Sinoseris (Crepidinae, Cichorieae, Asteraceae), a new genus endemic to China of three species, two of them new to science

Elucidating the systematic position of two Chinese species described originally as Lactuca hirsuta and L. scandens, of which only historical specimens from the late 19th and early 20th century were known, field work confirmed the occurrence of three different species. Molecular phylogenetic analysis of these species based on sequences of the nuclear ribosomal internal transcribed spacer (nrITS) region uncovered a hitherto unknown lineage in a phylogenetic backbone of the subtribe Crepidinae of the sunflower family tribe Cichorieae. Substantiated by comparative morphological studies, this lineage is described as genus new to science, named Sinoseris, endemic to the Chinese provinces Sichuan and Yunnan. Two of its three species are new to science, while the third is conspecific with both L. hirsuta and L. scandens.


Introduction
Investigations into the flora of China have considerably increased our knowledge of the various plant groups in this country during the last decades and they have also pointed out knowledge gaps including still insufficiently known taxa. This also applies to the members of tribe Cichorieae of the 2 largest flowering plant family Asteraceae (sunflower family). One of the least known species in this tribe was described as Lactuca hirsuta Franch. (Franchet, 1895) from Yunnan and has been known so far from only a few collections of the late 19th and early 20th century. Shih (1997)

assigned it to
Chaetoseris, which was later found to be a congener of Melanoseris (Shih & Kilian, 2011;Wang et al., 2013;Kilian et al., 2017). Doubts on this systematic position of L. hirsuta were already expressed by Shih & Kilian (2011). In the context of the general poverty of morphological features coupled with extensive parallel evolution in this tribe, which renders the recognition of lineages difficult (Kilian et al., 2009a), the scarcity of fruiting material available for this taxon and the absence of any recent collection of it so far have hindered re-addressing its systematic position.
The situation changed when the first author discovered new specimens that matched the description and historical specimens of L. hirsuta from Dayao County collected in the frame of the fourth national survey of traditional Chinese medicine resources. More in-dept studies, including special field trips guided by the re-evaluated historical collections disclosed the involvement of another taxon, described as Lactuca scandens C. C. Chang (1934) and not so far identified with any of the known species (Shih & Kilian, 2011), and brought to light that actually a separate lineage of three species, two of them hitherto unknown to science, is involved.
The aims of the study presented in this paper were to test the previous hypotheses of the systematic position of this taxon and its allies by comparatively studying their morphology and including them in a molecular phylogenetic analysis, and finally to draw the taxonomic conclusions from the phylogenetic evidence.

Plant material:
The study is based on life plants observed and documented in the field during two field trips made to Sichuan and Yunnan in 2017 and 2018 as well as on newly collected and historical specimens of Lactuca hirsuta and its allies. Historical specimens were studied from the herbaria of GH, LBG, P, PE and W. Newly collected specimens were deposited in KUN, with duplicates in B and PE. For comparative morphological studies of L. hirsuta and allies on one hand and related genera on the other hand the herbarium collections in B, KUN and PE were consulted. 3 Morphological studies: For scanning electron microscopy, achenes and pollens were directly mounted onto SEM stubs on double-sided sticky tape, coated with 20 nm Pt-Pd using a Cressington 108Auto sputter-coater and examined using a ZEISS SIGMA 300.
Sampling, DNA extraction, amplification, sequencing and phylogenetic analysis: Multiple samples of all three species were sequenced and included in the molecular phylogenetic analysis (see Table 1) based on the nuclear ribosomal internal transcribed spacer (nrITS) using otherwise already published sequences. The sampling was designed to represent all genera of the Crepidinae (except for the extremely rare monotypic Spiroseris of Pakistan of which no sequence data are available) and its sister subtribe Chondrillinae (see Kilian et al., 2009b+; for the up-do-date systematics). Launaea sarmentosa (Hyoseridinae) as used as outgroup and root of the phylogenetic trees. INSDC (International Nucleotide Sequence Database Collaboration) accession numbers of published sequences follow the taxon name in the tree (Fig. 1). Extraction of DNA and amplification and sequencing of the nrITS region was done as described by Wang et al. (2013). Sequences were aligned with MAFFT version 7 using default parameters (Katoh et al., 2013) and the alignment adjusted manually using PhyDE version 0.9971 . Indels were coded as binary characters using simple indel coding (Simmons & Ochoterena, 2000) implemented in SeqState v.1.40 (Müller, 2005a). The matrix was subdivided into four partitions: ITS1, 5.8s, ITS2, indels. Phylogenetic relationships were reconstructed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI). MP was performed with the parsimony ratchet using PRAP v.2.0 (Müller, 2004) with default parameters in combination with PAUP v.4.0b10 (Swofford, 2003); Jackknife (JK) support values were calculated in PAUP with 10,000 jackknife replicates using the TBR branch swapping algorithm with 36.788% of characters deleted and one tree held during each replicate. ML analyses were done with RaxML (Stamatakis, 2014) in the version of RAxMLHPC v.8 on XSEDE on the CIPRES Science Gateway (Miller et al., 2010), using rapid bootstrapping integrated with the ML search for the optimal tree applying the general time-reversible (GTR) + Γ model. The BI analyses were performed with the MPI version of MrBayes (Ronquist et al., 2012) on the Soroban high-performance computing system of the 4 Freie Universität Berlin. Instead of apriori testing, the optimal substitution model was sampled across the entire general time reversible (GTR) model space in the Bayesian MCMC analysis (Huelsenbeck et al., 2004). Two simultaneous runs of four parallel chains each were performed for 3 × 10 7 generations with a sample frequency of 1 tree per 2000 generations. Convergence of the runs was checked by making sure that the average standard deviation of split frequencies of the post-burn-in runs was below 0.01 and the effective sampling size (ESS) well above 200 in either run for all parameters. TreeGraph v.2 (Stöver & Müller, 2010) was used to visualise the trees with statistical node support.

Results
The nrITS phylogeny ( Fig. 1) places the species originally described as Lactuca hirsuta and its two allies separate from all other genera into a well-supported clade (JS = 98.1, PP = 1, BS = 70) of the large, chiefly E Asian Dubyaea-Nabalus-Soroseris-Syncalathium polytomy. Finding the three species nested in one clade agrees with the conspicuous overall morphological similarity of them. The sister group relationship (with full statistical support) of two of them ( Fig. 1 as Sinoseris scandens and S. triflora), also corresponds with the stronger morphological similarity between these two species compared to the third (Fig. 1, as S. changii), which is apparent in particular with respect to achene morphology and the larger capitula.
The three species of this clade are also morphologically clearly distinct from the other members of the Dubyaea-Nabalus-Soroseris-Syncalathium polytomy. Diagnostic for this clade is a synflorescence of secund, subspiciform to paniculiform paracladia, the involucre with very few, inconspicuous outer phyllaries, beaked achenes with more than two secondary ribs per main rib and a dirty white to pale brownish pappus of moderately coarse, scabrid bristles (Table 2).
Consequently, this lineage is best classified as an own genus, for which we have chosen the name Sinoseris (see Taxonomy, below). The genus is endemic to China, where its species are restricted to Yunnan and Sichuan (Fig. 2). Only the most widespread of the three species has been known to science so far. Its original name Lactuca hirsuta is illegitimate as a younger homonym of a name coined much earlier for a species of North America. The Chinese species was described a second time 5 40 years later by Chao Chien Chang (1900Chang ( -1972 as L. scandens, a name that has to be taken now as basionym for this species. The conspecifity of both taxa was blurred, however, by infraspecific variation and incorrectly described features in the protologue of the first name (see Taxonomy, below).
The second species (see Taxonomy below, under S. triflora), hitherto undescribed, was discovered by us first among the historical specimens from the early 20th century determined as L. hirsuta before we succeeded to recollect it from the wild. The third species (S. changii) was not known to us from historical herbarium material, but ITS sequences published under the name L. scandens (INSDC acc. no. KF732051 to KF732056, see Fig. 1) and included in our phylogenetic reconstruction were found nested in the same clade as our sequences of this undescribed species.

Discussion
The Asian-North American Dubyaea-Nabalus-Soroseris-Syncalathium polytomy, in which the clade with the species under study is nested, is one of six major terminal clades resolved in our phylogenetic backbone of the subtribe Crepidinae spanning all genera (Fig. 1). This seems surprising in view of their previous classification as members of Lactuca or the Lactucinae. It must be considered, however, that the generic concept of Lactuca in the late 19th and early 20th century was extremely wide and spanned members of several of the modern subtribes (Kilian et al., 2017). Disentangling the subtribes Lactucinae and Crepidinae has, moreover, proven particularly difficult due to the scarcity of nonhomoplastic morphological synapomorphies (Bremer, 1994).
The Dubyaea-Nabalus-Soroseris-Syncalathium clade has been resolved in several studies with varying partial representation of its members (Kilian et al., 2009a;Zhang et al., 2011;Liu et al., 2013;Kilian et al., 2017). Estimations divergence times for the Crepidinae by Zhang et al. (2011) revealed that the Dubyaea-Nabalus-Soroseris-Syncalathium clade is likely of Pliocene origin, with a crown age of around 5 myr only; for genera such as Soroseris or Syncalathium a crown age of less than 2 and 3 myr, respectively, have been estimated. The comparatively young age of this clade may be responsible for the shallow genetic differentiation among many of its members (Zhang et al., 2011), in particular within lineages such as Soroseris or the Nabalus trifoliatus lineage, as indicated by branch lengths in All three known species of the clade classified here as the new genus Sinoseris are annuals (to, perhaps, monocarpic biennials) and grow in the subtropical (warm-temperate) highlands of Sichuan and Yunnan, where they all seem to grow preferably in open rocky habitats. All other members of the Dubyaea-Nabalus-Soroseris-Syncalathium clade are, in contrast, perennials and confined to the higher montane or alpine zone (Dubyaea, Soroseris, Syncalathium, "Youngia" racemifera) or more continental and either colder (Nabalus, Hololeion) or more arid (Sonchella) climates. The Sinoseris lineage is therefore somewhat outstanding in this clade, which deserves further attention.
The species have been rarely collected so far, which appears surprising as they are not inconspicuous. Reasons may be their late flowering from late September onwards and their scattered occurrence, perhaps due to their preference of rocky habitats. Diagnostic features: Annual (to monocarpic biennial) herbs; stems, leaves and involucres with conspicuous hirsute indumentum; basal leaves distinctly petiolate; synflorescence of secund, subspiciform to paniculiform paracladia; capitula with 3-12 florets; involucre with inconspicuous outer phyllaries; achenes beaked; achene corpus with 5 main ribs alternating with (2-)3-4 secondary ribs; pappus dirty white to pale brown of moderately coarse scabrid bristles.

Taxonomy
Description: Annual (to monocarpic biennial?) herbs with leafy stem and conspicuous hirsute indumentum on stem, leaves and involucres, late flowering (Sep-Nov). Basal and lowermost cauline leaves with petiole-like portion as long as or longer than lamina. Capitula 3-12 flowered, in ± secund, subspiciform to narrowly paniculiform paracladia from the axils of the cauline leaves, pendent in bud, 7 subpendent at anthesis and pendent again at fruiting. Involucre narrowly cylindrical at anthesis, strongly differentiated into an equal inner and very inconspicuous outer phyllary series. Receptacle epaleate, glabrous and smooth. Florets with yellow corolla, styles greyish to blackish. Pollen echinolophate, tricolpate, of the Cichorium type (sensu Blackmore 1986) with polar areas either triangular, moderately extensive and each with c. 12 spines, or very extensive, approximately hexagonal and each with >20 spines, and with moderately narrow interlacunar gaps ( Fig. 4e-f, 6e-f, 8e-f). Achenes beaked; corpus somewhat flattened, ± subconical, with 5 main ribs (best discernable near base) alternating with (2-)3-4 secondary ribs (fully developed in middle third and then similar in shape to main ribs) dark brown, with appressed to spreading-erect (in distal portion of corpus) linear flattened acute papillae; beak slender, shorter than or as long as corpus. Pappus dirty white, caducous, of scabrid bristles similar in length and diameter, with 7-12(-14) rows of cells in cross section near base.
Etymology: The generic name is a compound of the Latin name "Sina" for China and "seris" (σέρις), the classical Greek name for salad (more precisely of Cichorium species).

Key to the species of Sinoseris
1 Involucre with usually 8 inner phyllaries; capitula with 8-12 florets; anther tube golden yellow to brownish; achenes abruptly contracted into a slender beak as long as the obconical and below the isolectotype P00750292! [marked as "isotype"]).
Description: Annual (to monocarpic biennial) herbs, 15-80 cm tall, strongly hirsute of eglandular reddish-purplish hairs, with a taproot. Stem solitary, or if branched right from the base, plants seemingly with several stems, erect or arched erect, branching, leafy. Basal and lower cauline leaves abruptly contracted into a petiole-like portion up to 15 cm long, its base semi-amplexicaul to, more often, winged and distinctly clasping the stem; lamina broadly triangular, ovate to broadly lanceolate, or oblanceolate, 3-14 cm long, 2.5-13.5 cm wide, entire to lyrately pinnatisect, with a broadly ovate to broadly triangular terminal lobe, cordate or obtuse to cuneate at base and acute at apex, and with 1-2 pairs of much smaller triangular to rhombic, acute to obtuse, or up to 5 pairs of very small ovate to elliptic lateral lobes; lamina margin variably shallowly or deeply sinuate-dentate, often irregularly so, and denticulate. Middle and upper cauline leaves oblanceolate or ovate to lanceolate, smaller, with winged or ± without petiole-like basal portion, otherwise similar to lower cauline leaves, base distinctly clasping. Synflorescence of a flowering stem in well-developed plants with several paracladia from the axils of the cauline leaves, all subspiciform to narrowly paniculiform and ± secund, each with a few to more than a dozen capitula pendent in bud, subpendent at anthesis and pendent again at fruiting. Capitula with (4-)5(-6) florets; peduncle in most cases less than 1 cm long.
Involucre narrowly cylindrical, 10-13 mm long; strongly differentiated into inner and outer phyllary series, the latter even very inconspicuous; phyllaries abaxially reddish hirsute as remainder of the 9 plant; outer phyllaries up to 3, very inconspicuous, narrowly linear, 0.9 mm × 0.2 mm; inner phyllaries usually 5, linear-lanceolate and similar in length, green, sometimes (partly) with a purplish tinge.
Receptacle epaleate, glabrous and smooth. Florets with bright yellow corolla; ligule broadly elliptical to obovate, ± horizontally spread, 14-16 mm long and up to 3 mm wide, tube c. 6 mm long; anther tube blackish, fertile part 4.6-4.8 mm long, apical appendages rounded, c. 0.2 mm long, basal appendages c. 0.6 mm long; style and style arms blackish. Pollen of the Cichorium type (sensu Blackmore 1986) with triangular, moderately extensive polar areas each with c. 12 spines and with moderately narrow interlacunar gaps (Fig. 4e-f). Achenes 8-9 mm long, corpus narrowly ellipsoidal to subconical, subcompressed, dark brown mottled white, covered with linear flattened acute antrorse papillae, shorter and appressed in the lower two thirds of the corpus, longer and spreading-erect in the upper third, with 5 main ribs (best discernible near base) alternating with (2-)3(-4) secondary ribs (fully developed in middle third and then similar in shape to main ribs), apex attenuate into a slender whitish beak of 3-4 mm ( Fig. 4a-d). Pappus c. 6-7 mm long, dirty white to pale brownish, caducous, bristles of similar length and diameter, near base of 8-14 rows of cells in cross section.

Distribution: NW Yunnan and SW Sichuan.
Habitat and ecology: The species occurs at altitudes between 1750 m and c. 3000 m on rocky slopes with open grassland vegetation. Fl. and fr. Sep-Nov.

Notes on typification and synonymy:
The description in the protologue is detailed and well corresponds to the type material. The eglandular hirsute reddish indumentum in combination with capitula of (mostly) 4 or 5 yellow florets, blackish anther tubes and styles, and an involucre of (4 or) 5 inner and very inconspicuous outer phyllaries diagnose the taxon perfectly. The author compares the species with L. hirsuta Franch. as its "nearest ally", stating that L. scandens differs from that by a red (versus "white", actually "dirty white" ["setis sordidis"]) hispid eglandular (versus mixed eglandularglandular) indumentum, a "white" (versus "dirty white") pappus, and undivided (versus lyrately pinnate) leaves. The description of the pappus of L. scandens is contradicting in the protologue: the 1 0 Latin description states "setae ... sordide albae", thus "dirty white" instead of "white", which agrees with our observation and also with the description of L. hirsuta. The leaves in L. hirsuta are lyrately pinnatisect but they are variable within populations, ranging from entire to pinnatisect. The indumentum in the type specimens of L. hirsuta looks in fact dirty white but this is likely only an effect of drying; the statement about presence of glandular hairs in the protologue of L. hirsuta seems erroneous. Also the number of 8 florets per capitulum given in the protologue of L. hirsuta is apparently erroneous. With the help of the curators in P their number has been confirmed as 5-6. The number of inner phyllaries is correctly and in agreement with L. scandens given as 5. It can safely be concluded that both taxa are conspecific. reproducing a map of the area drawn by P. Delavay). Technically the two sheets of the same gathering by Père Delavay of Lactuca hirsuta Franch. at P are syntypes so that a lectotype has to be designated.
We have designated the one marked as "holotype", with the original label of the collector and the determination in Franchet's hand.
Further specimens seen:  (IUCN 2001) for this species requires more data on its actual and historical distribution and population sizes in relation to land use changes. Although the scarcity of collections of this species may be partly due to its late flowering in the year, S. scandens seems to have a scattered distribution and be a rare species endemic to some small part of the two provinces. Its status should be thus of concern and addressed by further investigations. lyrately pinnatifid to pinnatisect, with a large orbicular to broadly triangular terminal lobe with cordate or obtuse to cuneate base and acute apex, and one to several pairs of (much) smaller ovate or elliptic to rhombic or ± triangular, acute to obtuse lateral lobes; lamina margin variably shallowly or deeply sinuate-dentate, often irregularly so, and denticulate. Middle and upper cauline leaves ovate to lanceolate, smaller, with winged or ± without petiole-like basal portion, otherwise similar to lower cauline leaves, base distinctly clasping the stem. Synflorescence of a flowering stem in well-developed plants with several paracladia from the axils of the cauline leaves, all subspiciform to narrowly paniculiform and ± secund, each with a few to more than a dozen capitula pendent in bud, subpendent at anthesis and pendent again at fruiting. Capitula with 3 florets; peduncles mostly shorter than the involucre. Involucre narrowly cylindrical, 10-12 mm long; strongly differentiated into inner and ± inconspicuous outer phyllary series; phyllaries abaxially pale to reddish hirsute as remainder of the plant; outer phyllaries 2, narrowly linear, 2-4 × 0.2-0.3 mm; inner phyllaries 3, linear-lanceolate and similar in length, green, sometimes (partly) with a purplish tinge. Receptacle epaleate, glabrous and smooth. Florets with yellow corolla; ligule elliptical, reflexed, 9-12 mm long and up to c. 2 mm wide, dorsally pale yellow, tube c. 5-6 mm long; anther tube blackish, fertile part 2.8-3.6 mm long, apical appendages rounded, c. 0.2 mm long, basal appendages c. 0.3 mm long; style and style arms blackish.

Sinoseris triflora
Pollen of the Cichorium type (sensu Blackmore 1986) with very extensive, approximately hexagonal polar areas, each with >20 spines, and with moderately narrow interlacunar gaps ( Fig. 6e-f). Achenes 7-9 mm long, corpus subconical, with 5 main ribs (best discernable near base) alternating with 3(-4) secondary ribs (fully developed in middle third and then similar in shape to main ribs), dark brown, with linear flattened acute antrose papillae, shorter and appressed in the lower two thirds of the corpus, longer and spreading-erect in the upper third; apex of corpus attenuate into a slender whitish beak of c.
Distribution: E Yunnan and SW Sichuan. Threat status: The species is known from two historical and three current localities. At these three populations in the Muli County only 10, 80 and 250 individuals were counted respectively, so that the population sizes seem in general smaller than the other two species. The species occurs in an only scattered way in its distribution area, similar to S. scandens, which may be due to its requirement of open rocky habitats. Nearly a third of the few-flowered capitula of many individuals was found infected in 2018 by some insects species and did therefore not produce fruits. The species is certainly rare and of localised distribution. Its status should be thus of concern and addressed by further investigations. with moderately narrow interlacunar gaps (Fig. 8e-f). Achenes 6-7 mm long, corpus subconical, with 5 main ribs (best discernible near base) alternating with 3(-4) secondary ribs (fully developed in middle third and then similar in shape to main ribs), dark brown, with linear flattened antrose papillae, spreading-erect at the apex and appressed and shorter in the rest, at its widest diameter abruptly contracted into a slender pale brown, basally appressed papillate beak of about the same length as the corpus, corpus below the beak broad-shouldered ( Fig. 8a-d). Pappus 5-6 mm long, dirty white to pale brownish, caducous, bristles of similar length and diameter, near base of 7-12 rows of cells in cross section.

Distribution: Central Yunnan
Habitat and ecology: The species is confined to open rocky habitats. Fl. and fr. Sep-Oct.
Etymology: We dedicate this species to the memory of Chao Chien Chang (1900Chang ( -1972 Threat status: The occurrence of this species is less scattered and rare compared to the other two species. Between Tanhua and Wanbi, for example, the species is present in sunny rocky slopes with high frequency, although the individual populations usually do not exceed a few hundred mature individuals. Considering its localised distribution in Central Yunnan, its status should be of concern and addressed by further investigations.