﻿Morphological and molecular evidence gives insight into the taxonomic position of Peucedanumpubescens (Apiaceae, Selineae)

﻿Abstract In this study, morphological and molecular evidences were combined to determine the taxonomic position of Peucedanumpubescens Hand.-Mazz. Morphologically, Peucedanumpubescens is similar to the species of the genus Ligusticopsis in having fibrous remnant sheaths at the stem base, pinnate and linear coexisted bracts, strongly compressed dorsally mericarps, filiform median and lateral ribs, winged marginal ribs, numerous vittae in each furrow and commissure, but can also be easily distinguished from members of Ligusticopsis by its hispid fruit and linear-lanceolate bracteoles. Molecular phylogenetic analyses based on the single-copy protein-coding sequences (CDS) of plastomes and internal transcribed space (ITS) region showed that Peucedanumpubescens nested in the genus Ligusticopsis. As both morphological and molecular data supported the inclusion of Peucedanumpubescens within Ligusticopsis, the species is here transferred and the new combination, Ligusticopsispubescens (Hand.-Mazz.) J.J.Deng, C.K.Liu & X.J.He, made.


Introduction
Peucedanum sensu lato was previously characterized by dorsally compressed mericarps with slightly prominent dorsal ribs, narrowly winged lateral ribs, as well as a broad commissure (Sheh 1992;Spalik et al. 2004;Sheh and Watson 2005). As such it was one of the largest genera of Apiaceae, comprising 100-120 species with 33 endemics to the territory of China, and with a distribution in Eurasia, South Africa, and sometimes Australia (Spalik et al. 2004;Sheh and Watson 2005;Cieśla et al. 2009). However, Peucedanum sensu lato has been a taxonomically confusing genus due to its great heterogeneous characteristics (Solov'eva et al. 1985;Reduron et al. 1997;Downie et al. 2000Downie et al. , 2010Winter et al. 2008;Zhou et al. 2014), exhibiting a wide variety of life forms, leaf and fruit structures, and chemical compositions (Shneyer et al. 2003). Moreover, several molecular phylogenetic studies based on DNA fragments and plastomes indicated that Peucedanum sensu lato was not a monophyletic group (Downie et al. 2000;Spalik et al. 2004;Valiejo-Roman et al. 2006;Feng et al. 2009;Zhou et al. 2009Zhou et al. , 2020Liu et al. 2022). Consequently, the circumscription of the genus has been greatly reduced with Peucedanum sensu stricto, recognized by ternate leaves, linearsubulate or filiform bracteoles, one vitta in each furrow and two vittae on commissure in mericarp (Kadereit and Bittrich 2018) and several members of Peucedanum sensu lato were separated to restitute or establish genera or transfer into other genera (Reduron et al. 1997;Winter et al. 2008;Ostroumova et al. 2016;Pimenov et al. 2016;Pimenov 2017). However, the previous studies mainly focused on those species distributed in Europe and South Africa, and the taxonomic position of Chinese endemic species of this genus was still unresolved.
Ligusticopsis Leute was described by Leute in 1969 with Ligusticopsis rechingeriana Leute as its type species. The taxonomy of genus has been controversial since its establishment, due to its close morphology to Ligusticum (Zhou et al. 2008(Zhou et al. , 2009Sun et al. 2010); e.g. Flora Reipublicae Popularis Sinicae and Flora of China treated Ligusticopsis as the synonym of Ligusticum and "Ligusticum in the broad sense", respectively (Sheh 1992;Sheh and Watson 2005), whereas the genus was recognized by Pimenov et al. (2001Pimenov et al. ( , 2003. Recently, a phylogenetic study based on morphological and molecular data confirmed the monophyly of Ligusticopsis and nine "true species of Ligusticopsis" were recognized (Li et al. 2022); the members of the genus are characterized by the following diagnostic characters: stem base clothed in fibrous remnant sheaths, pinnate bracts, pinnate bracteoles longer than rays of umbellule, mericarps strongly compressed dorsally, median and lateral ribs filiform or keeled, marginal ribs winged, and numerous vittae in each furrow and commissure.
Peucedanum pubescens Hand.-Mazz. (1933: 728) was described based on a collection (E00002620) from Yunnan, China, and was an endemic species to China (Sheh and Watson 2005;Pimenov 2017). Due to dorsally compressed mericarps with slightly prominent dorsal ribs and narrowly winged lateral ribs, P. pubescens was recognized as a member of Peucedanum sensu lato (Handel-Mazzetti Heinrich 1933). However, after examination of the type specimen and protologue, field observation, and morphological and micro-morphological research into it, we found this species was characterized by stem base clothed in fibrous remnant sheaths, pinnate leaves, linear and pinnate coexisted bracts, strongly dorsally compressed fruits, numerous vittae in each furrow and commissure, and these features are significantly similar to members of Ligusticopsis. To determine the taxonomic position of Peucedanum pubescens, we performed morphological and molecular analyses.

Morphological observation
The morphological features of Peucedanum pubescens were observed in field. Then, mericarp of this species was observed and photographed using a stereomicroscope, Nikon SMZ 25 (Japan). Furthermore, morphological diagnoses of nine "true species of Ligusticopsis" were obtained from type specimens from K, P, E, WU, BM, GH, KUN, and HNWP, Flora of China (Sheh and Watson 2005), and analysis performed by Li et al. (2022). The Herbarium code refers to Thiers (2015).

DNA extraction, ITS amplifying and sequencing
Total genomic DNA was extracted from silica-dried leaves with plant genomic DNA kit (Cwbio Biosciences, Beijing, China). The universal primers ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') and ITS5 (5'-GGA AGT AAA AGT CGT AAC AAG G-3', White et al. 1990) were used to amplify the entire internal transcribed sequences (ITS). Amplification was undertaken using a volume of 30 µl with 15 µl 2 × Taq Mas-terMix (CWBIO, China), 10 µl ddH2O, 1.5 µl forward primer, 1.5 µl reverse primer, and 2 µl total DNA. The amplification of the ITS region was obtained by initial denaturation for 3 min at 94 °C, followed by 30 cycles of 45 s at 94 °C, 70 s at 54 °C, and 90 s at 72 °C, and then a final extension of 10 min at 72 °C. All PCR products were separated using a 1.5% (w/v) agarose TAE gel and sent to Sangon (Shanghai, China) for sequencing.

Plastome sequencing and assembly
The extracted total DNA was fragmented into 400 bp to construct the pair-end library, following the manufacturer's protocol (Illumina, San Diego, CA, USA). The DNA libraries were sequenced on the Illumina NovaSeq platform at Personalbio (Shanghai, China). Quality control of the raw reads was performed using fastP v0.15.0 (-n 10 and -q 15) (Chen et al. 2018), produced at least 5GB clean reads per species. De novo genome assembly from the clean data was accomplished utilizing NOVOPlasty v2.6.2 (Dierckxsens et al. 2017), with a kmer length of 39 bp and a sequence fragment of the rbcL gene from Ligusticopsis brachyloba (Franch.) Leute (Genebank no. MN204661) as the seed sequence. The assembled complete plastome was annotated initially by using PGA (Qu et al. 2019) and then examined using Geneious v9.0.2 (Kearse et al. 2012).

Phylogenetic analyses
To confirm the phylogenetic position of Peucedanum pubescens, phylogenetic trees were reconstructed based on single-copy protein-coding sequences (CDS) of 34 plastomes and 36 ITS sequences (Table 1). Chamaesium mallaeanum Farille & S. B. Malla and Chamaesium viridiflorum (Franch.) Wolff ex Shan were selected as outgroups according to the result of a previous study (Li et al. 2022). Plastome CDs and ITS sequences were respectively aligned using MAFFT v7.221 (Katoh and Standley 2013), and then manually adjusted in MEGA7.0 (Kumar et al. 2016) to obtain plastome CDs and ITS datasets. The two alignments were subjected to Maximum-Likelihood (ML) analyses and Bayesian Inference (BI). For ML analyses, the software RAxML v8.2.8 (Stamatakis 2014) was used to construct the phylogenetic trees with the GTR+G+I model and 1000 bootstrap (BS) replicates. Bayesian inference (BI) analyses were conducted by MrBayes version 3.2.7 (Ronquist et al. 2012) with the best-fit substitution model (GTR+G+I) determined by Modeltest v3.7 (Posada and Crandall 1998). Markov Chain Monte Carlo (MCMC) search was performed for 1 × 10 6 generations, sampling every 100 generations. The first 25% of trees were discarded as burn-in and the remainder was used to generate the consensus tree. Results of phylogenetic analyses were visualized and edited in FigTree v1.4.2 (Rambaut and Drummond 2015).

Plastome feature of Peucedanum pubescens
The plastome of Peucedanum pubescens is a typically quadripartite structure, including a large single copy region (LSC), a small single copy region (SSC), and a pair of inverted repeat regions (IR) (Fig. 1)

Phylogenetic analyses
The phylogenetic trees based on plastome CDs and ITS were given in Fig. 4 and Fig. 5, respectively. Both tree topologies strongly supported that Peucedanum pubescens nested in the genus Ligusticopsis (PP = 1.00 & BS = 100%; PP = 0.99 & BS = 88%). Although the phylogenetic position of this species could not be resolved in ITS tree, phylogenetic tree constructed based on plastome CDs showed that Peucedanum pubescens was sister to the clade that included the species L. rechingeriana (type species of the genus Ligusticopsis) and L. involucrata with high support (PP = 1.00 & BS = 99%).

Discussion
Peucedanum sensu stricto and Ligusticopsis both belong to the Selineae tribe of Apiaceae, and members of these two genera are similar in the dorsally compressed fruits with filiform dorsal ribs, and winged marginal ribs (Spalik et al. 2004;Sheh and Watson 2005;Li et al. 2022), but the former genus can be distinguished significantly from the latter by having ternate leaves, linear-subulate, caducous or lacking bracts, one vitta in a furrow and two vittae in commissure in mericarp (Kadereit and Bittrich 2018), while the latter can also be distinguished from the former by possessing pinnate leaves, pinnate bracts, numerous vittae in each furrow and in commissure (Li et al. 2022). Peucedanum pubescens is more similar to the genus Ligusticopsis in having pinnate leaves, linear and pinnate coexisting bracts, numerous vittae in each furrow and in commissure (Table 2), rather than Peucedanum sensu stricto. This result was further supported by the molecular phylogenetic analyses that Peucedanum pubescens nested in Ligusticopsis. As a result, Peucedanum pubescens is here transferred to Ligusticopsis as an independent species and a new combination in Ligusticopsis made, so that this genus now includes ten recognized species. The species is easily distinguished from other members of Ligusticopsis by the hispid fruit and linear-lanceolate bracteoles.  Type. China. Yunnan centralis: In regionis calide temperatae ad orientem fluminis Dsolin-ho, declivibus siccis inter vicos Mabou schan et Bölu, ad elevationem 1900-2000m, 9 November 1916 (lectotype: WU! (WU0029560); isolectotypes: E (E00002620), W!).