Cotoneaster densiflorus, a new species of Rosaceae from western Sichuan, China, is described and illustrated. Morphologically, we inferred that the new species belongs to Cotoneaster Ser. Salicifolii sensu Yü et al. (1974) in the Flora of China and Fryer and Hylmö (2009). This species is most similar to C. salicifolius, but differs in its leaf blade of ovate-lanceolate to obovate shape (vs. elliptic-oblong to ovate-lanceolate), smaller length-width ratio of 2.37 ± 0.31 (vs. 3.17 ± 0.32), slightly conduplicate (vs. not conduplicate), less lateral veins of 6–8 pairs (vs. 12–16 pairs), upper surface slightly rugose (vs. rugose), leaf margin plane (vs. revolute), lower surface densely grey tomentose (vs. grey tomentose, with bloom), greater corolla diameter of 7–9 mm (vs. 5–6 mm), styles 2 (vs. 2–3), pyrenes 2 (vs. 2–3), larger pollen grains P/E values of 2.05 ± 0.12 (vs. 1.19 ± 0.05) and leaf epidermis type W (vs. type I). Based on phylogenetic analysis of the whole chloroplast genome, C. densiflorus is sister to C. rhytidophyllus, but distantly related to C. salicifolius.

Anatomical, chloroplast genome, leaf epidermis, palynological, Ser. Salicifolii

Cotoneaster Medik. (Rosaceae, Maloideae) is a morphologically highly variable genus that is naturally distributed in Europe, North Africa and the temperate areas of Asia except Japan. The Himalayas and neighboring mountains in Yunnan and Sichuan of China are species diversity and distribution centers for this genus (Fryer and Hylmö 2009). Due to the frequent occurrence of hybridization and polyploidisation, together with apomixis, its infrageneric classification is controversial and unstable (Campbell et al. 2007; Lo and Donoghue 2012; Li et al. 2014, 2017a; Meng et al. 2021). The number of species in the genus ranges from about 50 to more than 400 according to different (and often contradictory) species concepts and traits (Flinck and Hylmö 1966; Yü et al. 1974; Phipps et al. 1990; Lu et al. 2003; Fryer and Hylmö 2009; Dickore and Kasperek 2010). In the Flora of China, the genus was divided into three sections (Sect. Densiflos, Sect. Cotoneaster and Sect. Uniflos), based on the number of flowers in the inflorescence (Yü et al. 1974). However, more studies supported the conclusion of two major sections/subgenera: Chaenopetalum and Cotoneaster (Koehne 1893; Flinck and Hylmö 1966; Phipps et al. 1990; Fryer and Hylmö 2009), which was also proved by molecular phylogenetic studies, based on nuclear ITS, chloroplast or low-copy nuclear genes in the last decade (Li et al. 2012, 2014; Lo and Donoghue 2012; Meng et al. 2021). For species in Subg. Chaenopetalum, the flowers in a cyme open simultaneously, within white spreading (rarely pink) petals, white filaments and purple to black anthers. While for species in Subg. Cotoneaster species, the flowers in a cyme open continuously over an extended period, within red (rarely pink) erect (less suberect) petals, red, pink or white filaments and white anthers.

Within the sections/subgenera, the further division into 7–39 series was mainly based on morphology of stems, branches, leaves, number of pyrenes and resistance (Flinck and Hylmö 1966; Yü et al. 1974; Phipps et al. 1990; Fryer and Hylmö 2009). The Ser. Salicifolii included 5–7 species and about three varieties, whose name was derived from the mostly lanceolate and willow-like leaves. In Fryer and Hylmö (2009) and Flora of China (Yü et al. 1974), the important morphological features that distinguished the Ser. Salicifolii species from other series include evergreen or semi-evergreen shrub, leaf blade leathery, mostly lanceolate, abaxially persistently densely tomentose, veins impressed, white spreading petals, and pyrenes 2–4 (–5). Multiple phylogenetic trees of Cotoneaster, based on low-copy nuclear and chloroplast genes showed that these series, as smaller taxonomic units under subgenera, were not monophyletic (Lo and Donoghue 2012; Meng et al. 2021), which agreed with our recent phylogenetic study results on Ser. Salicifolii (unpublished study).

During our field survey in western Sichuan Province, an interesting population that shares morphological affinities with Ser. Salicifolii species was discovered. These affinities are based on evergreen or semi-evergreen shrub, leathery leaf blade, dense inflorescence and white spreading petals. However, this taxon was not completely similar to any species that has been described worldwide. Furthermore, the individuals of this species were distributed in Baoxing County, which is located in Siguniang and Jiajin Mountains at the eastern edge of the Hengduan Mountains, a biodiversity hotspot in southwest China (Zhang and Ma 2008, 2009; Li et al. 2017b). Interestingly, this county was also the type specimen collection site of C. salicifolius and C. moupinensis. Over the past 10 years, approximately 10 new plant species were discovered and illustrated in Baoxing County, such as Youngia baoxingensis Y.S. Chen (Chen 2018), Primula luteoflora X.F. Gao & W.B. Ju (Ju et al. 2018), Berberis jinwu Y.K. Li, Harber, Y.W. Xing & C.C. Yu (Li et al. 2022) and several others. From 2016 to 2023, after detailed morphological examination, specimen collection and comparison with herbarium specimens sampled in this region, we identified this shrub as a new species and determined its phylogenetic position in the genus of Cotoneaster through whole chloroplast genome data. Our study not only enriched the diversity of Cotoneaster species in China, but also highlighted the importance of the basic survey of biodiversity in this area of Sichuan and the Hengduan Mountains.

Plant morphological features and habits were recorded and photographed in the field during the flowering and fruiting periods of the putative new species. The characteristics and measurements were compared with those of its related species naturally occurring in Sichuan Province (i.e. C. salicifolius and C. rhytidophyllus) as described in the Flora of China (Yü et al. 1974) and related taxonomic literature of Cotoneaster (Fryer and Hylmö 2009). Voucher specimens were deposited in the Herbarium of SunYat-sen University (SYS) in China.

For scanning electron microscopy (SEM) observations, pollen grains of this putative new species and two related species were collected from specimens (M.W. Li 20230617007, Q.Fan 15682-01 and Q.Fan 15643 (SYS)). For scanning electron microscopy (SEM) observation, the pollen grains were transferred onto metal stubs with double-sided adhesive tape and sputter-coated with technical gold (Li et al. 2017b; Xiong et al. 2019). Approximately 20 randomly selected pollen grains were scanned and photographed by the SEM (S-3400, Hitachi, Tokyo, Japan) at 5 kV accelerating voltage and SE detector, then the pollen grains were measured and the polar axis (P), equatorial axis (E) and the ratio of polar axis length to equatorial axis length (P/E) were calculated. The nomenclature for pollen morphology mainly followed Erdtman (1943, 1952) and the terminology of ornamentation mainly followed Ueda and Tomita (1989).

Leaf epidermal materials were prepared from mature leaves and macerated in 1:1 (by volume) hydrogen dioxide solution and glacial acetic acid and then were boiled in a water bath for 1.5–2 h. After being rinsed with water, leaf materials were transferred to Schultze’s solution for 30 minutes. Finally, pieces of leaf epidermis were stained with a solution of 1% safranin prior to mounting in glycerine gel. Prepared cuticles were observed using a SY100 light microscope and JSM-6330F SEM. The nomenclature of stomatal types and leaf epidermis is mainly based on the descriptions of Wilkinson (1979), Prabhakar (2004) and Ding et al. (2008).

Total genomic DNA was extracted using the Plant Genomic DNA Kit (DP305, Tiangen Biotech Co., Ltd., Beijing, China) and DNA quality was measured using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). The qualified DNAs (≥50 ng) were sent to Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) for paired-end (PE) library construction and genome-skimming sequencing. The generated reads were assembled by the GetOrganelle (Jin et al. 2020) pipeline. In this pipeline, the chloroplast genome of C. salicifolius (KY419943.1; Zhang et al. (2017)) was set as a reference. The genome annotation was performed with CpGAVAS (Liu et al. 2012), then the inverted repeat (IR) boundaries were manually adjusted and confirmed on geneious prime2023.0.4 (https://www.geneious.com/). In order to determine the phylogenetic position of this species in Cotoneaster, complete chloroplast genomes of 64 accessions downloaded from NCBI and seven unpublished Ser. Salicifolii taxa were obtained to reconstruct the phylogenetic trees with Rhaphiolepis bibas and Rhaphiolepis prinoides as outgroups (Table 1). The sequences were aligned using MAFFT version 7 (https://mafft.cbrc.jp). The best-fit nucleotide substitution model was determined by ModelFinder (Kalyaanamoorthy et al. 2017). The maximum-likelihood (ML) phylogenetic tree was constructed using RAxML-HPC Blackbox Software (Stamatakis 2014) with the GTRGAMMAI model and 1000 bootstrap replicates to assess the support for each branch. Bayesian inference (BI) was conducted using MrBayes v.3.2.7 (Ronquist et al. 2012) with Markov chains for at least 10,000 generations and sampled every 10 generations. After the average standard deviation of split frequencies (ASDFs) was assessed and reached < 0.01, the first 25% trees were discarded as burn-in.

Flow cytometry was used to estimate the genome size and to determine the ploidy level. Samples were prepared by a modified method according to Rothleutner et al. (2016). Approximately 1 cm² of fresh leaf tissue was chopped with maize as the internal standard using a blade in cold (4 °C) Otto I buffer (0.1 M citric acid, 0.5% (v/v) Tween 20) for 90 s before being filtered through a 50-μm nylon mesh. The suspension was stained with Otto II buffer (0.4 M Na₂HPO₄·12H₂O), β-mercaptoethanol, Rnase and PI fluorochrome. An Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with 488-nm laser, was employed with a sample flow rate 14 µl min⁻¹. Fluorescence measurements were obtained using the FL2 (585/40 nm) optical filter, capturing 10 000 events and utilizing the FL2-A values for the 2C peak.

We described and illustrated a new species of Cotoneaster genus (Rosaceae) in western Sichuan Province of China and provided evidence for its phylogenetic position through whole chloroplast genome data. After detailed field research, we found Cotoneaster densiflorus M.W. Li, Q. Fan & W. B. Liao, sp. nov. is distributed in a narrow range of Baoxing County, which is located in the Hengduan Mountains. Only one large population of nearly 60 individuals was observed, with about 40 densely distributed individuals and 20 scattered shrubs on a steep slope of sunny sparse forest along the National Highway. Morphologically, this shrub is most similar to C. salicifolius, but obviously differs in leaf upper surface rugose state, margin revolute state, number of lateral veins, styles and pyrenes, pollen grains P/E values and leaf epidermis type. Our study not only enriched the diversity of Cotoneaster species in China, but also highlighted the importance of the basic survey of biodiversity in this area of Sichuan and the Hengduan Mountains.