Taxonomic resurrection of Saxifraga lancangensis (Saxifragaceae)

Background Accurate species delimitation is fundamental for testing evolutionary theory and provides essential implications for conservation management. The arctic-alpine genus Saxifraga L. (Saxifragaceae) is taxonomically complex and many species have not been critically assessed. The taxonomic and phylogenetic status of Saxifraga lancangensis Y.Y.Qian, considered as a synonym of Saxifraga mengtzeana Engl. & Irmsch. in previous studies, is re-evaluated in light of new evidence presented here. Results Evidence from morphological comparison and sequencing of plastid genome indicate that S. lancangensis belongs to Saxifraga sect. Irregulares Haw., and is closely related to Saxifraga geifolia Balf.f., and S. mengtzeana . However, S. lancangensis can be diagnosed by its petals with red and clawless base, leaf blade orbicular and leaf margin shallowly dentate. Conclusions The morphological and molecular evidence support the resurrection of S. lancangensis as a distinct species. An updated morphological description based on protologue and fresh material, diagnostic characters, and original photographs of the resurrected species are presented.

In China, fifteen species of S. sect.Irregulares were recorded, including eight recently reported new species (Chen et al. 2022;Zhang et al. 2017Zhang et al. , 2018Zhang et al. , 2019Zhang et al. , 2021Zhang et al. , 2022Zhang et al. , 2023b;;Zhao et al. 2019), and a resurrected species (Zhang et al. 2023a).Most members of this section are confined to small areas (Magota et al. 2021;Pan et

Background
The species-rich genus Saxifraga L. comprises more than 400 species that are mainly distributed in arctic and mountainous regions of the Northern Hemisphere (Ebersbach et al. 2017;Pan et al. 2001;Tkach et al. 2015).Saxifraga is taxonomically difficult for their remarkable morphological variation, hybridization and Taxonomic resurrection of Saxifraga lancangensis (Saxifragaceae) Xin-Jian Zhang 1,2 , Kang-Zheng Jiang 1,2 , Xin-Yuan Kuai 1,3 , Jun-Tong Chen 1,2 , Peng-Rui Luo 1,2 , Hang Sun 1* and Tao Deng 1* al. 2001), and many of them are known only from type specimens or a handful of herbarium collections.More investigations are needed to clarify the patterns of variation and the delimitation of these species.
In 2021, during an examination of Saxifraga specimens in the herbarium of the Kunming Institute of Botany (KUN), the first author found a specimen of Saxifraga identified as Saxifraga mengtzeana Engl.& Irmsch.(KUN 1,238,150).However, the locality of this specimen was Lancang County (Yunnan Province, China), which is out of the distribution range of S. mengtzeana, but coincides with the type locality of Saxifraga lancangensis Y.Y.Qian (Qian 1995;Zhang et al. 2023a).In 2022, Dr. Guo Yongjie shared a plant photograph of Saxifraga from the above locality with us.Recently, we conducted a fieldwork and collected living material of this plant from the above locality.After a comprehensive morphological comparison of the type specimens and herbarium materials and review of the relevant taxonomic literature, we confirmed that this plant represented S. lancangensis, which is considered as a synonym of S. mengtzeana in the Flora of China (Govaerts et al. 2021;Pan et al. 2001).The purpose of this study was to re-evaluate the taxonomic status of S. lancangensis.Based on the protologue and type specimens, S. lancangensis differs from S. mengtzeana by its petals with red base and orbicular leaf blade (Qian 1995).Geographically, the type locality of S. lancangensis is some 500 km distant from the type locality of S. mengtzeana.The flowering time of S. lancangensis is December to January of the next year, while the flowering time of S. mengtzeana is September to November (Zhang et al. 2023a).These differences suggest that the taxonomic status of S. lancangensis should be re-examined.

Morphological comparison
Morphological data were recorded based on both fresh and dried plant samples from field collections and herbarium specimens.Voucher specimens of our collections were deposited in the herbarium of the Kunming Institute of Botany (KUN), Kunming, China.Herbarium specimens of Saxifraga sect.Irregulares from the herbaria CDBI, CSFI, GXMG, HITBC, IBSC, KUN, PE, SYS, and WUK (Thiers, (updated continuously)) were examined either through direct examination of the specimens or by viewing their digital images provided by the National Plant Specimen Resource Center (https://www.cvh.ac.cn/index.php)and the JSTOR Global Plants web portal (https://plants.jstor.org/).Voucher specimens for the morphological observations of S. lancangensis are cited under Additional specimens examined in the Taxonomic treatment.

Phylogenetic reconstruction
We collected 20 samples representing S. lancangensis, S. mengtzeana and related taxa (Table 1), which included 12 species of S. sect.Irregulares.Saxifraga sinomontana J.T.Pan & Gornall from Saxifraga sect.Ciliatae Haw. was selected as the outgroup based on previous molecular studies (Li et al. 2019;Tkach et al. 2015;Zhang et al. 2020bZhang et al. , 2023a)).Leaf materials were collected from both field (silica-gel-dried leaves) and dry herbarium specimens.Molecular analyses were conducted using genome skimming data.The procedures for DNA extraction, library preparation, and sequencing were carried out at Novogene (Beijing, China).Sequencing libraries were generated using the NEB Next® Ultra DNA Library Prep Kit for Illumina® (NEB, USA).The prepared libraries were sequenced on an Illumina Hiseq 4000 platform with 150 bp paired-end reads.The GetOrganelle pipeline was used to assemble plastid genome data (Jin et al. 2020).Complete plastid genome was annotated in batches using the web application GeSeq (https://chlorobox.mpimpgolm.mpg.de/geseq.html)(Tillich et al. 2017).A concatenation-based approach was conducted for shared protein coding genes (PCGs) regions of the plastid genome, and sequences were aligned in MACSE v2 and trimmed by Gblocks 0.91b implemented in Phylosuite v1.2.3 (Ranwez et al. 2018;Talavera and Castresana 2007;Zhang et al. 2020a).Phylogenetic reconstruction was performed using both maximum likelihood (ML) and Bayesian inference (BI) methods.The GTR + I + G model of sequence evolution was selected as identified by jModeltest ver.

Morphology
The asymmetric flowers and absence of stolons (Figs.Irregulares.It is notable that the peltate feature is plastic in S. lancangensis, and there are intermediates with the basifixed phenotypes within the same population, which is consistent with our observations on S. mengtzeana and S. daqiaoensis (Zhang et al. 2023a).Besides, Balfour (1916) described Saxifraga henryi Balf.f. on account of its peltate leaves, but it was now accepted as part of the variation encompassed by S. mengtzeana (Pan et al. 2001;Zhang et al. 2023a).S. lancangensis is distinct from the latter two chiefly in its petals with a red and clawless base, and its orbicular leaf blade.The morphological comparisons of these species are presented in Table 2. Geographically, the distribution ranges of these three species are isolated from each other (the type locality of S. lancangensis is about 500 km distant from the type locality of S. mengtzeana, and some 1300 km distant from the type locality of S. daqiaoensis) (Fig. 4).

Phylogenetic analyses
A total of 13 taxa were included in the phylogenetic analysis using 73 shared protein coding genes (PCGs) regions of the plastid genome of 20 samples.The resulting concatenated matrix dataset contained 60,472 bp.The 50% majority-rule consensus tree (Fig. 5) based on maximum likehood bootstraps (ML) and Bayesian posterior probability (PP) of PCGs both revealed a sister relationship between S. lancangensis and Saxifraga geifolia Balf.f. with strong supports (ML = 100, PP = 1).The monophyletic clade of S. lancangensis and S. geifolia grouped together with Saxifraga viridiflora X.J.Zhang, T.Deng, J.T.Chen

Discussion
Saxifraga lancangensis was listed among the synonyms of S. mengtzeana in the Flora of China, which are widely accepted in reference lists such as POWO (https://powo.science.kew.org/) and Tropicos (https://tropicos.org/home).In the current study, the taxonomic status of S. lancangensis was re-evaluated by conducting morphological comparison and phylogenetic analysis of this species and related species.Morphological (Table 2) and molecular (Fig. 5) evidence are consistent in distinguishing S. lancangensis from S. mengtzeana.Overall, S. lancangensis is easily distinguished from any other species of Saxifraga sect.Irregulares by its petals with a red, clawless base.Additionally, S. lancangensis differs markedly from others species in S.

Phenology
Flowering was observed from December to January of the next year, and fruiting from January to March.

Distribution and habitat
Saxifraga lancangensis is native to Zhutang Town, the eastern parts of Lancang County, Yunnan Province.It grows in limestone at altitudes 1780-2000 m under evergreen broad-leaved forest dominated by Neocinnamomum delavayi (Lecomte) H.Liu, Cornus oblonga Wall., Piper sarmentosum Roxb.and Elatostema salvinioides W.T.Wang.

Comparison with close Saxifraga species
Peltate-leaved feature was found in S. lancangensis.There are only two known species with peltate petiole insertions in S. sect.Irregulares-S.daqiaoensis and S. mengtzeana.S. lancangensis differs from latter two species in flower color (petals white to greenish, base red vs. petals white, base with yellow spots), petals shape (base of small petals clawless vs. base of small petals contracted into a claw), nectary disc (greenish vs. yellow).S. lancangensis is also distinct from S. mengtzeana in leaf shape (orbicular vs. triangular-ovate), leaf margin (shallowly dentate vs. crenate-dentate), flowering time (December to January of the next year vs. September to November).

Conservation significance
Saxifraga lancangensis is an endemic species with narrow geographical distribution only known from Zhutang Town, Lancang County of Yunnan, China.It grows in limestone under forest close by the road, which can be easily disturbed.We estimate that the population size at the type locality comprises less than 300 individuals.Due to its limited range, fragile habitat and low population size, S. lancangensis should be considered as a protected plant in China.
1 and 2) of Saxifraga lancangensis indicate a position in Saxifraga sect.Irregulares ser.Rufescentes J.T. Pan (Pan 1991; Pan et al. 2001).Morphological comparison of S. lancangensis and known species of S. sect.Irregulares indicates that S. lancangensis resembles S. mengtzeana and Saxifraga daqiaoensis F.G.Wang & F.W.Xing in having leaf blade with a peltate petiole insertion (Figs. 2 and 3), which distinguish them from other species in S. sect.

Fig. 2
Fig. 2 Saxifraga lancangensis from type locality.A leaf blade; B petiole base; C pedicel with white glandular pubescent; D inflorescence; E adaxial surface of flower; F abaxial surface of flower; G rhizomes; H&I plant and and habitat sect.Irregulares not only by morphological characteristics and geographical distance, but also by flowering time.Most species of S. sect.Irregulares bloom in summer or autumn, while S. lancangensis flowers in winter.The differentiation in flowering time may play an important role in the reproductive isolation of the species in S. sect.Irregulares.Taxonomic treatment Saxifraga lancangensis Y.Y.Qian, Acta Bot.Austro Sin.10: 70-72 (1995) (Figs. 1 and 2).Type CHINA.Yunnan Province, Lancang County, Zhutang Town, alt.2000 m, 10 Dec. 1991, Qian Yiyong, 2409 (Holotype HITBC!, isotype IBSC! ).

Table 1
Voucher information and genbank accessions for phylogenetic analysis

Table 2
(Zhang et al. 2023b)related to S. geifolia(Zhang et al. 2023b), however, S. cataphracta morphologically differs from other species of S. sect.Irregulares by its abaxial leaf surface covered with white or virescent streaks.The capsule beaks of S. cataphracta is winged when mature (vs.capsule beaks divergent in S. geifolia Diagnostic characters of Saxifraga lancangensis and comparison with other related species of Saxifraga sect.Irregulares