﻿Neottiabifidus (Orchidaceae, Epidendroideae, Neottieae), a new mycoheterotrophic species from Guizhou, China

﻿Abstract Neottiabifidus, a new mycoheterotrophic orchid, found in Maolan National Nature Reserve in Guizhou Province, China, is described and illustrated here. The new species is close to N.nidus-avis, N.kiusiana and N.papilligera but differs in having a finely pubescent rachis with fewer flowers, a finely pubescent pedicel, and a fishtail-shaped lip that is deeply bilobed to the middle of the lip, with the lobes diverging at an acute angle (45°) to each other and mesochile with many papillae. Additionally, N.bifidus is well supported as a new species by molecular phylogenetic results based on ITS and chloroplast genome. The chloroplast genome of the novelty, which contains an LSC region of 33,819 bp, SSC region of 5,312 bp and IRs of 46,762 bp was assembled and annotated. A key to mycoheterotrophic Neottia species in China is also provided.


Introduction
The genus Neottia Guett. comprises 81 accepted species, including 63 autotrophic species and 18 mycoheterotrophic species (https://powo.science.kew.org, Mu et al. 2017;Chen and Jin 2021), distributed widely in north temperate areas with a few species extending into alpine regions in the mountains of tropical Asia (Govaerts et al. 2019;Chen and Jin 2021). East Asia is one of the diversity centers for this genus with more than 70% of Neottia species occurring in this region (So and Lee 2020). Formerly, Neottia was divided into Listera and Neottia (Bentham 1881;Pfitzer 1887;Schlechter 1926;Brieger et al. 1974;Dressler 1981;Rasmussen 1982) by the distinct morphological differences possessed by autotrophic plants (Listera) with two opposite leaves (sometimes three or more) in the middle of the stem, while mycoheterotrophic plants are achlorophyllous and possess densely fleshy bird's nest like roots. In 2003, Govaerts cited another genus in Tribe Neottieae Lindl., Holopogon, as a synonym of Neottia (Govaerts 2003).
There are 52 species and one variation of Neottia in China, amongst which 14 species are mycoheterotrophic (https://powo.science.kew.org, Mu et al. 2017;Chen and Jin 2021). During our fieldwork in the Maolan National Nature Reserve, Libo County, Guizhou Province, China in 2021, an unknown species of mycoheterotrophic Neottia was found in the evergreen broad-leaved forest. Based on morphological characters and molecular evidence, it was considered as a new species of Neottia and is described below.

Materials and methods
Morphological characteristics of the new species were observed, measured and photographed, based on living plants in Maolan National Nature Reserve, Guizhou. The studied specimens are deposited at The National Orchid Conservation Center of China and the Orchid Conservation & Research Center of Shenzhen. The general morphology was derived from fresh specimens and photographs were taken with a DSLR camera. To investigate the systematic position of the new species, the plastid genome and the nuclear ribosomal internal transcribed spacers (nrITS) marker were used in molecular phylogenetic analysis. Total genomic DNA was extracted from fresh flowers and stems (voucher specimens J.B.Chen 00599) using a plant genomic DNA kit and then sent to Novogene (Beijing, China) for the library (350 bp) preparation for genome skimming sequencing. Paired-end (150 bp) sequencing was conducted on the Illumina Hiseq 6000 platform (San Diego, CA), producing approximately 8 Gb reads. The plastid genome was assembled using GetOrganelle (Jin et al. 2020) with the chloroplast genome of Neottia camtschatea (L.) Rchb. F.(NC_030707) and Neottia listeroides Lindl. (NC_030713) as the reference sequences. After assembly, the obtained scaffolds and contigs were annotated by Geneious Prime (Biomatters Ltd., Auckland, New Zealand) (Kearse et al. 2012) and Plastid Genome Annotator (Qu et al. 2019). The annotated complete chloroplast genome was deposited in GenBank with accession number OP279442. nrITS were also sequenced for the new species in this study. The PCR reactions and Sanger Sequencing were performed by Sangon Biotech (Shanghai, China). The primers used in this study are presented in Table 1. In total, 70 species (incl. 29 species of Neottia) from seven genera were used for molecular phylogenetic analyses ( Table 2). The nrITS dataset consists of six genera and 66 species and the plastid genome dataset consists of five genera and 27 species, respectively. Five species of Cionisaccus, Ophrys and Serapias were selected as outgroup taxa based on Li et al. (2016). All plastid genomes were aligned by MAFFT 7.3 (ffT-NS-i × 1000 strategy) after removing one inverted repeat (IR) region of each sample (Katoh and Standley 2013). Poorly-aligned regions were removed by trimAl 1.2 with default settings before phylogenetic analyses (Capella-Gutiérrez et al. 2009). Maximum Likelihood (ML) analyses were conducted in IQTREE 1.6 using the SH-aLRT test and ultrafast bootstrap (UFBoot) feature (-alrt 1000 -bb 1000 -nt AUTO) (Nguyen et al. 2015;Hoang et al. 2018

Results
The whole chloroplast genome of N. bifidus showed a typical quadripartite structure containing a pair of inverted repeats (IRs) separated by a large single-copy (LSC) region and a small single-copy (SSC) region (Fig. 1). The complete plastid genome sequence of N. bifidus was 85,893 bp in length containing an LSC region of 33,819 bp, SSC region of 5,312 bp and IRs of 46,762 bp. The chloroplast genome contained 72 genes, including 36 protein-coding genes, 28 tRNA genes and eight rRNA genes ( Table 3). The overall GC content is 35%. The phylogenetic analyses indicated that this unknown species is far from other autotrophic species, but has a better clustering relationship with leafless holomycotrophic species in Neottia. The phylogenetic tree, based on the plastid genome, indicated that it is close to N. kiusiana T.Hashim. & S.Hatus. (KT338757) with high support (SH-aLRT 100%, UfBoot 100%) and then sister to N. nidus-avis (L.) Rich. (JF325876) also with strong support (SH-aLRT 100%, UfBoot 100%) (Fig. 2). The phylogenetic tree, based on nrITS, showed that the new species is sister to N. kiusiana and N. papilligera Schltr. with high support (SH-aLRT 100%, UfBoot 100%) (Fig. 3).

Group of genes Gene
Photosystem I -   Diagnosis. Neottia bifidus is morphologically similar to N. nidus-avis, N. kiusiana and N. papilligera but differs in having a finely pubescent rachis, with fewer flowers; finely pubescent pedicel; and fish-tail-shaped lip, deeply 2-lobed to the centre of mid-lip, lobes diverging at an acute angle (45°) to one another, mesochile with many papilloses (Table 4).
Etymology. The species epithet refers to the fish-tail-shaped lip of the new species.
Distribution and habitat. Neottia bifidus is currently known only from the type locality in Libo, Guizhou, China. It grows in humus-rich soil under broad-leaved forests at elevations of 700-900 m and is found growing with Miliusa sinensis  Conservation status. During our fieldwork, only one population with less than 10 individuals was discovered in Maolan National Nature Reserves (213 km 2 ). Most individuals were found growing along the roadside and are easily disturbed by human activities. According to the guidelines for using the IUCN Red List Categories and Criteria (IUCN 2022), the new species should be temporarily assigned as 'Critically Endangered' by its limited populations, localities and vulnerable habitats.  Rachis sparsely glandular hairy, with 10-28 flowers.
Rachis glabrous or pubescent, with much more than 20 flowers.
Rachis glabrous, with much more than 20 flowers.
Note. Neottia bifidus is morphologically -related to three species, namely, N. nidus-avis, N. kiusiana and N. papilligera, but it is readily distinguished from them, based on morphological characters given in Table 4.