High-throughput sequencing yields the complete plastid genome of the endemic species Phragmipedium kovachii (Orchidaceae) from northeastern Peru

Abstract Phragmipedium kovachii is a species of orchid endemic to the Amazonas and San Martín regions. Unfortunately, its excessive extraction has made it a critically endangered species. In this study, we performed next-generation sequencing of P. kovachii (GenBank accession number OR348669) and assembled its complete chloroplast genome. The complete chloroplast genome of P. kovachii is A + T-rich (64.3%), measuring 152,918 bp in length. This plastid genome contains a total of 124 genes (77 protein-coding genes, 39 tRNAs, and eight rRNAs) and five pseudogenes, including a pair of inverted repeats (IRs) 25,116 bp in size and separated by a large single-copy (LSC) region of 89,216 bp and a small single-copy (SSC) region of 13,470 bp. This genome has a typical quadripartite organization following the structure of other Orchidaceae plastomes. Phylogenetic analyses revealed the close relationship between P. kovachii and P. besseae. This study contributes to the understanding of the phylogenetic relationships of the monophyletic group Cypripedioideae.


Introduction
Orchidaceae is one of the largest families of flowering plants, representing approximately 10% of seed plants (Roberts and Dixon 2008;Wang et al. 2019).One of the most representative groups of this family is the monophyletic subfamily Cypripedioideae, commonly known as slipper orchids, which are characterized by flowers with a lip and a column (Cameron et al. 1999;Chase et al. 2003;Yen et al. 2022).This group is composed of approximately 200 species distributed in the five genera Cypripedium, Mexipedium, Paphiopedilum, Phragmipedium, and Selenipedium (Guo et al. 2012;Unruh et al. 2018).An orchid with one of the most beautiful flowers in the world is Phragmipedium kovachii, which is 10-20 cm in size with pink to purple petals and large lips up to 7.5 cm long and 4 cm wide (Atwood et al. 2002;Cribb 2007).P. kovachii is an endemic species of northeastern Peru restricted to the Amazonas and San Mart� ın regions (Mill� an et al. 2007).
This species was first described as P. kovachii by Atwood et al. (2002) based on material that was illegally introduced to the US and deposited in the Selby Botanical Gardens.A few days later, this species was described as Phragmipedium peruvianum by Christenson (2002).The oldest name given to a plant is the correct name according to the norms of priority of the International Code of Botanical Nomenclature, with later names being relegated to synonymy (Turland et al. 2018).Accordingly, P. peruvianum is now a synonym of P. kovachii (Cribb 2007).The discovery of P. kovachii led to uncontrolled extraction of many specimens, in turn reducing its population size and distribution (Mill� an et al. 2007).Currently, P. kovachii is classified as critically endangered according to the Red List criteria (IUCN criterion and guideline) since its geographical distribution is less than 100 km 2 , while its population is restricted to 25 individuals (Cribb 2007).This will likely result in its extinction in the wild in the next 10 years (Rankou 2016).
In Peru, 11 species of the genus Phragmipedium are recognized, and P. kovachii is currently categorized as a threatened species according to Supreme Decree N � 043-2006-AG (SERFOR 2020).Additionally, P. kovachii is a protected species according to Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES 2017).This protection is on the basis of the IUCN criterion and guideline.Most studies on P. kovachii are limited to morphological analyses (Ruiz S� anchez 2021), and studies at the molecular level are insufficient (Leitch et al. 2009).Genetic and genomic analyses provide information about species gene transfer, cloning, domestication, and evolutionary information (Vu et al. 2020;Song et al. 2022).Accordingly, the aim of this study was to decodify the plastid genome of the critically endangered P. kovachii using next-generation sequencing technology in order to (i) give insights about plastome composition and (ii) better understand the evolutionary history of the species.
Genomic DNA was extracted from P. kovachii (specimen voucher KUELAP988) using a NucleoSpin Kit (Macherey-Nagel, D€ uren, Germany) following the manufacturer's instructions.The DNA was fragmented and ligated to unique adapters with the Swift 2S Turbo DNA Library Prep Kit (Swift Bioscience, Ann Arbor, MI).The 150 bp PE Illumina library was constructed and sequenced using the NovaSeq platform from Macrogen (Seoul, South Korea).The genome was assembled using default de novo settings in MEGAHIT (Li et al. 2021) and Geneious Prime 2023.2 (https://www.geneious.com)to close gaps.Sequencing depth and coverage were calculated following Yang et al. (2023).Genes were manually annotated using blastx, NCBI ORFfinder, and tRNAscan-SE 2.0 (Lowe and Chan 2016).The plastid genome of P. kovachii was aligned with other plastomes using MAFFT (Katoh and Standley 2013).Phylogenetic analysis was performed with RAxML-NG (Kozlov et al. 2019) using the GTR þ gamma model and 1000 bootstraps (Tineo et al. 2022).The tree was visualized with TreeDyn 198.3 at Phylogeny.fr(Dereeper et al. 2008).

Phylogenetic analysis
The presence of highly supported nodes in the phylogenetic analysis of plastid genome sequences from specimens within the subfamily Cypripedioideae confirmed that the genus Phragmipedium is sister to the clade comprising the genera Cypripedium and Mexipedium (Figure 3).This analysis also revealed that P. kovachii is a sister species of P. besseae.

Discussion
Currently, 21 species of the genus Phragmipedium are accepted (D� ıas-Morales et al. 2019), and the sixth complete plastid genome of this genus was assembled in the present study.The plastome of P. kovachii is highly conserved in length, content, and organization compared to that of other species assigned to Phragmipedium (Table 1).The plastid genome of P. kovachii was larger than that of the other members of Phragmipedium (Table 1).

Figure 1 .
Figure 1.Morphology of Phragmipedium kovachii (KUELAP988).(A) Habit, showing a plant with a single flower.(B, C) Prominent flower with petals and a lip.All images were obtained from Yoiner Kalin Lapiz from the plant nursery Kgory Thika, Yambrasbamba, Bongar� a, Amazonas.

Figure 2 .
Figure 2. Schematic map of the general characteristics of the chloroplast genome of Phragmipedium kovachii.The map contains six tracks by default.From the center outward, the first track shows the scattered repeats connected with arcs.The second track shows the long tandem repeats as short bars.The third track shows the short tandem repeats or microsatellite sequences as short bars.The small single-copy (SSC), inverted repeat (IRa and IRb), and large single-copy (LSC) regions are shown in the fourth track.The GC content along the genome is represented in the fifth track.The genes are shown in the sixth track.Optional codon usage bias is shown in parentheses after the gene name.Genes are coded according to their functional classification.The transcription directions of the inner and outer genes are clockwise and counterclockwise, respectively.The functional classification of the genes is shown in the lower left corner.