The complete chloroplast genome of Chrysoglossum ornatum (Epidendroideae, Orchidaceae) and its phylogenetic analysis

Abstract Chrysoglossum ornatum Blume, the type species of Chrysoglossum Blume, belongs to the tribe Collabieae of the subfamily Epidendroideae of Orchidaceae. In this study, we sequenced, assembled, and analyzed the complete chloroplast genome of C. ornatum. The result showed that the complete chloroplast genome of C. ornatum was 158,175 bp in size, consisting of a large single-copy (LSC) region of 87,235 bp, a small single-copy (SSC) region of 18,384 bp, and a pair of inverted repeats (IRs) of 26,278 bp. The chloroplast genome encoded 113 unique genes, comprising 80 protein-coding genes, 29 tRNA genes, and four rRNA genes. Phylogenetic analysis inferred from the complete chloroplast genome indicated that Chrysoglossum was closely related to Collabium Blume. This study provides genomic resources helpful for further phylogenetic and biodiversity research on Chrysoglossum.


Introduction
Orchidaceae is one of the largest families of flowering plants, with approximately 736 genera and 28,000 species (Christenhusz and Byng 2016).Molecular phylogenetic studies have recognized five monophyletic subfamilies within Orchidaceae, namely Apostasioideae, Vanilloideae, Cypripedioideae, Orchidoideae, and Epidendroideae (Chase et al. 2015).Chrysoglossum Blume, a small genus with four species (Chrysoglossum assamicum Hook.f., Chrysoglossum ensigerum W. Burgh & de Vogel, Chrysoglossum ornatum Blume, and Chrysoglossum reticulatum Carr), belongs to the tribe Collabieae of the subfamily Epidendroideae of Orchidaceae and mainly ranges from tropical Asia to New Guinea and the Pacific islands (Xiang et al. 2014;Zhou et al. 2023).Chrysoglossum is morphologically similar to the genus Collabium Blume and was once treated as a synonym of Collabium (Seidenfaden 1983).Phylogenetic analyses based on four plastid markers demonstrated that Collabium and Chrysoglossum should be maintained as two distinct genera (Xiang et al. 2014).However, the phylogenetic resolution among closely related species was very low (Xiang et al. 2014).
Compared with nuclear and mitochondrial genomes, the chloroplast genome has great advantages for genetic and phylogenetic analyses, owing to its stable genetic structure, relatively small size, and moderate nucleotide substitutions (Daniell et al. 2016).However, to date, the chloroplast genomes of Chrysoglossum are still lacking.In this study, we sequenced, assembled, and analyzed the complete chloroplast genome of Chrysoglossum ornatum, which will contribute to further phylogenetic and biodiversity studies of Chrysoglossum.

Phylogenetic analyses
Phylogenetic tree was constructed based on the complete chloroplast genome using maximum-likelihood (ML) and Bayesian inference (BI) methods in CIPRES Science Gateway (https://www.phylo.org/).All sequences were aligned using MAFFT v.7.388 with auto strategy (Katoh and Standley 2013).The GTRGAMMA model was employed to infer the ML tree, with 1000 bootstrap (BS) replicates.The best-fitting model of BI analysis was selected according to Akaike information criterion (AIC) (David and Buckley 2004).The BI tree was constructed under the GTR þ F þ I þ G4 model using Markov Chain Monte Carlo (MCMC) algorithm, with 2,000,000 generations independently and every 1000 generations for tree sampling.Finally, we edited these results in the iTOL (Letunic and Bork 2007).
In total, 33 species were included in phylogenetic analyses, of which 30 species were from the subfamily Epidendroideae, with the other three species from the subfamily Orchidoideae as the outgroups (Figure 3).The phylogenetic trees inferred from ML and BI yielded identical topologies.As a result, only the ML tree was presented with ML bootstrap support (BS) and BI posterior probability (PP) values indicated.Phylogenomic analysis showed that Chrysoglossum was sister to Collabium with a full support value (BS ¼ 100, PP ¼ 1.00).

Discussion and conclusions
Similar to most angiosperms (Daniell et al. 2016), the complete chloroplast genome of Chrysoglossum ornatum displayed a typical quadripartite structure.Generally, the size of the chloroplast genome in the land plants ranges from 120 to 160 kb encoding 110 to 130 unique genes (Green 2011).Chrysoglossum ornatum showed relative conservation in genome size and gene content.In Flora Reipublicae Popularis Sinicae, Chrysoglossum and Collabium were placed in the subtribe Collabiinae, and Risleya was placed in the subtribe Risleyinae, all of which belonged to the tribe Epidendreae.Using four DNA markers, however, Chrysoglossum was sister to Risleya and clustered together with Collabium, in which the three genera were retreated into the tribe Collabieae (Xiang et al. 2014).The treatment was followed by Chase et al. (2015).Nevertheless, Li et al. (2020) based on the complete plastid data suggested that the genus Risleya should be placed in the tribe Epidendreae.In this study, phylogenetic analyses demonstrated that Chrysoglossum was closely related to Collabium with strong support (BS ¼ 100, PP ¼ 1.00) and also supported the treatment of Risleya from Collabieae to Epidendreae.It is worth mentioning that phylogenetic analysis based on the complete chloroplast genome generated a higher resolution than that inferred from a few DNA markers.Overall, our study provides a molecular resource for phylogenetic and evolutionary studies of Chrysoglossum.

Figure 2 .
Figure 2. The chloroplast genome map of Chrysoglossum ornatum.From the center outward, the map consists of six rings.The first circle represents the forward and reverse repeats connected with red and green arcs, respectively.The second circle shows the tandem repeats marked.The third circle displays the microsatellite sequences.The fourth circle indicates the sizes of feature regions, including a large single-copy (LSC), a small single-copy (SSC), and two inverted repeats (IRa and IRb).The fifth circle exhibits the GC content.The sixth circle presents the genes with different functions.