DOI: 10.5061/dryad.02v6wwq90
title: Spiranthes stylites is another long overlooked Asian Spiranthes with an unusual Sylvian habitat

description: We reexamine the taxon Spiranthes stylites, currently synonymized under S.
 australis, finding multiple lines of evidence from molecular
 phylogenetics, phenology, morphology, and habitat differences to support
 its distinction from other members of the Spiranthes sinensis species
 complex. Compared to its closest relative S. australis, S. stylites blooms
 earlier (June-early July vs. mid July-September), has consistently white
 tubular flowers (vs. commonly pink and pseudocampanulate), a distinctly
 long-clawed labellum (vs. shortly clawed), slender labellum nectar
 callosities (vs. globose), a proportionally larger column foot (ca. 50% of
 the length of the entire column vs. ca. 25%), and occurs in closed canopy
 woodlands (vs. open graminoid habitats). Spiranthes stylites is the only
 Spiranthes in the Eastern Hemisphere to have a woodland habitat. The
 recognition of S. stylites helps support white colored flowers as the
 plesiomorphic and most common floral condition in the complex, and
 indicates pink colored flowers likely arose independently in S. australis
 and S. sinensis s.s.
description: <strong><em>Sampling</em></strong>—Sampling incorporated and expanded upon Dueck et al. (2014), Surveswaran et al. (2018), and Pace et al. (2019), including publicly available samples downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Newly collected material for molecular analysis was collected from Zhejiang Province, China by P. Li (Appendix 1). New molecular data / loci were annotated and uploaded to GenBank, and data matrices uploaded to Dryad (Dryad submission citation will be added after review, Appendix 1). <strong><em>Molecular Techniques, Plastid Assembly, and Phylogenetic Analysis</em></strong>—Genomic DNA was extracted from silica-gel-dried leaves with modified CTAB reagent (Plant DNAzol, Shanghai, China) according to the manufacturer’s protocol. DNA were sequenced with 150 bp paired-end reads using the BGISEQ platform at China National GeneBank (CNGB, Shenzhen, China). The complete plastome and nrITS sequences were de novo assembled by GetOrganelle (Jin et al., 2020) with default settings. The matK, trnF-L intron, trnS-fM, ycf1 sequences were mined from the complete plastome. Manual annotation and modifications were conducted in Geneious (Kearse et al., 2012). The plastid genome map of S. stylites was drawn by online tools OGDraw (http://ogdraw.mpimp-golm.mpg.de/).           Our molecular data matrices were analyzed as distinct datasets: (1) nrITS; (2) combined chloroplast markers, and (3) combined total evidence nrITS+chloroplast. Analyses were performed under Bayesian inference (MrBayes on XSEDE 3.1.2) implemented through the CIPRES Portal V. 3.3 (Miller et al., 2010). The GTR+G (general time-reversible with a gamma distribution) model was implemented for all datasets and partitions. Bayesian analyses were run for 10,000,000 generations, with a sample frequency of 100 000, nruns = 2, nchains = 4, temp = 0.2 and a burnin of 500,000. Phylogenetic inference of the 50% majority-rule consensus tree was constructed using the ‘sumt’ option based on the remaining trees. We visualized the topology of these trees in FigTree (Rambaut, 2014). <strong><em>Taxonomic and Morphological Examination</em></strong>—Herbarium specimens of the S. sinensis s.l. species complex were reviewed from AMES, BM, BNU, CDBI, E, F, GMNHJ, HAST, HEAC, HIB, HO, IBK, K, KPM, KUN, L, LE, LINN, MO, MU, NY, NYS, P, PE, RENZ, SING, TKPM, TNS, and US (acronyms follow Thiers 2023), either by examining the specimens in person, or electronically through GBIF, (https://www.gbif.org/species/2805315), the Global Plants web portal (https://plants.jstor.org/), or the individual virtual herbarium of the institution. Efforts were made to examine the type specimen(s) and protologues of all proposed Spiranthes described from Asia and Australasia. A few plants collected by co-author Li in eastern China were examined from live material. Other individuals were examined via rehydrated flowers taken from herbarium specimens. Flowers were examined from a broad selection of specimens that cover the full spectrum of geographic, phenological, floral color, and habitat variation expressed in this complex. Individual flowers were judiciously removed from the lower third of an inflorescence, placed into beakers with a solution of ammonia, water, and glycerin, and then examined under a dissecting microscope. Our taxonomic framework follows Pace et al. (2019) and Suetsugu and Hsu (2023). We do not recognize the recently described <em>S. hachijoensis</em> Suetsugu or <em>S. minamitaniana</em> Suetsugu as distinct (Suetsugu et al. 2023a, 2023b), and include them within the morphological variation of <em>S. sinensis</em>. Other than a reduction of the column (a feature commonly found in apomictic or autogamicitc populations across many <em>Spiranthes</em> species), the morphologies of <em>S. hachijoensis</em> and <em>S.minamitaniana</em> entirely correspond to that of <em>S. sinensis</em>. A similar reduction of the column occurs in plants previously identified as <em>S. novae-zelandiae</em> Hook.f., which is now considered to be a synonym of <em>S. australis.</em> 
description: # Spiranthes stylites is Another Long Overlooked Asian Spiranthes with an
 Unusual Sylvian Habitat. [https://doi.org/10.5061/dryad.02v6wwq90]
 Description of the data and file structure Nexus concatenated data set of
 nrITS, matK, trnL-F, and trnS-M loci. (in this order)