﻿Resurrection of Perilimnastes (Sonerileae, Melastomataceae) with description of a new species P.nana

﻿Abstract Recent research has indicated that the Phyllagathis (raphides) clade (Sonerileae, Melastomataceae) is only distantly related to the type of Phyllagathis and should be separated as a distinct genus. Phylogeny of this clade is here reconstructed with expanded taxon sampling. Four strongly supported subclades have been identified. The possible affinities of taxa that were not sampled in the analysis are discussed, based on morphological data. Perilimnastes is resurrected as the generic name of the Phyllagathis (raphides) clade. A generic description, colour figures, map of distribution, a list of included species and a key are provided for Perilimnastes. Fifteen new combinations are made plus the description of a new species. As interpreted here, Perilimnastes consists of twenty species and two varieties.


Introduction
The genus Perilimnastes Ridl.was initially established based on P. fruticosa (Ridl.)Ridl.(Ridley 1918(Ridley , 1922)).Nayar (1974) followed Ridley's concept of Perilimnastes and described a second species in the genus, namely P. rupicola M.P.Nayar.The two species show clear similarities in the morphology of leaves, calyx lobes, stamens, and capsules.However, subsequent authors did not recognise Perilimnastes (Maxwell 1982(Maxwell , 1989;;Cellinese 2002Cellinese , 2003)).Both species are currently treated in a broadly defined Phyllagathis Blume.Previous molecular phylogenetic studies have revealed the polyphyletic nature of Phyllagathis (Zeng et al. 2016;Zhou et al. 2018;Zhou et al. 2019a, b, c;Liu et al. 2022;Zhou et al. 2022).The species currently treated under Phyllagathis were found to be nested within 17 lineages of Asian Sonerileae (Liu et al. 2022;Zhou et al. 2022).Although P. fruticosa, the generic type of Perilimnastes, was not sampled in these studies, species that are quite similar to it were identified as belonging to the Phyllagathis (raphides) clade.Members of this clade are often shrubs or shrublets with cuneate to rounded leaf bases, umbellate or cymose inflorescences (sometimes reduced to a single flower), isomorphic stamens, dorsally spurred connectives, crowned capsules, horned placental column and thready PhytoKeys 238: 11-31 (2024), DOI: 10.3897/phytokeys.238.116168 Ying Liu et al.: Resurrection of Perilimnastes (Sonerileae, Melastomataceae) placentas.Some of them (Fig. 1) are also characterised by the presence of raphide crystals in various parts of the plant.Based on these diagnostic features as well as strong resemblance between sampled and unsampled species, Zhou et al. (2022) estimated that the Phyllagathis (raphides) clade might contain 20 species in southernmost China, Vietnam, the Malay Peninsula and Borneo.This clade should be removed from Phyllagathis and treated as a distinct genus, since it is only remotely related to the type of Phyllagathis.As a result, Perilimnastes should be re-instated as the generic name (Zhou et al. 2022).
This work aims to formalize the taxonomic treatment of the Phyllagathis (raphides) clade.To this end, we reconstructed the phylogeny of this clade with expanded taxon sampling, using a nuclear genomic dataset assembled by mapping the genome resequencing reads to the draft genome of Bredia hirsuta Blume.We also discussed putative affinities based on morphological data for species that were not sampled in the phylogenetic analysis.Perilimnastes is resurrected as the generic name of the Phyllagathis (raphides) clade.A generic description, colour figures, map of distribution, a list of included species and a key are provided for Perilimnastes.Fifteen new combinations are made plus the description of a new species from southern China.Perilimnastes, as we here delimit it, now consists of twenty species and two varieties.
For DNA extraction, library preparation, whole genome resequencing and quality control of the raw reads, methods employed in this study followed the protocols outlined in Zhou et al. (2022).The genomic single nucleotide polymorphism (SNP) dataset was assembled by mapping the genome resequencing data to the draft genome of Bredia hirsuta, which can be accessed at https://doi.org/10.17632/s85vv6yyjs.1.High-quality reads were mapped to the reference genome using BWA-MEM (Li and Durbin 2010).SNPs and short insertions/deletions (InDels) were identified using HaplotypeCaller in GATK v.4.1.8.1 (McKenna et al. 2010) under the GVCF mode for each sample separately.Next, we conducted hard filtering to minimise false positives by applying the following parameters: (1) QUAL < 30.0; (2) DP < 15.0; (3) QD < 2.0; (4) FS > 60.0; (5) MQ < 50.0; (6) SOR > 3.0; (7) MQRankSum < -12.5; (8) ReadPosRankSum < -8.0; (9) InbreedingCoeff < -0.5.VCFtools v.0.1.16(Danecek et al. 2011) is used to exclude SNPs with a missing rate exceeding 15% and those with minor allele frequencies (MAF) below 0.05.The SNPs obtained were pruned, based on their linkage disequilibrium (LD) patterns using the -indep-pairwise option in PLINK (Purcell et al. 2007).Only one SNP was retained for each SNP pair with an r 2 value above 0.5 within a sliding window of 50-SNPs (advanced by 5 SNPs each).Maximum Likelihood analysis of the genomic dataset was performed using a partitioned approach in IQ-TREE v.2.0.3 (Nguyen et al. 2015).Phyllagathis rotundifolia was designated as the outgroup taxon.The selection of best fitting substitution model was conducted using ModelFinder (Kalyaanamoorthy et al. 2017) based on the Bayesian Information Criterion (BIC).The genomic dataset was partitioned into bins of equal length, each containing 2,000 SNPs.TVMe+ASC+R2 was selected as the best fitting substitution model for all partitions.Node support was accessed using 1000 replicates of the UFBS and SH-aLRT test.

Morphological comparison
Morphological data were obtained through fieldwork, herbarium records, literature survey and observation of living plants in the facilities of Sun Yat-sen University.We examined specimens or their high-resolution photos of the relevant species from the following herbaria: A, BM, C, E, G, GXMI, IBSC, IBK, K, KUN, NY, P, PE, SYS and US.Species delimitation mainly followed Chen (1984a), Hansen (1992), Cellinese (2002Cellinese ( , 2003) ) and Chen and Renner (2007).
Four well-supported lineages were identified within the Phyllagathis (raphides) clade, but relationships amongst them were only moderately supported (SH-aL-RT test = 100%, UFBS = 92%; SH-aLRT test = 100%, UFBS = 92%).Liu & Ying Liu, P. ternata C.Chen, P. ovalifolia H.L.Li and P. calisaurea C.Chen (currently synonymised under P. ovalifolia).These species have raphides and share obvious similarities in the inflorescences with 1-3.5 cm long peduncles and purple anthers with a short dorsal spur and without ventral appendages.Zhou et al. (2022) found that the crystal type exhibits the lowest level of homoplasy amongst 14 characters they tested.The shift from druses to raphides took place on only three occasions within Asian Sonerileae (Zhou et al. 2022), one in Fordiophyton and two in two subclades of the Phyllagathis (raphides) clade.The presence of raphides, therefore, is a useful diagnostic character for these lineages.

Species without molecular data
Perilimnastes fruticosa, Phyllagathis guillauminii H.L.Li, Phyllagathis brookei M.P.Nayar and Perilimnastes rupicola M.P.Nayar, four putative members of the Phyllagathis (raphides) clade, have never been included in phylogenetic studies.Nevertheless, they can be easily referred to specific lineages within this clade, based on compelling morphological evidence.Perilimnastes fruticosa from the Malay Peninsula closely resembles P. multisepala from subclade 1 and P. stenophylla and P. suberalata from subclade 2. The four species are shrubs characterised by somewhat oblong-lanceolate, 3-veined leaf blades, few-flowered inflorescences, narrow calyx lobes and the presence of druses.Moreover, they grow in similar habitats, specifically on rocks along streams in dense forests.Perilimnastes fruticosa is possibly a member of subclade 1 or subclade 2. Raphides have been found in the tissues of P. guillauminii (southern Vietnam), P. brookei (Borneo) and P. rupicola (Borneo).The three species can be confidently referred to subclade 3 since all Vietnamese and Bornean species with raphides were consistently recovered as members of this subclade in phylogenetic analyses (Zhou et al. 2022;this study).Phyllagathis guillauminii resembles P. uniflora from subclade 3 in 3-veined leaves with cuneate base and somewhat acuminate apex and narrow calyx lobes.The close relationships amongst P. dispar, P. elliptica, P. brookei and P. rupicola had been proposed by Cellinese (2003).Their caulescent and erect stems, small leaves, few-flowered umbels, as well as crystal type make them a distinct group that is morphologically very different from other Bornean species treated under Phyllagathis (Cellinese 2003).
Another species, P. marumiaetricha (Guillaumin) C.Hansen, was listed as a putative member of the Phyllagathis (raphides) clade by Zhou et al. (2022).It resembles P. setotheca from subclade 1 in the inflorescences with large basal bracts, petals and flowers.However, the huge leaves, distinctive hypanthial emergences and the peculiar sepals of this species readily distinguish it from members of the Phyllagathis (raphides) clade.As its generic affiliation remains to be further tested, no taxonomic treatment is proposed here.

Conclusion
Molecular phylogenetic data and morphological evidence support the Phyllagathis (raphides) clade as a distinct lineage encompassing species distributed in southernmost China, Vietnam, the Malay Peninsula and Borneo.Perilimnastes is, therefore, resurrected below as the generic name for this clade.For a comparison of Perilimnastes [the Phyllagathis (raphides) clade] and other lineages of Asian Sonerileae, please see table S9 in Zhou et al. (2022).
Description.Erect shrubs, erect/ascending shrublets or caulescent herbs, sometimes with raphides in many parts.Stems terete, obtusely 4-sided or ribbed, with uniseriate or multiseriate, appressed or spreading hairs, rarely glabrous.Leaves opposite, equal, subequal or unequal in a pair, petiolate, rarely sessile (in P. sessilifolia); leaf blades elliptic, ovate, elliptic-lanceolate, obovate, oblanceolate or suborbicular, submembranous, papery or stiffly papery, 3-7-nerved, base cuneate, acute, rounded, subcordate to broadly cordate, margin entire or inconspicuously serrulate or denticulate.Inflorescences usually terminal (rarely axillary) umbels subtended by two or more bracts, many-to few-flowered, sometimes reduced to a single flower.Flowers 4-merous; hypanthia ± campanulate, cup-shaped or funnel-shaped; calyx lobes triangular, ± attenuate to ligulate or linear; petals white, pink or purplish, obovate, ovate, oblong, or elliptic, more or less oblique, apex acute or acuminate; stamens 8, equal or subequal; anthers isomorphic, yellow, pinkish or purplish, narrowly ovate to lanceolate, curved to ventral side, connectives ventrally inappendiculate and dorsally spurred, or basally forming a collar with two ventral auricles/lobes/ridges and a dorsal spur; ovary half inferior, ovoid, 4-celled, crown of four partly or fully connate lobes; style filiform.Old capsule cup-shaped, campanulate, quadrangular, crown persistent and enlarged, enclosing an obpyramidal space; placental column 4-horned; placentas thready.Seeds numerous, minute, cuneate.(Figs 2-4) Distribution.Twenty species and two varieties, eight species (seven endemic) and two varieties in southernmost China (Guangdong, Guangxi, Hainan, Yunnan), eight (seven endemic) in Vietnam, one on the Malay Peninsula and four in Borneo (Fig. 5).Notes.When publishing A. fruticosus, Ridley (1908) designated L.Wray and H.C.Robinson 5453 as the type without citing a particular herbarium, only stating that the whole collection made by Robinson's expedition should be sent to the British Museum (BM).Nayar revised Perilimnastes in 1974 and noted the specimen in BM as holotype of this species.This was probably only a speculation rather than deliberate lectotypification.In the revision of Phyllagathis, Cellinese (2002) chose a duplicate sheet in K as the lectotype, but did not include the phrase "designated here" in the typification statement, as required by Art. 7.11 of the Code (Turland et al. 2018).The specimen sheet in BM [BM000565932] is here designated as the lectotype to eliminate any uncertainty.
Type: Cochinchine.Bien Hoa, Bao Chiang, L.Pierre s.n.(lectotype, designated by Hansen [1992] Notes.Phyllagathis calisaurea was described, based on specimens collected in western Guangxi, China (Chen 1984b).Subsequent authors did not recognise it as a distinct species and synonymised it within P. ovalifolia (Hansen 1992;Chen and Renner 2007).Phyllagathis calisaurea and P. ovalifolia have adjacent distribution ranges (Guangxi vs. Yunnan, China).They are morphologically quite similar, with the only differences being leaf size (6.5-11.5 × 2-3.7 cm vs. 9-18 × 3-8.5 cm), leaf shape (ovate lanceolate vs. ovate to elliptic) and indumentum of the stems and leaves.Nonetheless, they failed to form a monophyletic group in both the previous (Zhou et al. 2022) and current phylogenetic analyses (Fig. 1).As only one accession of P. ovalifolia was included in these analyses, the boundary between P. ovalifolia and P. calisaurea needs to be further investigated using multiple accessions from across the distribution range.For the time being, we adhere to the species delimitation proposed by Hansen (1992) and Chen and Renner (2007).
Distribution.Perilimnastes nana is currently known from Taishan County, Guangdong Province, China (Fig. 8).It grows amongst rocks along streams in the forest, at 200-300 m elevation.
Notes.During a survey of herbarium specimens of Phyllagathis in IBSC, a collection (Ze-xian Li et al. 516) from Taishan, Guangdong, China caught our attention.This plant (P.nana) closely resembles P. stenophylla from Hainan Island in the oblong-lanceolate leaf blades and was misidentified as the latter species.Closer inspection reveals that it has strictly 1-flowered inflorescences and broadly obovate calyx lobes, which distinguishes it from P. stenophylla.Field trips in 2022 and 2023 revealed other differences between the two species, such as plant size and peduncle length.Perilimnastes nana is phylogenetically closest to P. setotheca, a species found in Guangdong, Guangxi and Hainan China (Fig. 8).However, they differ markedly in plant size, leaf shape and size and number of flowers per inflorescence.As a result, P. nana is quite distinct from its closest relatives, prompting us to describe it as a new species.
Subclade 1 contains Perilimnastes multisepala J.H.Dai, T.V.Do & Ying Liu from central Vietnam, Phyllagathis setotheca H.L.Li from southern China and a new species from Guangdong, China, namely Perilimnastes nana C.Y.Zou & Ying Liu.The three species are characterised by large flowers (> 20 mm in diameter), large anthers (> 8 mm long) and the presence of druses (instead of raphides).Subclade 2 consists of two species from Hainan Island, China [Phyllagathis stenophylla (Merr.& Chun) H.L.Li and P. melastomatoides (Merr.& Chun) W.C.Ko] and two from central Vietnam (P.suberalata C.Hansen and P. sessilifolia C.Hansen).Species in this subclade varied in the morphology of leaves and flowers, but they all have druses and yellow connectives that produced into a collar at the anther base.Subclade 3 comprises two species from Borneo [Phyllagathis dispar (Cogn.)C.Hansen and P. elliptica Stapf] and three newly-published species from central and southern Vietnam (Perilimnastes setipetiola J.H.Dai, T.V.Do & Ying Liu, P. uniflora J.H.Dai, T.V.Do & Ying Liu and P. banaensis J.H.Dai, T.V.Do & Ying Liu).These species are morphologically quite different, yet all of them have raphide crystals, somewhat elliptic leaf blade and at least some have terminal and axillary umbels with very short or no peduncles.Subclade 4 consists of five taxa mainly distributed in southern China, viz.Phyllagathis deltoidea C.Chen, P. elegans Hai L.Chen, Yan

Figure 1 .
Figure 1.The partitioned Maximum Likelihood (ML) phylogenetic tree inferred from the genomic SNP dataset using IQ-TREE, showing the four subclades within Perilimnastes [Phyllagathis (raphide) clade].For the nodes without full support, values from SH-aLRT test (left) and ultrafast bootstrap (right) are given at the nodes.The new species is indicated with a star.Lineages with raphides are noted with solid circles.

Figure 7 .
Figure 7. Perilimnastes nana A habitat B habit C close-up of a branchlet D adaxial leaf surfaces E abaxial leaf surfaces F a flowering branch showing an inflorescence with a single flower and two large bracts G lateral view of a flower H longitudinal section of a flower showing stamen morphology I lateral view of an old capsule with one persistent bract removed J longitudinal section of an old capsule showing enlarged ovary crown and morphology of the placental column and placentas.Scale bars: 5 mm (G-I); 3 mm (J).All from Chun-yu Zou 3608 (IBK, PE).