A peer-reviewed open-access journal PhytoKeys 226: 89-100 (2023) & doi: 10.3897/phytokeys.226. 100062 4ePh y toKe y S https:/ / Pp hyto keys -pen soft.net Launched to accelerate biodiversity research Independent origins of Spiranthes *kapnosperia (Orchidaceae) and their nomenclatural implications Matthew C. Pace! | New York Botanical Garden, 2900 Southern Blvd., Bronx, New York, 10348, USA Corresponding author: Matthew C. Pace (mpace@nybg.org) Academic editor: Timothée Le Péchon | Received 8 January 2023 | Accepted 28 April 2023 | Published 19 May 2023 Citation: Pace MC (2023) Independent origins of Spiranthes xkapnosperia (Orchidaceae) and their nomenclatural implications. PhytoKeys 226: 89-100. https://doi.org/10.3897/phytokeys.226. 100062 Abstract Spiranthes Rich. (Orchidaceae) is a commonly encountered but systematically and nomenclaturally chal- lenging component of the North American orchid flora. Here, the evolutionary history and hybrid origin of the recently described S. sheviakii Hough and Young are critically examined. The available molecular data unambiguously support a hybrid origin of S. cernua (L.) Rich. x S. ochroleuca (Rydb.) Rydb. for S. sheviakii, the same parentage as the priority name S. xkapnosperia M.C. Pace. As hybrid formulas can have only one correct name, S. sheviakii is a synonym of S. xkapnosperia. It is likely that S. xkapnosperia evolved independently at least twice in at least two widely disjunct locations. Keywords hybrid speciation, Interior Lowlands, nomenclatural priority, species complex, Spiranthes cernua, Spiranthes ochroleuca Species complexes continue to present some of the most impenetrable systematic chal- lenges for evolutionary biology and conservation biology, and the challenges associated with their study are amplified when species within a complex hybridize (e.g., Arnold 2001; Fu et al. 2020), with challenging implications for nomenclature. Although Orchi- daceae have been long seen as a model family for pre-zygotic barriers to hybridization, primarily due to documented or inferred pollinator specificity (Ackerman et al. 2023), a growing body of literature makes clear that reproductive barriers are often porous, and that hybridization plays an important role in the speciation of many orchid genera (e.g., Dactylorhiza Neck. ex Nevski (e.g., Pillon et al. 2007), Epidendrum (e.g., Pinheiro et al. 2010), Ophrys L. (e.g., Soliva and Widmer 2003), Orchis Tourn. ex L. (e.g., Jacquemyn Copyright Matthew C. Pace. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 90 Matthew C. Pace / PhytoKeys 226: 89-100 (2023) et al. 2012), Platanthera Rich. (e.g., Wettewa et al. 2020), and Yolumnia Raf. (e.g., Ackerman and Galarza-Pérez 1991)), making for ‘fuzzy’ species boundaries. Spiranthes is one such orchid genus where renewed systematic attention has sup- ported many previous hypotheses of hybridization (e.g., Dueck et al. 2014), in addition to the discovery of new hybrid taxa (e.g., Pace and Cameron 2017). Of the 44 currently accepted Spiranthes species (including nothospecies), 10 have molecular evidence to support a hybrid origin (Sun 1996; Arft and Ranker 1998; Szalanski et a. 2001, Dueck et al. 2014; Pace 2015, 2021; Pace and Cameron 2017, 2019; Surveswaran et al. 2018; Pace et al. 2019). These hybrid species do not only occur within complexes of closely related species (e.g., S. xstellata M.Br., Dueck and K.M.Cameron), but between clad- es of species complexes that are often distantly related (e.g., S. diluvialis Sheviak). The S. cernua (L.) Rich. species complex has traditionally been regarded as systematically “intractable” (Sheviak 1982, 1991; Sheviak and Brown 2002), primarily due to the frequency of hybridization and cryptic speciation (Pace and Cameron 2017) and the variability of all taxa involved. Within the S. cernua species complex, the identity of S. ochroleuca (Rydb.) Rydb. has contributed significantly to systematic and nomenclat- ural challenges. For example, primarily due to the nature of the Gyrostachys ochroleuca Rydb. holotype (Mrs Long s.n., drawing, NY barcode 9463, Fig. 1), and morphological similarities to other members of the complex, S. ochroleuca has either been treated as a synonym or variety of S. cernua for much of the last 90 years (e.g., Gleason and Cron- quist 1963). It was only after the detailed work of Sheviak and Catling (1980), that S. ochroleuca was widely accepted as a species fully distinct from S. cernua (e.g., Pace and Freudenstein 2018). Despite this distinction, S. cernua s.s. and S. ochroleuca were still hypothesized to engage in frequent and widespread hybridization and introgres- sion (Sheviak 1982; Sheviak and Brown 2002). Pace and Cameron (2017) presented the first molecular evidence for hybridization between S. ochroleuca and S. cernua s.s. in the southern Appalachians, describing this hybrid taxon as S. xkapnosperia M.C. Pace. The name S. sheviakii Hough and Young (2021) was recently described as a spe- cies of hybrid origin distributed from central New York to the greater Ohio River Valley, but Hough and Young (2021) were unusually vague about the parentage of S. sheviakii, writing “[this is] apparently the result of hybridization of S. ochroleuca with another member of the S. cernua species complex” (pg. 47). They included compari- sons to S. cernua, S. ochroleuca, and S. xkapnosperia in the diagnosis and throughout the discussion, noting that S. sheviakii is “intermediate in form” between S. cernua and S. ochroleuca (pg. 37), but did not give a full parentage to their newly proposed species. Curiously, Hough originally identified the type specimens of S. sheviakii as “S. xkapno- speria, S. cernua x S. ochroleuca” (Fig. 1), indicating he was aware of its full parentage, or that he thought these plants were morphologically similar to S. xkapnosperia. After reviewing the relevant type specimens (Fig. 1) and the publicly available molecular data presented in Pace and Cameron (2017) and Hough and Young (2021), it is clear that both S. xkapnosperia and S. sheviakii share a hybrid ancestry of S. cernua x S. ochroleuca, although the genetic patterns are differently expressed in the resulting regional hybrids. Appalachian S. xkapnosperia displays a discord- ance between nuclear and chloroplast datasets: the chloroplast data (including ndh/) Spiranthes xkapnosperia nomenclature 91 THAI ALANA CORTON a8 ini Figure |. Comparison of type specimens A holotype of Spiranthes xkapnosperia (M.C. Pace 1030, NY) B holotype of Spiranthes sheviakii (M. Hough and M.A. Young s.n., BH) C holotype of Gyrostachys ochro- leuca Rybd. (Mrs Long s.n., NY); this image is a composite of two images to show the front and back of the drawing plate D lectotype of Ophrys cernua L. (P. Kalm s.n., LINN) A and C courtesy of the C. V. Starr Virtual Herbarium (http://sweetgum.nybg.org/science/vh/) B courtesy of the Liberty Hyde Bailey Hortorium, Cornell University D courtesy of the Linnean Society of London. 92 Matthew C. Pace / PhytoKeys 226: 89-100 (2023) indicates a maternal parent of S. ochroleuca, whereas the nuclear data (ACO and nrITS) indicate a paternal parentage of S. cernua (Table 2; Pace and Cameron 2017). The ACO dataset for Appalachian S. xkapnosperia lacks major nucleotide ambiguities at points of differentiation between the parental species, sharing all of the unique molecular synapomorphies of S. cernua vs. S. ochroleuca. Spiranthes sheviakii also dis- plays a discordance between nuclear and chloroplast data, although the discordance is slightly different than in Appalachian S. xkapnosperia. The available ndh/ data for S. sheviakii clearly indicate a maternal parentage of S. cernua, as the samples share all of the same nucleotide patterns as S. cernua and are clearly different from S. ochro- leuca (or any other member of the S. cernua species complex). However, S. sheviakii displays ACO nucleotide ambiguities at all of the exact and unique points of mo- lecular differentiation between S. cernua and S. ochroleuca (Fig. 2). These ambiguities in the ACO dataset indicate a hybrid origin between S. cernua and S. ochroleuca for Table |. Comparison of Spiranthes cernua, S. xkapnosperia, and S. ochroleuca. Labellum Taxon Distribution Flower color es peas rere aes al position | (Adaxial / position Abaxial) S. cernua Maritime Canada s. to Perpendicular] White to | Conical, | Sepal base held n. FL, west to central to stem to | very pale | reduced | in-line with the PA, the interior lowland strongly yellow / profile of the plateaus, and e. Texas nodding white flower, sepals ascending S. xkapnosperia| Southern Appalachian White to Nodding | Pale yellow} Rounded | Sepal base held Mountains of NC, SC, ivory to slightly / pale just below the and TN; southern Great ascending yellow profile of the Lakes Basin from central flower, sepals NY to IL, s. to Ohio ascending River Valley S. ochroleuca Maritime Canada s. to Ivory to Slightly to Deep Rounded | Sepal base held w. NC, west through the | ochroleucous | strongly yellow / | (sometimes| below the profile Southern Great Lakes ascending |deep yellow| reddish) of the flower, Basin, disjunct in s. IL, held low against IN, central KY, and TN the profile of the flower, downwardly falcate to ascending Table 2. Inferred genetic contributions for S. xkapnosperia sensu nov. Chloroplast regions include matK, ndhj, trnL-F, trnS-M, and ycfl 3° (Pace and Cameron 2017). The only chloroplast region sampled in Hough and Young (2021) for a priori S. sheviakii is ndhJ. Hybrid taxa ACO (nuclear) orITS Chloroplast S. xkapnosperia (Appalachian S. xkapnosperia) S. cernua s.s. S. cernua $.s. S. ochroleuca S. sheviakii (Interior Lowland S. xkapnosperia) S. cernua s.s. + S. ochroleuca [not sampled] S. cernua 8.8. Spiranthes xkapnosperia nomenclature Te eee rer per ser get 105. S.magnicamporum sm7h NM CCCATTTGTMATCACCTGTMAGGATTAAGTTG@TTAAAGTTTTCATCGTCATCAAATCTCTAAC GIT AAGC TTTTTTT 106. S.magnicamporum_sm8a_VA CCCATTTGTEATCACCTGTBAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTIT 107. S.magnicamporum_15e_TX CCCATTTGTEATCACCTGTBAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT e C® 108. S.magnicamporum_1Sf_WI CCCATTTGTEATCACCTGTHAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT 109. S.magnicamporum_sm11a_VA CCCATTTGTEATCACCTGTBAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT 110. S.magnicamporum_sm15a_Ontario CCCATTTGTEATCACCTGTMAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTIT 111. S.magnicamporum_sm12a_GA CCCATTTGTEATCACCTGTBAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTIT 112. $.magnicamporum_sm13a_Ontario CCCATTTGTEATCACCTGTBAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTIT 113. S.magnicamporum_sm17a_TN CCCATTTGTEATCACCTGTMAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT Ce 114, S.magnicamporum_sm19a_IL CCCATTTGTEATCACCTGTGAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTIT tM 115. S.magnicamporum_sm22¢_WI CCCATTTGTEATCACCTGTGAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT Ce 116. S.magnicamporum_sm25z_IL CCCATTTGTEATCACCTGTBAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTIT ( 117. S.magnicamporum_sm26k_IL CCCATTTGTEATCACCTGTBAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT Ce 118, S.magnicamporum_sm28h_IN CCCATTTGTEATCACCTGTBAGGATTAAGTTGATTAAAGTTTTICATCGTCATCAAATCTCTAAC GTT) AAGC TITTTIT Ce 119. S.magnicamporum_sm23e¢_IL CCCATTTGTEATCACCTGTGAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT Ce 120, S.incurva_4v_OH CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTIT Ce 121. S.incurva_4t_WI CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT Ce 122. S.incurva_sm1c_Wi CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTIT (® 123, S.ineurva_sm21r_WI CCCATTTGTBATCACCTGTGAGGATTAAGTTGATTABAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT Ce 124. S.incurva_sm23b_IL CCCATTTGTBATCACCTGTGAGGATTAAGTTGATTABAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT CM 125. S.incurva_s¢33a_Ontario CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT ( 126, S.incurva_soch10a_VT CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT 127. S.cernua_4ee_SC CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT 128. S.cernua_4dd_NC CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT 129. S.cernua_4L_TX CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT 130. S.cernua_4m_FL CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT 131. 5.cernua_sc1b_DE CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT Ce 132. S.cernua_AR29_AR CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT 133. S.cernua_sc9a_VA CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTT TICATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT ao se sciSb GA CCCATITGTGATCACCTGT ERA SUR ADD SURE ee ee pas GTT _AAGC_TTTTTTT CCCATTTGTGATCACCTG) TGAGGATTAAGTT GATTABIAGT TTTCATC GTCATHAAATCTC TAAC CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTABAGTTTTCATCGTCATEIAAATCTCTAAC CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTABAGTTTTCATCGTCATBIAAATCTCTAAC CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTABAGTTTITCATCGTCATHIAAATCTCTAAC CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTABAGTTTTCATCGTCATRIAAATCTCTAAC CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAAAGTTTTCATCGTCATCAAATCTCTAAC TTTTTTT TTTTTIT TTTTTTT TITTTIT TITTTTT TITTTIT TTTTTIT . 5. xkap! te 136. 5. hapsheviak 526 C® 137. S. xkapsheviakii_s33 Ce 138. S, xkapsheviakii_s36 Ce 139. S. xkapsheviakii_s38 CM 140. S. xkapsheviakii_s40 Ce 141. S.xkapnosperia_sc20a_NC Ce 142. S.xkapnosperia sh2 NC 43. 5.ochroleuca_4n_NY CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAAAGTTTTCATCGTCATMAAATCTCTAAC Mir aAaGcc 144. S.ochroleuca_4wx_OH TITTTT= 145. S.ochroleuca_16b_VA CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAAAGTTTTCATCGTCATMAAATCTCTAAC MTT =AAGC TTTTTTIT 146. S.ochroleuca_16c_NH CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAAAGTTTTCATCGTCATMAAATCTCTAAC MTT AAGC TTTTTTT 147. S.ochroleuca_16f_NH CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAAAGTTTITCATCGTCATBAAATCTCTAAC MTT AAGC TTTTTTT 148. S.ochroleuca_16g_Ml CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAAAGTTTTCATCGTCATBAAATCTCTAAC MITT AAGC TTTTTTT 149. S.ochroleuca_16h_NY CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAAAGTTTTCATCGTCATBAAATCTCTAAC MIT AAGC TTTTTTT 150. S.arcisepala_4u_OH CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT 151. S.arcisepala_sc30a_OH CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTTT C* 152. S.arcisepala_4y_NY CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTIT Ce 153. S.arcisepala_4z_OH CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTIT 154. S.arcisepala_NY1_NY CCCATTTGTGATCACCTGTGAGGATTAAGTTGATTAGAGTTTTCATCGTCATCAAATCTCTAAC GTT AAGC TTTTTIT 4€C Crsesi Is Kal PCCATTTATCATCACCT ET CAGCGATTAALTTOATTABACTTTTCATOCOTCATEAAATOCTCTAAS ETT AAG TITTTTT 105. S.magnicamporum_sm7h_NM ‘TICTTTG = CEITCC:- TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 106. S.magnicamporum_sm&a_VA TTCTITG =CHTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 107. S.magnicamporum_15e_TX TTTCTITG = CTC. TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC (Ce 108. S.magnicamporum_15f_WI TICTITG «= CEITCC. TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC oe 109, S.magnicamporum_sm11a_VA TTCTTTG CGETCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 110, S.magnicamporum_sm15a_Ontario STHCTTTG = CETCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 111. S.magnicamporum_sm12a_GA TTCTITG =«CEITCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 112, S.magnicamporum_sm13a_Ontario TTCTTTG ==CETCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 113. S.magnicamporum_sm17a_TN STTCTTTG CETCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC Ce 114. S.magnicamporum_sm19a_IL TICTTTG CETCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC Ce 115, S.magnicamporum_sm22c. mt TTCTTITG CETCC TCCTCTTCATTETTCACCA GCTCAGGCGCC GGGAAGATCACHGCGTC GCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC C+ 116, Smagnicamporum_sm2: TICTTTG CETCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC C+ 117. Sumagnicamporum_sm26k_I TICTITG CETCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC Ce 118, S.magnicamporum_sm28h_ iH TTCTTTG CHTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC C+ 119, S.magnicamporum_sm23e_IL ‘TICTTTG CHTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC Ce 120. S.incurva_4v_OH TICTITG ccTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC Ce 121. S.incurva_4t_Wl TTCTTITG § §=6CCTOC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC Oe 122. S.incurva_smic WI ‘TTCTTTG ecTcC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCT GGGTTIGTAGAAGGAAGCGATGGACATGCGGTTGC TICTTTG ecTcc TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC STTCTTTG CHTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTC GCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC STICTTTG CHTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC Ce 126. Siincurva_ soch10a_VT TICTTITG CcTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GOGAAGATCACGGCGTCGCTGCCT GGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 127. S.cernua_dcec_SC TTCTTITG eCcTcc TECTCTTCATTCTTCACCA GCTCAGGCGCC GOGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 128. S.cernua_4dd_Nc STICTTTG ecTccC TCCTCTTCATTCTTCACCA GCTCAGGOGCC GGGAAGATCACGGCGTCGCTGCCT GOGTTGTAGAAGGAAGCGAT GGACATGCGGTTGC 129. S.cernua_4L_TX TICTTTG CCTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 130. S.cernua_4m_FL TTCTTTG eccTec TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGAT GGACATGCGGTTGC 131. S.cernua_sclb_DE :TTCTTTG ecTcC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCT GGGTTGTAGAAGGAAGCGAT GGACATGCGGTTGC Ce 132. S.cernua_AR29_AR TICTTTG CCTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTC GCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC TTCTTTG CCTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 133. S.cernua_sc9a_VA. : Broa b GA TCT TTS ccTcc —_——————————_ SC TCAGGCGCC —— SS TCCTCTTCATTET TEACCA. GCTCAGGCGCC acilaacaTcaceacaTeacTace TEGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC TCCTCTTCATT@TTCACCA GCTCAGGCGCC GGRAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC TCCTCTTCATTG@TTCACCA GCTCAGGCGCC GGRAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC TCCTCTTCATTGTTCACCA GCTCAGGCGCC GGHAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC TCCTCTTCATTBTTCACCA GCTCAGGCGCC GGHAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC POSTCIACAL TAN: TCACCA GCTCAGGCGCC SEEMBAL CAC GSC ET EGC IEE TGGGTTGTAGAAGGAAGCGAT GGACATGCGGTTGC CTTTsG cTTTG cTTTG CTTTG CcTTTG TICTTITG TICTTTG 8. S. xkapsheviakii_s36 139. S. xkapshevial 140, S. xkapsheviakii_s40 141, S.xkapnosperia_sc20a_NC 10 144, Scchroleuca™4vx OH TICTTITG caTcc TCCTCTTCATTETTCACCA GCTCAGGCGCC GGHAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 145, S.ochroleuca_16b_VA STTCTTTG CETCC TCCTCTTCATTGTTCACCA GCTCAGGCGCC GGHAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 146. S.ochroleuca_16¢_NH TICTITG CBTCC TCCTCTTCATTG@TTCACCA GCTCAGGCGCC GGHAAGATCACGGCGTCGCTGCCT GGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 147. S.ochroleuca_16f_NH TICTTITG CBTCC TCCTCTTCATTGTTCACCA GCTCAGGCGCC GGBAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGAT GGACATGCGGTTGC 148, S.ochroleuca_16g_ MI TICTTTG CBTCC TCCTCTTCATTSTTCACCA GCTCAGECGCC GGBAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 149. S.ochroleuca_16h_N¥ ‘TTCTTTG CETCC TCCTCTTCATTTTCACCA GCTCAGGCGCC GGBAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 150. S.arcisepala_4u_OH TcTTITG CBTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 151, S.arcisepala_se30a_QH rctiTs CBTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC C+ 152. S.arcisepala_4y_NY WTcTTTG CETCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGEAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC Ce 153, S.arcisepala_4z_OH WTecTTTs CHTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGGAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTGC 164 Carrisenala NW NY ret. CMTCC TCCTCTTCATTCTTCACCA GCTCAGGCGCC GGMAAGATCACGGCGTCGCTGCCTGGGTTGTAGAAGGAAGCGATGGACATGCGGTTG( Figure 2. Examples of ACO gene sequence concatenations for selected Spiranthes. Samples 135-140 labeled “S. xkapsheviakii” represent a priori interior lowland S. sheviakii from Hough and Young (2021), all other samples are from Pace and Cameron (2017). Samples 141 & 142 represent Appalachian S. xka- pnosperia. Samples of S. cernua s.s. are included immediately above the highlighted box and samples of S. ochroleuca are included immediately below the highlighted box A examples of ambiguities in a priori S. sheviakii that correspond to nucleotide differences between S. cernua and S. ochroleuca (e.g., G, R, A) B examples of nucleotide states that are shared with S. ochroleuca but not S. cernua (e.g., left-most high- lighted A & G), and additional examples of ambiguous states in a priori S. sheviakii that correspond to nucleotide differences between S. cernua and S. ochroleuca (e.g., G, R, and A). S. sheviakii. Additionally, the ACO nucleotide ambiguity patterns for S. sheviakii are distinct from those of regionally sympatric S. incurva (Jenn.) M.C. Pace, S. magni- camporum Sheviak, or any other member of the S. cernua species complex (Fig. 2), indicating these species are not involved in the evolution of S. sheviakii. The nucleo- 94 Matthew C. Pace / PhytoKeys 226: 89-100 (2023) tide ambiguity and nuclear/chloroplast discordant patterns are consistent across all samples of S. xkapnosperia and S. sheviakii included in Pace and Cameron (2017) and Hough and Young (2021). Thus, S. xkapnosperia and S. sheviakii share the same ancestral hybrid parentage of S. cernua x S. ochroleuca, but this parentage is expressed differently within the genomes of the two resulting regionally distinct hybrid popula- tions (Table 2). Nomenclaturally, per The Code (Article H.4.1): When all the parent taxa can be postulated or are known, a nothotaxon is cir- cumscribed so as to include all individuals recognizably derived from the crossing of representatives of the stated parent taxa (i.e. not only the F1 but subsequent filial generations and also back-crosses and combinations of these). There can thus be only one correct name corresponding to a particular hybrid formula; this is the earliest legitimate name (Art. 6.5) at the appropriate rank (Art. H.5), and other names cor- responding to the same hybrid formula are synonyms of it. Thus, any recognizably intermediate individual or population that results from the hybridization of S. cernua and S. ochroleuca must be recognized by the priority name S. xkapnosperia, even if different hybridization events between the parental species occurred at different geologic times, in different places, resulting in different genomic expressions, and different morphologies. Based on the available ndh] and ACO molec- ular data of Hough and Young (2021), S. sheviakii is unambiguously of hybrid origin between S. cernua and S. ochroleuca, and is thus synonymous with S. xkapnosperia. It should be noted that species and nothospecies are the same nomenclatural rank, and the use of the multiplication symbol (x) is simply to emphasize the hybrid origin of nothospecies. Because Hough and Young (2021) appear to have been aware of the full hybrid parentage of their newly proposed name when they described S. sheviakii (based on the label of the type specimens, Hough s.n., Fig. 1), this name is likely superfluous, although it is not illegitimate as they did not include the type of S. xkapnosperia within the circumscription of S. sheviakii. The evolutionary history of S. xkapnosperia in its newly expanded understand- ing (S. xkapnosperia sensu nov.) is perhaps one of the more unusual within the en- tire genus, having formed from the same two parental species (at least) two times, in widely disjunct locations, displaying different molecular signals between the parents. Additionally, the parental species likely played different maternal vs. paternal roles in the formation of the regionally disjunct S. xkapnosperia populations (Table 2). The resulting differences in ambiguity patterns (or the lack of ambiguities) are likely due to differences in the hybridization and introgression histories of these regional popula- tions. As Appalachian S. xkapnosperia lacks ACO and ntITS ambiguities, it may be the result of chloroplast capture. This is a process through which an initial F1 hybridiza- tion event between paternal S. cernua and maternal S. ochroleuca is then followed by several backcrossing events with S. cernua as the pollen (paternal) parent, until the entire nuclear genome is only represented by S. cernua, but the chloroplast genome re- tains the original chloroplast contribution of S. ochroleuca. By contrast, the ambiguities present in the ACO locus of Interior Lowland S. xkapnosperia (previously referred to as S. sheviakii) indicate it likely resulted from an initial F1 hybridization without exten- Spiranthes xkapnosperia nomenclature wie) sive (or only limited) backcrossing. Elsewhere in Spiranthes, Arft and Ranker (1998) hypothesized that at least two separate hybridization events between S. magnicampo- rum and S. romanzoffiana Cham. led to the formation of S. diluvialis, with subsequent localized dispersal in Utah and Colorado. However, the examined molecular signals from all sampled populations were the same (at the time of their study S. diluvialis was known from Colorado, Montana, Nevada, Utah, and Wyoming, but their study focused on samples from Colorado and Utah; Arft and Ranker 1998). Additional mo- lecular phylogenetic study has not found major molecular differentiation between dif- ferent populations of S. diluvialis (Dueck et al. 2014; Pace 2015). Spiranthes xkapnosperia was originally known to occur diffusely over a small region of the greater Smoky Mountain region and southern Blue Ridge Mountains, in the southern Appalachian Mountains of North Carolina, South Carolina, and Tennessee (Pace and Cameron 2017). The expanded understanding of S. xkapnosperia sensu nov. discussed here extends the known distribution of this nothospecies throughout the distributional contact zone between S. cernua and S. ochroleuca along the northern limit of S. cernua in the area of the Interior Lowlands, Ohio River Valley, and south- ern Great Lakes Basin, an area that was not heavily sampled in the molecular work of Pace and Cameron (2017). Ecologically, populations in the southern Appalachians occur in more mesic sites vs. less mesic habitat of the Interior Lowlands populations; habitat variability is not uncommon across the genus. Morphologically, both disjunct populations are readily identifiable as intermediate hybrids of S. cernua x S. ochroleuca. However, they display slightly different morphological affinities to their parents, with southern Appalachian S. xkapnosperia being more similar to S. ochroleuca, and Interior Lowland S. xkapnosperia being more similar to S. cernua. The flowers of southern Appalachian S. xkapnosperia are generally slightly ascending (as is common in S. och- roleuca). lhe flowers of Interior Lowlands S. xkapnosperia are generally very similar in overall size and appearance to S. cernua s.s., commonly with a nod to the flowers, but sharing the yellowish labellum coloration and rounded abaxial labellum glands with S. ochroleuca (Table 1). Within the S. cernua species complex, molecular data have supported hybridiza- tion as a strong driver of speciation, with four of the seven non-hybrid species within the complex involved in the evolution of six species of hybrid origin or nothospecies (Table 3, Pace and Cameron 2017). Spiranthes cernua is the most frequently involved species, giving rise to the evolution of four hybrid taxa, and is typically the inferred maternal parent. The frequent involvement of S. cernua in the evolution of hybrid taxa may be due to its broad geographic distribution, stretching from Maritime Canada south to northern Florida and west through the mid- and southern-Appalachian Mountains to Texas. The repeated evolution of hybrid taxa such as S. xkapnosperia, in addition to the cryptic morphological nature of the species within the complex, has contributed to the systematic and nomenclatural challenges commonly associated with the genus. The repeated evolution of S. xkapnosperia and complicated hybridiza- tion history of the wider S. cernua species complex also highlight the need for slow and careful study when deciding to name and describe new taxa within the genus (Ames 96 Matthew C. Pace / PhytoKeys 226: 89-100 (2023) Table 3. Ancestry of known hybrid taxa derived from members of the S. cernua species complex, as sup- ported by combined molecular and morphological evidence. Hybrid taxa Inferred paternal species Inferred maternal species Literature source S. bightensis S. odorata S. cernua Pace (2021) S. casei S. lacera (var. lacera) S. ochroleuca Pace (2015); Pace unpublished data S. diluvialis S. magnicamporum S. romanzoffiana Arft and Ranker (1998) S. incurva S. magnicamporum S. cernua Pace and Cameron (2017) S. xkapnosperia S. cernua (southern Appalachian S. cernua (Interior Lowland Pace and Cameron sensu nov. populations) or S. ochroleuca populations) or S. ochroleuca (2017); Hough and (Interior Lowland populations) (southern Appalachian populations) Young (2021) S. niklasii S. cernua S. ovalis Pace and Cameron (2017) 1921), with particular attention given to nomenclatural rules and priority. Although they do not provide any molecular data, Hough and Young (2021), using terminol- ogy from Sheviak (1982), also place emphasis on the potential distinction of “low prairie race” and “southern prairie complex” populations currently contained within the circumscription of S. cernua s.s. Moving forward, researchers should keep in mind the priority names for taxa that involve hybridization between S. cernua and other members of the S. cernua species complex (Table 3). Additionally, not all individu- als or populations of hybrid ancestry should be named, as genomic data increasingly shows complex hybridization and introgression patterns make for cryptically complex genetic ancestries and species relationships in groups with porous reproductive barriers (e.g., Evans et al. 2023). A few additional notes related to Hough and Young (2021) are discussed here: 1) Hough and Young (2021) discuss the “holotype for S. incurva.” As detailed in Pace and Cameron (2017), the name S. incurva is a nomenclatural combination based on the basionym Jbidium incurvum Jenn.: Since Jennings selected a suite of specimens, “Aug. 24-26, 1905”, housed at CM as “the type specimens”, and not a specific specimen, collection number, or sheet, the specimen designated by Catling as the holotype, via an annotation label, is more prop- erly designated as the lectotype. All other specimens collected on Aug. 26, 1905 must then be isolectotypes, and all other specimens collected within “the type specimens” collection range designated as syntypes. The lectotype of L. incurvum is the Jennings s.n. specimen collected on 26 August 1905, from Fog Whistle (CM). However, this specimen is not discussed or examined in Hough and Young (2021), which only discusses the remaining syntypes. 2) Hough and Young (2021) note “at least within the range of this study, we have not observed S. incurva growing in xeric sites. The typical habitats appear to be mostly moist to wet and mediacid to calcareous.” It should be noted that S. incurva is found in a wide variety of habitats, from hot, dry, sandy lake beach dunes, old fields, and roadside embankments, to standing in shallow water of fens and lake beach dune swales (Pace and Cameron 2017). This is inclusive of locations within the study range of Hough and Young (2021). 3) Hough and Young (2021) make much of apparent ambiguities Spiranthes xkapnosperia nomenclature vii FWO S.cernua_4l-wis240.ab1 TEnTCGGTCATCAAATC TCTARCG GILAAGE TTT TT rTGAGTT TTCG PD AK ARAYA aN Oa BoD Sees A er es a REY §.cernua_4m-wis239.ab1 TOATGCOTLOCA TC AA AG TOC TAAC GT MAAGEe Ft b ho Gke i) tr Cos Ce REY S.arcisepala_S4 TCATCGTCATBBAAATCTCTAACHITTAAGCTTTTMITTGAGTTTTCG INP OXKPAAALN AN AAM AAA ALNAA JS AAAA LON FWD S.arcisepala_4u_OH TCATCGTCATCAAATCTCTAACGTTAAGCTTTTETTGAGTTTTCG FwO S.arcisepala_4y_NY Fu S.arcisepala_4z_OH FWD S.ochroleuca_4wx-wis240,ab1 REV S.ochroleuca_4wx-wis239.ab1 REV §.cernua_4m-wis239.ab1 TT CTGCCGGACTTGGAACCATAACTGTCAAATGAAGT'® Ce REV S.arcisepala_S4 TTCTGCCGGACTTGGAACCATAACTGTCAAATGAAGT' FWO S.arcisepala_4u_OH TTCTGCCGGACTTGGAACHHATAACTGTCAAATGAAGT= FWD S.arcisepala_4y_NY TTCTGCCGGACTTGGAACHHATAACTGTCAAATGAAGT' FWD S.arcisepala_4z_OH TTCTGCCGGACTTGGAACHHATAACTGTCAAATGAAGT™ Fu S.ochroleuca_4wx-wis240.ab1 7 Tolcc os GA SOT Ty G S AA We oar 7s T A HANDS A ATGA chi 1 Figure 3. Examples of ACO gene nucleotide ambiguities for selected Spiranthes. The sample “S.arcisepala_S4” is from Hough and Young (2021), all other samples are from Pace and Cameron (2017) A examples of ambi- guities present in Hough and Young (2021), but not present in Pace and Cameron (2017) (Y and R), and an ambiguity present in Hough and Young (2021) but overlooked in Pace and Cameron (2017) (K) B example of ambiguity present in Pace and Cameron (2017) (M), but not present in Hough and Young (2021). in the nuclear ACO data for their accessions of S. arcisepala M.C. Pace, claiming that Pace and Cameron (2017) misinterpreted their data. After comparing the GenBank data of Hough and Young (2021) to the raw sequence data of Pace and Cameron (2017) (Fig. 3), there was a single instance where Pace and Cameron (2017) missed an ambiguity that is present in the data of Hough and Young (2021). However, the overwhelming majority of supposed S. arcisepala ambiguities present in Hough and Young (2021) are simply not present in our data, which are unambiguously a single nucleotide. Furthermore, I found an additional ambiguity in the ACO data of Pace and Cameron (2017) that is not present in the ACO data of Hough and Young (2021), and this point of ambiguity does not correspond to a point of molecular differentia- tion between any other member of the S. cernua species complex (Fig. 3). Based on this reexamination and comparison, I reassert that S. arcisepala is not of hybrid origin, although it may be an autopolyploid. Genomic examination of hybridization across the genus is obviously needed, and is currently underway. 98 Matthew C. Pace / PhytoKeys 226: 89-100 (2023) Nomenclature Spiranthes xkapnosperia M.C. Pace [S. cernua x S. ochroleuca], Syst. Bot. 42: 659. 2017. = Spiranthes sheviakii M. Hough and M.A. Young. Native Orchid Conf. J. 18.3: 35. 2021. Type: U.S.A. New York: Onondaga County, town of Lysander, Three Riv- ers WNA, 19 Sep 2020, M. Hough and M.A. Young s.n. (holotype: BH! [BH 000298396]; isotype: CORT! (x2)). Type. U.S.A. North Carolina: Transylvania County, Great Smoky Mountains, Pisgah National Forest, ca 7.5 km NW of Balsam Grove, north side of 215, below a steep seeping cliff, growing in moss and lichen hummocks, 2 Oct 2016, MC. Pace 1030 (holotype: NY! [01392730]; isotype: NCU! [NCU00332163], US!). Acknowledgements Thank you to Kanchi Gandi (Harvard University) for helpful conversations regarding the nomenclature of hybrids while this manuscript was in preparation; two anony- mous reviewers for their helpful comments; Anna M. Stalter (BH) and Michael Hough (CORT) for sending images of the S. sheviakii types. James (Jim) Fowler (1947-2021) provided samples that led to the description of S. xkapnosperia. 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