Danxia landform. We inferred the phylogenetic position of the new species based on sequences from three nuclear markers, namely internal transcribed spacer (ITS), external transcribed spacer (ETS), and chloroplast glutamine synthetase gene (nepGS). The results demonstrated a close relationship among the new species and members of I. sect. Lioprinos. The inferred phylogenetic position of the new species is also supported by morphological evidence that all of these species are characterized by having axillary cymes inflorescences and abaxially 1-sulcate pyrenes. However, the new species can be distinguished from others by having puberulent branchlets, petioles, peduncles, and pedicel; (1–)2–3-flowered cymes of male inflorescences; 1-flowered cymes of female inflorescences; ciliate sepals, 4-merous flowers, pink or white female flowers; and 4 pyrenes.
... Chloroplast markers were not chosen due to their poor resolution and the conflicts of the chloroplast gene tree with the species tree (Manen et al., 2010;Xu et al., 2022). Details of DNA extraction, primers, PCR amplification and sequencing can be found in Jiang et al. (2017) and Yang et al. (2018Yang et al. ( , 2020. Raw chromatograms were evaluated in Sequencher 5.4.6. ...
Resolving the infrageneric classification of species‐rich genera has been challenging in plant taxonomy. Ilex L. is a sub‐cosmopolitan genus with over 600 species of dioecious trees and shrubs. Many classification systems based on morphological data have been proposed during the past 250 years. However, these systems (such as Loesener's and Galle's systems) may not truly reflect Ilex's evolutionary trajectories because most of those system's infrageneric hierarchies are not monophyletic. In this study, we reconstructed a phylogeny of Ilex L. comprising fifteen moderately to highly supported clades using rigorously identified samples (202 species) and closely authenticated gene sequences of three nuclear genes (ITS, ETS, and nepGS). The newly generated phylogenetic tree essentially resembles that of the nuclear tree of Manen et al. (2010) but shows conspicuous topological differences with the phylogeny of Yao et al. (2021). Closely scrutinizing morphological variation and distributional patterns of 202 species, this study found that most lineages of Ilex identified herein are well‐defined by a particular trait or a combination of morphological and distributional traits, displaying phylogeny‐morphology‐distribution conformity that has seldom been uncovered in previous studies. Given the general phylogeny‐morphology‐distribution conformity revealed in this genus, we put forward an updated sectional classification system for Ilex that temporarily contains fourteen sections. The new classification will provide a robust framework for studying the evolution and diversification of this ecologically and economically important genus. This article is protected by copyright. All rights reserved.
... In the past two decades, advances in sequencing technology and analytical methods have contributed to greater phylogenetic resolution within Ilex. Several loci from both the nuclear and plastid genomes, including rbcL, trnL-trnF, atpB-rbcL, nuclear ribosomal DNA internal transcribed spacers (nrITS), and chloroplast glutamine synthetase (nepGS), have been used to estimate phylogenetic relationships within the genus [10][11][12][13][14][15][16][17]. However, a broad and representative sample of Ilex species has not yet been achieved in any phylogenetic study; thus the phylogeny of Ilex remains largely unresolved [13,16]. ...
... However, a broad and representative sample of Ilex species has not yet been achieved in any phylogenetic study; thus the phylogeny of Ilex remains largely unresolved [13,16]. Furthermore, recent phylogenetic studies have revealed substantial incongruence between the nuclear and plastid topologies [10,[13][14][15]. Recent molecular phylogenies did not support traditional classifications of Ilex based on morphological features [18,19]; however, these studies used only a few plastid or nuclear gene fragments and had generally poor resolution due to high conservation of plastid genes. ...
... Here, we expand Ilex genetic resources by newly sequencing the chloroplast genomes of seven species: I. dasyphylla, I. fukienensis, I. lohfauensis, I. venusta, I. viridis, I. yunnanensis, and I. zhejiangensis. Three of which, Ilex fukienensis, I. venusta, and I. zhejiangensis, are known to have a very narrow distribution in China [15,25], while the other four species are widely distributed in China and adjacent regions. We aimed to (i) investigate the structural and compositional variations of Ilex chloroplast genomes, (ii) identify highly variable regions useful for resolving interspecific relationships and species delimitation, and (iii) test the cyto-nuclear discordance by reconstructing high-resolution phylogenetic trees. ...
Background: Ilex (Aquifoliaceae) are of great horticultural importance throughout the world for their foliage and decorative berries, yet a dearth of genetic information has hampered our understanding of phylogenetic relationships and evolutionary history. Here, we compare chloroplast genomes from across Ilex and estimate phylogenetic relationships.
Results: We sequenced the chloroplast genomes of seven Ilex species and compared them with 34 previously published Ilex plastomes. The length of the seven newly sequenced Ilex chloroplast genomes ranged from 157,182bp to 158,009bp, and contained a total of 118 genes, including 83 protein-coding, 31 rRNA, and four tRNA genes. GC content ranged from 37.6 to 37.69%. Comparative analysis showed shared genomic structures and gene rearrangements. Expansion and contraction of the inverted repeat regions at the LSC/IRa and IRa/SSC junctions were observed in 22 and 26 taxa, respectively; in contrast, the IRb boundary was largely invariant. A total of 2146 simple sequence repeats and 2843 large repeats were detected in the 41 Ilex plastomes. Additionally, six genes (psaC, rbcL, trnQ, trnR, trnT, and ycf1) and two intergenic spacer regions (ndhC-trnV and petN-psbM) were identifed as hypervariable, and thus potentially useful for future phylogenetic studies and DNA barcoding. We recovered consistent phylogenetic relationships regardless of inference methodology or choice of loci. We recovered fve distinct, major clades, which were inconsistent with traditional taxonomic systems.
Conclusion: Our fndings challenge traditional circumscriptions of the genus Ilex and provide new insights into the evolutionary history of this important clade. Furthermore, we detail hypervariable and repetitive regions that will be useful for future phylogenetic and population genetic studies.
... In the past two decades, advances in sequencing technology and analytical methods have contributed to greater phylogenetic resolution within Ilex. Several loci from both the nuclear and plastid genomes, including rbcL, trnL-trnF, atpB-rbcL, nuclear ribosomal DNA internal transcribed spacers (nrITS), and chloroplast glutamine synthetase (nepGS), have been used to estimate phylogenetic relationships within the genus [10][11][12][13][14][15][16][17]. However, a broad and representative sample of Ilex species has not yet been achieved in any phylogenetic study; thus the phylogeny of Ilex remains largely unresolved [13,16]. ...
... However, a broad and representative sample of Ilex species has not yet been achieved in any phylogenetic study; thus the phylogeny of Ilex remains largely unresolved [13,16]. Furthermore, recent phylogenetic studies have revealed substantial incongruence between the nuclear and plastid topologies [10,[13][14][15]. Recent molecular phylogenies did not support traditional classifications of Ilex based on morphological features [18,19]; however, these studies used only a few plastid or nuclear gene fragments and had generally poor resolution due to high conservation of plastid genes. ...
... Here, we expand Ilex genetic resources by newly sequencing the chloroplast genomes of seven species: I. dasyphylla, I. fukienensis, I. lohfauensis, I. venusta, I. viridis, I. yunnanensis, and I. zhejiangensis. Three of which, Ilex fukienensis, I. venusta, and I. zhejiangensis, are known to have a very narrow distribution in China [15,25], while the other four species are widely distributed in China and adjacent regions. We aimed to (i) investigate the structural and compositional variations of Ilex chloroplast genomes, (ii) identify highly variable regions useful for resolving interspecific relationships and species delimitation, and (iii) test the cyto-nuclear discordance by reconstructing high-resolution phylogenetic trees. ...
Background
Ilex (Aquifoliaceae) are of great horticultural importance throughout the world for their foliage and decorative berries, yet a dearth of genetic information has hampered our understanding of phylogenetic relationships and evolutionary history. Here, we compare chloroplast genomes from across Ilex and estimate phylogenetic relationships.
Results
We sequenced the chloroplast genomes of seven Ilex species and compared them with 34 previously published Ilex plastomes. The length of the seven newly sequenced Ilex chloroplast genomes ranged from 157,182 bp to 158,009 bp, and contained a total of 118 genes, including 83 protein-coding, 31 rRNA, and four tRNA genes. GC content ranged from 37.6 to 37.69%. Comparative analysis showed shared genomic structures and gene rearrangements. Expansion and contraction of the inverted repeat regions at the LSC/IRa and IRa/SSC junctions were observed in 22 and 26 taxa, respectively; in contrast, the IRb boundary was largely invariant. A total of 2146 simple sequence repeats and 2843 large repeats were detected in the 41 Ilex plastomes. Additionally, six genes ( psaC , rbcL , trnQ , trnR , trnT , and ycf1 ) and two intergenic spacer regions ( ndhC - trnV and petN - psbM ) were identified as hypervariable, and thus potentially useful for future phylogenetic studies and DNA barcoding. We recovered consistent phylogenetic relationships regardless of inference methodology or choice of loci. We recovered five distinct, major clades, which were inconsistent with traditional taxonomic systems.
Conclusion
Our findings challenge traditional circumscriptions of the genus Ilex and provide new insights into the evolutionary history of this important clade. Furthermore, we detail hypervariable and repetitive regions that will be useful for future phylogenetic and population genetic studies.
... In the past two decades, the advances in sequencing technology and analytical methods have contributed to the phylogenetic reconstruction of the genus Ilex using several plastid DNA fragments, including rbcL, trnL-trnF, and atpB-rbcL as well as the nuclear ribosomal DNA internal transcribed spacers (nrITS) and chloroplast glutamine synthetase gene (nepGS) sequences [8][9][10][11][12][13][14]. Although a large taxon sampling of Ilex has been achieved, the phylogeny of Ilex is still not completely resolved [11,14]. ...
... Although a large taxon sampling of Ilex has been achieved, the phylogeny of Ilex is still not completely resolved [11,14]. Substantial incongruence between the nuclear phylogeny and the plastid phylogeny of the genus Ilex has been found in recent phylogeny studies, but only a few plastid or nuclear gene fragments were used in these studies and the resolution was generally poor due to their relatively conserved features in some plastid genes [8, [11][12][13]. Furthermore, the ndings obtained from molecular phylogenetic analysis did not complement to those previously proposed traditional classi cation of Ilex based on morphological features [15,16]. ...
... In the effort to expand the current genetic resource of Ilex at chloroplast genome-scale, herein, we newly sequenced the chloroplast genomes of seven species of Ilex, including I. dasyphylla, I. fukienensis, I. lohfauensis, I. venusta, I. viridis, I. yunnanensis, and I. zhejiangensis. Three of them, Ilex fukienensis, I. venusta, and I. zhejiangensis, were known to have a very narrow distribution in China [13,22], while the other four species are widely distributed in China and adjacent regions. We aimed: (i) to investigate the structural and compositional variations of the chloroplast genomes in genus Ilex by involving more lineages; (ii) to identify highly variable regions in the chloroplast genomes that could be used as potential markers in resolving the interspeci c relationship and species delimitation of Ilex; and (iii) to reconstruct a high resolution phylogenetic tree based on the complete chloroplast genome sequences that is further tested for its cyto-nuclear discordance by comparing to existing nuclear-based phylogenetic tree of the genus Ilex. ...
Background
Despite many species of Ilex (Aquifoliaceae) are of horticultural importance and are widely grown in parks and gardens throughout the world for their foliage and decorative berries, limited genetic information has greatly hampered our understanding of the chloroplast genome evolution and phylogenetic relationships within the genus. This study attempted to address these problems by comparing the chloroplast genomes and analyzing phylogenetic relationships within the genus.ResultsIn this study, analyses of chloroplast genome structure, codon usage, GC content, gene rearrangement, nucleotide diversity, inverted repeats (IR) boundary, repeat sequence, and SSR component were conducted by comparing 41 chloroplast genomes of Ilex . The results showed that these Ilex chloroplast genomes were evolutionary conserved at the genome level and no rearrangement of the complete cp genome in the 41 Ilex genomes was recorded. On the contrary, there were still a few mutational hotspots identified from these chloroplast genomes, which were considered as hypervariable regions useful for future phylogenetic studies and DNA barcoding. Using the complete chloroplast genome sequences, we reconstructed a highly supported phylogeny of Ilex and well-resolved the complicated relationships among the different lineages within Ilex .Conclusion
The present study increased our understanding of the chloroplast genome evolution and phylogenetic relationships within Ilex . The availability of these genetic resources will be helpful for the future studies in DNA barcoding, species delimitation, phylogenetic reconstruction as well as the hybridization and introgression events between distantly related lineages within Ilex .
... ITS and ETS, were employed for the phylogenetic analyses. The detailed information of primers and protocols for DNA extraction, PCR amplification, and DNA sequencing followed that of Jiang et al. (2017) and Yang et al. (2018). Voucher information and GenBank accession numbers for all sequences used in the present study were shown in Appendix S1. ...
Based on the results of phylogenetic reconstructions employing two nuclear ribosomal DNA loci (ITS and ETS), as well as morphological observation and comparisons of branches, leaves, inflorescences/infructescences and pyrenes, a taxonomic revision of three species of Ilex from China is conducted and presented herein. Ilex fargesii is subdivided into I. fargesii subsp. fargesii and I. fargesii subsp. melanotricha, with I. fargesii var. angustifolia and I. fargesii var. brevifolia newly treated as synonyms of the type subspecies. The split of the two subspecies of I. fargesii in the molecular phylogeny is conformed with their divergence in distribution, which is clearly separated by the Tanaka-Kaiyong Line. Similar geographic disjunction is also revealed in the two subspecies of I. wattii, i.e. I. wattii subsp. wattii and I. wattii subsp. marlipoensis. Meanwhile, I. gintungensis and I. venosa newly reduced to the synonyms of I. wattii subsp. wattii, and I. marlipoensis and I. cupreonitens to I. wattii subsp. marlipoensis. Furthermore, we confirm I. pubifructa to be a newly recorded species in China and I. denticulata to be excluded from China. Both our molecular and morphological analyses do not support the synonymization of I. chartaceifolia (including I. chartaceifolia var. glabra) and I. micropyrena with I. fargesii. However, the specific status of I. chartaceifolia and I. micropyrena is not resumed in this study because of their intricate and unclear relationships with I. dunniana, I. intermedia and I. subodorata.
... Ten Ilex and one Helwingia chloroplast genomes from Aquifoliaceae were used for constructing neighbor joining (bootstrap repeat is 10,000) and maximum likelihood (bootstrap repeat is 1,000) phylogenetic trees using MEGA X (Kumar et al. 2018) after aligning whole chloroplast genome sequences using MAFFT 7.388 (Katoh and Standley 2013). Phylogenetic trees show that I. cornuta is clustered with Ilex latifolia and new species of Ilex (Section Ilex in Figure 1; Yao et al. 2016), agreeing with previous studies (Cu enoud et al. 2000;Jiang et al. 2017) in Section Ilex ). In addition, Ilex dumosa and Ilex paraguariensis originated from South America are not in basial clade of Ilex genus with high bootstrap supports (Figure 1), disagreeing with the previous study . ...
Ilex cornuta Lindl. & Paxton is a species distributed in eastern China and Korea, utilized as traditional medical plants as well as horticultural species. Here, we completed chloroplast genome of I. cornuta. Its length is 157,224 bp long and has four subregions: 86,610 bp of large single copy (LSC) and 18,429 bp of small single copy (SSC) regions are separated by 26,092 bp of inverted repeat (IR) regions including 131 genes (86 protein-coding genes, eight rRNAs, and 37 tRNAs). The overall GC content of this chloroplast genome is 37.7% and those in the LSC, SSC, and IR regions are 35.7%, 31.9%, and 42.9%, respectively. Phylogenetic trees show that I. cornuta is clustered in Section Ilex clade clearly. In addition, two Ilex species from South America are not in a basal clade, which is different from previous phylogenetic study, suggesting more Ilex chloroplast genomes are required to understand phylogenetic relationships of Ilex species.
... Ilex (see Figure S1). The detailed protocols of DNA extraction, PCR amplification, and DNA sequencing followed that of Jiang et al. (2017). Voucher information and GenBank accession numbers of sequences newly generated in present study (55 sequences) and downloaded online (4 sequences) are shown in Table 1. ...
Ilex formosana and I. tetramera, both occurring in subtropical forests of China, are easily confused in morphology and distribution. To unravel their identities, phylogenetic analyses of multiple accessions of the two species and their possible relatives using two nuclear DNA loci (ITS and ETS) are conducted in present study. Our results show that I. formosana (sampled from type locality) is closest to I. matanoana and I. mertensii, and I. tetramera (sampled from type locality) is sister to I. nuculicava, while the remaining samples, which were originally identified as I. formosana or I. tetramera, form a strongly supported clade sister to I. cinerea, representing a cryptic new species, which is named here as I. shukunii. Despite the homogeneity in morphology, the divergence in the distribution of I. formosana, I. shukunii, and I. tetramera also supports our phylogenetic results. Insulated by the Taiwan Strait, the re-defined I. formosana is restricted to Taiwan Island. Separated by the Tanaka-Kaiyong Line, a major phytogeographic boundary of Sino-Himalayan and Sino-Japanese Floras, I. shukunii occurs from southeast Yunnan to southeast mainland of China, whereas the re-defined I. tetramera is endemic to southwest Yunnan.
... In addition, comparing of the genetic diversity patterns across the main lineages in Quercus can provide more information to infer the driving forces which might contribute to their divergence. Furthermore, the knowledge on biodiversity inventory in East Asian subtropics is far from enough, and new species are continuously being identified in recent years (e.g., Xiao et al., 2015;Liang et al., 2016;Jiang et al., 2017;Wang C.W. et al., 2017). DNA barcoding of keystone species in this region can provide crucial insight into the mechanism of plant community assembly and evolutionary trajectory of the East Asian regional biota. ...
The East Asian subtropics mostly occupied by evergreen broad-leaved forests (EBLFs), is one of the global diversity centers for evergreen oaks. Evergreen oaks are keystone canopy trees in EBLFs with important ecosystem function and crucial significance for regional biodiversity conservation. However, the species composition and diversity of Asian evergreen oaks are poorly understood. Here, we test whether the four chloroplast markers atpI-atpH, matK, psbA-trnH, and ycf1, can discriminate the two evergreen oak sections in Asia – Cyclobalanopsis and Ilex. Two hundred and seventy-two individuals representing 57 species were scanned and 17 species from other oaks sections were included for phylogenetic reconstruction. The genetic diversity of the Quercus sections was also compared. Overall, we found that universal chloroplast DNA (cpDNA) barcoding markers could resolve two clades in Quercus, i.e., subgenus Cerris (Old World Clade) and subgenus Quercus (New World Clade). The chloroplast markers distinguished the main sections, with few exceptions. Each cpDNA region showed no barcoding gap and none of them provided good resolution at the species level. The best species resolution (27.78%) was obtained when three or four markers were combined and analyzed using BLAST. The high conservation of the cpDNA and complicated evolutionary patterns, due to incomplete lineage sorting, interspecific hybridization and introgressions may hinder the ability of cpDNA markers to discriminate different species. When comparing diversification pattern across Quercus sections (Cyclobalanopsis, Ilex, Cerris, Quercus, and Protobalanus), we found that section Ilex was the most genetically diverse, and section Cyclobalanopsis was lower genetically diverse. This diversification pattern may have resulted from the interplay of the Eurasia Cenozoic tectonic movements, climate changes and different niches of their ancestral lineages.
