The complete chloroplast genome of Iris speculatrix Hance, a rare and endangered plant native to Hong Kong

Abstract Iris speculatrix is a rare and endangered plant first discovered in and native to Hong Kong. The whole chloroplast genome of I. speculatrix is 152,368 bp in length. It contained a large single copy region (82,003 bp), a small single copy region (17,941 bp), and two inverted repeats (26,212 bp). Phylogenetic analysis of 17 species of Iridaceae was conducted. I. speculatrix was found to be sister to a group of 12 Iris species, including I. setosa, I. lacteal, and I. uniflora. The sequenced chloroplast whole genome would be useful to understand the phylogeny and to conservation of I. speculatrix.

Iris speculatrix; Iridaceae; rare and endangered plant; phylogenetic analysis; chloroplast genome Content Iris speculatrix Hance (Iridaceae) (Hance 1875), a herb with attractive purplish flower, was first discovered and published in Hong Kong and is a rare and precious plant of Hong Kong scheduled under the Forestry Regulations (Hu et al. 2003). Members of the genus Iris are well known for their attractive flowers and are widely applied in horticulture (Kang et al. 2020).
The sample of I. speculatrix was collected in Tai Tam, Hong Kong, in collaboration with Agriculture, Fisheries and Conservation Department (AFCD), The Government of the Hong Kong Special Administrative Region (HKSAR) as part of the Rare and Endangered Species project. The species is scheduled under protective legislation (and exact locality details are restricted). Hence, it is required by AFCD, The Government of the Hong Kong Special Administrative Region that the GPS should be kept confidential. The voucher specimens with collector number Rare and endangered plants (SW Shek, KW Lam, DTW Lau, and JYY Lau) 009 were deposited in the Hong Kong Herbarium, AFCD, HKSAR (https://www.herbarium.gov.hk/en/home/index.html, Dr. LAU Yuen Yung, Jenny, herbarium@afcd.gov.hk) and the Shiu-Ying Hu Herbarium, School of Life Sciences, The Chinese University of Hong Kong (https://syhuherbarium.sls.cuhk.edu.hk/, David Tai Wai Lau, syhuherbarium.sls@cuhk.edu.hk).
Fresh leaves of Iris speculatrix (60 mg) was used for the extraction of total genomic DNA. The sample was extracted using i-genomic Plant DNA Extraction Mini Kit (iNtRON Biotechnology, Seongnam, Gyeonggi, Republic of Korea) The manufacturer's protocol was used. The concentration of the extract was checked using NanoDrop Lite (Thermo Fisher Scientific, Massachusetts, USA). Then, 1% agarose gel electrophoresis was used to visualize and check DNA quality. The sample was sequenced using NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA) by Novogene Bioinformatics Technology Co., Ltd. (https://en.novogene. com/, Beijing, China). A 150 bp paired-end (PE) library was constructed by Illumina sequencing. CLC Assembly Cell package v5.1.1 (CLC Inc., Denmark) was used to trim poor-quality reads (Phred score < 33).
CLC de novo assembler in CLC Assembly Cell package and SOAPdenovo v3.23 were used to assemble reads into contigs under default parameters. Gapcloser module in SOAP package was used to close gaps. After that, the contigs were aligned to the reference genome Iris rossii (NC_056180.1), and assembled into complete chloroplast genome. Gene annotation was obtained from GeSeq platform with reference to complete chloroplast genomes of Iris domestica (NC_050833.1) and Iris gatesii (NC_024936.1). A few adjustments were made manually for protein-coding genes, start and stop codons. The annotated genome was deposited in GenBank with the accession number OK274247.
The complete chloroplast genome of I. speculatrix was 152,368 bp in length, containing a large single copy (LSC) region (82,003 bp), a small single copy region (17,941 bp), and two inverted repeats (26,212 bp). The GC content was 38.04%. The chloroplast genome contained 112 unique genes, including 79 protein-coding genes, 29 tRNA genes, and 4 rRNA genes.
To investigate the taxonomic position of I. speculatrix, the chloroplast genome was aligned with 15 other species in the genus Iris, and the Sisyrinchium angustifolium (NC056184) within Iridaceae was used as an outgroup. The complete chloroplast genomes were aligned using MAFFT 7.48 (Katoh et al. 2019). A maximum likelihood (ML) tree was constructed using MEGA X (Kumar et al. 2018) based on the best fit model GTR þ G and 1000 bootstrap replicates. The phylogenetic tree was labeled with species and series (Figure 1). I. speculatrix was found to be sister to a group of 12 Iris species, including I. setosa, I. lactea and I. uniflora. In previous study of Iris phylogeny based on trnL-trnF and rps4 data, I. speculatrix was sister to a group of 17 Iris species including I. xiphium and I. latifolia, while the group was sister to another group of Iris containing I. setosa, I. lactea and I. uniflora . From Tillie's study, I. speculatrix was more distantly related to the Chinenses group of Iris including I. rossii. However, from our phylogenetic analysis, I. speculatrix was sister to a larger group of Iris, which included I. rossii and I. koreana from series Chinenses, I. setosa from the series Tripetalae. The species was more distantly related to I. tectorum from series Tectores and I. dichotoma from Pardanthopsis.
It was proposed that Iris from the series Chinenses should be classified to the new genus Zhaoanthus nom. provis. (Mavrodiev et al. 2014). Although it was suggested that I. speculatrix should be moved to Zhaoanthus based on morphological characters (Crespo et al. 2015;Wilson 2020), the treatment was not accepted by The World Checklist of Vascular Plants (Govaerts et al. 2021), where Zhaoanthus speculatrix was treated as a synonym of I. speculatrix.
In another study, I. speculatrix was also found to be outside of the Chinenses group (Guo and Wilson 2013). Tillie had suggested that I. speculatrix was highly sequence-divergent compared to other members of the group including I. xiphium and I. latifolia. Previous study had also suggested that I. speculatrix was sister to a group that included series Spuriae and Tenuifoliae . I. speculatrix was also found to be outside of series Chinenses from our study. Further study is required to resolve the taxonomic placement of I. speculatrix between different series among the genus Iris.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by Wu Jieh Yee Charitable Foundation Limited.