﻿Eriobotryacrassifolia (Rosaceae), a new species from Yunnan Province, China

﻿Abstract The new species Eriobotryacrassifolia, collected from Yunnan Province, China, is characterised and illustrated. A phylogeny based on chloroplast genomes supported its closest affinity with E.tengyuehensis, while a phylogeny based on 197 single-copy nuclear genes supported its closest affinity with E.fragrans and E.deflexa. Morphologically, however, it resembles E.angustissima. Nevertheless, it can be easily distinguished from E.angustissima by its abaxially retroflexed and sharply serrate leaf margins, densely rusty tomentose inflorescences, and oblong or elliptic leaves.

Recently, intergeneric delimitation between Eriobotrya and Rhaphiolepis has been greatly disputed, based on the questions of whether they are both monophyletic or whether the latter is nested within the former. Liu et al. (2020a) argued that Eriobotrya and Rhaphiolepis should be merged into one genus, based on the evidence from chloroplast genomes, as well as the entire nrITS, which strongly supported the paraphyly of Eriobotrya, with Rhaphiolepis nested within it. However, based on the partial ITS region, the phylogenetic trees inferred from Bayesian Inference (BI) and Maximum Likelihood (ML) demonstrated that Eriobotrya is monophyletic with robust support and could be distinct from Rhaphiolepis (Kang et al. 2021). In the latest research (Dong et al. 2022), with increased sampling and well-supported phylogenies, based on nrITS datasets, the monophyly of Eriobotrya, as well as Rhaphiolepis, was also supported. Based on our recent work (Chen et al. 2022), the plastome data, indeed, agree with the paraphyly of Eriobotrya, while simplified haplotype networks of three nuclear genes of PXMP2, TPP2 and C23H indicated that all haplotypes of Rhaphiolepis formed one distinct clade and the phylogenetic tree constructed from 197 nuclear genes revealed that R. philippinensis, as a representative of Rhaphiolepis, was placed outside all Eriobotrya species with high support. Besides, morphologically, the (circular) annular ring after sepal senescence could only be observed in Rhaphiolepis regardless of the difference in persistent sepals of Eriobotrya (Robertson et al. 1991;Gu and Spongberg 2003). Interspecific and intergeneric chloroplast capture might be frequent in Maleae (Liu et al. 2020b;Chen et al. 2022). Therefore, we suggest that ancient chloroplast capture once occurred in the ancestor of Rhaphiolepis, which captured the chloroplast of Eriobotrya species, thus causing the cyto-nuclear discordance. Here, we propose that both Eriobotrya and Rhaphiolepis are monophyletic and these two genera should not be merged.
Previously, we described a new species of Eriobotrya laoshanica that flowers in autumn in the Laoshan Natural Reserve, Malipo County, south-eastern Yunnan Province, China (Chen et al. 2020). Afterwards, through our numerous field investigations, we identified another new sympatric Eriobotrya species that flowers in spring. After careful comparison and verification, we confirmed it as a new species, which is described and illustrated here.

Materials and methods
Field investigations and observations were conducted during the flowering and fruiting periods of the putative new species. Fresh leaves were collected and stored in silica gel for molecular experiments. Herbarium specimens were used for further morphological comparisons. Voucher specimens were deposited in the herbarium of Sun Yat-sen University (SYS). Total DNA extraction, sequencing and data analyses were conducted following procedures described by Chen et al. (2022).
We retrieved all available chloroplast genomes of Eriobotrya and Raphiolepis from the NCBI public database (https://www.ncbi.nlm.nih.gov/). In total, we downloaded 58 accessions representing 24 Eriobotrya species, eight Raphiolepis species and five related species as outgroups. Chloroplast genome sequences were aligned using MAFFT v.7 (Katoh and Standley 2013). The alignment was then trimmed using trimAl v.1.2 by setting the "gappyout" model (Capella-Gutiérrez et al. 2009). The Maximum Likelihood (ML) tree was constructed using IQ-TREE v.1.6.8 under 5,000 replicates of the SH approximate likelihood ratio test (SH-aLRT) and 10,000 replicates of ultrafast bootstraps (UFBS) (Nguyen et al. 2015). For the phylogenetic tree based on nuclear genes, we referred to the results of Chen et al. (2022), in which 197 single copy nuclear genes were used and one accession of the new species, 35 accessions of other Eriobotrya species, one accession of Rhaphiolepis and five accessions of outgroups were included.

Results
The alignment length of the chloroplast genome was 156,859 bp, amongst which 1,736 sites were parsimony-informative. According to the Bayesian Information Criterion (BIC), the best-fit nucleotide substitution model was GTR+F+I+G4.
The phylogenetic tree inferred from chloroplast genomes demonstrated that this new species is sister to E. tengyuehensis with high support (SH-aLRT = 98; UFBS = 100, Fig. 1), but distantly related to E. fragrans and E. deflexa. However, based on the 197 single copy nuclear genes, the species tree showed that this new species was closely related to E. fragrans and E. deflexa with low support (quartet score, QS = 38; local posterior probabilities, Astral-PP = 0.74) ( fig. 5b in Chen et al. 2022), while the divergence time estimation indicated that it was sister to the sympatric species of E. laoshanica ( fig. 8b in Chen et al. 2022), in which this new species was marked with E. sp1. All this molecular evidence identified that E. angustissima was distantly related with the new species although they were similar in morphology.

Discussion
Based on the PhyloNet analysis of Chen et al. (2022), we considered that E. tengyuehensis might originate from the hybridisation of E. obovata and this new species, with the latter as its female parent. It is for this reason that E. tengyuehensis and the new species formed a well-supported sister clade in the chloroplast phylogeny. Based on the species tree, this new species was closely related with E. fragrans and E. deflexa, but the support values were very low. As concluded by Chen et al. (2022), extensive ancient hybridisation events occurred in this genus during species diversification. Hence, this new species might also be involved in ancient hybridisation events and its parents were not found in extant species. Morphological traits of the parents were also unknown and we supposed morphological similarity between this new species and E. angustissima Figure 1. The phylogenetic tree, based on 58 complete chloroplast genomes. Numbers near nodes indicated SH approximate likelihood ratio test (SH-aLRT) and ultrafast bootstrap support values (UFBS), respectively. The symbols "*" imply that SH-aLRT ≥ 95% or UFBS ≥ 95%. The symbols "-" imply that SH-aLRT < 50% or UFBS < 50%. The new species was highlighted in bold.
was just a coincidence. Besides, population genomics of Eriobotrya species, especially for those distributed in Yunnan Province, need to be further conducted to explore the potential causes of those discordances.
Geographically, E. fragrans is distributed in Guangdong and Guangxi Provinces, China and E. deflexa is distributed in Guangdong and Taiwan Provinces, China. Additionally, they are also significantly different from this new species in morphology. Though this new species shares the same distribution regions with E. laoshanica, they are distinctly different, both in phenology and morphology. The new species is morphologically similar to E. angustissima and E. tengyuehensis. However, by our comprehensive comparisons, it could be easily distinguished from E. angustissima by its oblong or elliptic (vs. narrowly oblong) and thickly (vs. thinly) coriaceous leaves; inflorescence densely rusty tomentose (vs. glabrous or glabrescent) and 10-17 lateral veins (vs. 8-10). Though this new species shares a few characteristics with E. tengyuehensis, for example, thickly coriaceous leaves, similar number of lateral veins and styles, it could be easily distinguished from the latter by its abaxially retroflexed and sharply serrate margin (vs. not retroflexed and entire basally, serrate apically) and much smaller leaves (9-11 × 2.5-3.5 cm vs. 12-17 × 5-7 cm) ( Table 1).  (Figs 2, 3). Diagnosis. The new species resembles E. angustissima, but differs from the latter by its leaf shape and texture, the number of lateral veins and the indumentum of inflorescence.
Phenology. Flowering from March to April, fruiting from June to August. Etymology. Latin crassus, thick, and folia, leaved, alluding to leaf thickness Distribution and habitat. Presently, Eriobotrya crassifolia is known from a single locality, Laoshan Natural Reserve, Malipo County, south-eastern Yunnan Province, China. It is distributed in thick forests on the slopes of limestone hills at altitudes of 1502-1684 m a.s.l.
Conservation status. Only two populations were found with no more than 200 mature individuals. Thus, the species status could be considered as Endangered (