Lysimachia huangsangensis (Primulaceae), a New Species from Hunan, China

A new species, Lysimachia huangsangensis (Primulaceae), from Hunan, China is described and illustrated. The new species is closely related to L. carinata because of the crested calyx, but differs in the leaf blades that are ovate to elliptic and (3–)4.5–9 × 2–3.4 cm, 2–5-flowered racemes, and the calyx lobes that are ovate-lanceolate and 5–6 × 3–4 mm. The systematic placement and conservation status are also discussed.

In the course of field work in SW Hunan Province, China, a distinct species of Lysimachia with a crested calyx was found. Further studies showed that it represented a new species belonging to ser. Drymarifoliae Hand.-Mazz. in subgen. Lysimachia sensu Chen & Hu [8]. Ser. Drymarifoliae includes about 10 species in China and is characterized by having the flowers arranged in umbel-like, axillary, 1-several-flowered racemes and filaments connate at the base [9]. In this series, the species can be divided into two groups according to whether the calyx has crested ridges or not. The new species belongs to the group having a crested calyx.

Ethics statement
The new species reported in this work was collected from Huangsang Natural Reserve and Yunshan Natural Reserve, Hunan, China, which are protected by the Forestry Bureau of Hunan Province. The Forestry Bureau permits research in these reserves and no specific permits are required for the present study. The field studies did not involve endangered or protected species.

Morphological observations
The morphological description of the new species was based on the examination of fresh and pressed specimens. The morphological comparison with related species, L. carinata Y. I. , was based on studies of herbarium specimens and information gathered from literature searches. The specimens examined were deposited in the following herbaria: CDBI, IBSC, K, NAS, P, PE and WUK. The herbarium acronyms follow the Index Herbariorum [47].
The pollen grains and seeds were directly mounted on aluminium stubs coated with gold in a sputter coater and examined using scanning electron microcopy (SEM). The polar (P) axis and equatorial (E) diameter were measured by imaging analyzer (Smile View 2.1; JEOL Tokyo, Japan). Pollen terminology follows Erdtman [48] and Bennell & Hu [40]. Seed terminology follows Oh et al. [28].

Nomenclature
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Taxon sampling
In order to determine the systematic position of the new species, we proceeded to create a phylogenetic analysis by adding published sequences of Chinese Lysimachia species. We retrieved a total of 66 sequences for 25 species downloading those published in a barcoding study of Chinese Lysimachia [16] from GenBank, In addition, three samples representing the new species were included in this study.

Molecular markers
Total genomic DNA was extracted from silica-dried plant leaves using a modified CTAB protocol [49], and then target DNA regions including the two core barcodes (rbcL and matK), trnH-psbA and ITS were amplified with common DNA barcoding primers. Primer pairs, PCR amplification and sequencing conditions following Zhang et al. [16] and Chen et al. [50]. GenBank accession numbers for all the DNA sequences and voucher information are given in S1 Table. Sequence alignment and phylogenetic analysis Sequence alignment was initially performed using with MUSCLE 3.8.31 [51] in the multiple alignment routine followed by manual adjustment in Se-Al v2.0a11 (http://tree.bio.ed.ac.uk/ software/seal/). Given that the plastid genome behaves as a single linked region, the three plastid markers (rbcL, matK and trnH-psbA) were concatenated a priori. Congruence between the combined plastid fragments and nuclear marker ITS was tested with the incongruence length difference (ILD) test [52], which was conducted using PAUP Ã version 4.0b10with 100 replicates of the heuristic search (default setting) [53]. The 1% level of significance was chosen as described in [54]. The ILD value in this study was 0.02 and we therefore,decide to combined all the datasets (rbcL, matK,psbA-trnH, and ITS) in our analysis using Maximum Likelihood (ML) analysis. We first determined the best-fitting model of sequence evolution for the total data matrix using ModelTest 3.7 [55]. Results of the Akaike information criterion (AIC, [56]) indicated that the TIM + I + G model was the best-fitting model for the combined data matrix. The ML analysis was performed using GARLI Web Service [57,58] with the best-fitting model (http://www.molecularevolution.org/software/phylogenetics/garli). Default parameters were used in this analysis, and 10 independent search replicates were run with each replicate run. Bootstrap support values for nodes on the ML topology were computed with GARLI by running 500 bootstrap replicates.
Bayesian inference (BI) was conducted using MrBayes version 3.2.1 [59] with the best-fitting model (TIM + I + G). The Bayesian Markov chain Monte Carlo (MCMC) algorithm was run for 5000000 generations with four incrementally heated chains starting from random trees and sampling one out of every 1000 generations. A conservative burn-in (25%) was applied after checking for stability on the log-likelihood curves and split variances less than 0.01. A majority rule consensus tree was calculated from the remaining trees. The internodes support was determined by Bayesian Posterior Probabilities.

Phenology
Found in flower from May to June and in fruit from September to October.

Conservation status
Lysimachia huangsangensis has only been collected in 2014 from two protected areas in southwest Hunan, China. However, it is not excluded that this species could be found in other localities and therefore it is classified as Data Deficient (DD) [60] according to the guidelines for using the IUCN Red List Criteria [61].

Taxonomic position
According to the classification of Chen and Hu [8,9], the new species belongs morphologically to Ser. Drymarifoliae Hand.-Mazz. [22]. Ser. Drymarifoliae can be divided into two groups based on calyx characters. The new species belongs to a group having crested calyx lobes together with L. carinata, L. pterantha, L. baoxingensis, L. pteranthoides and L. crista-galli (specimens examined are listed in S1 Text). Lysimachia huangsangensis is most similar to L. carinata and differs in the character of leaf blades, inflorescence and calyx-lobes. The new species can be distinguished from allied species by the following key.

Phylogenetic relationship
In the current study we conducted ML and BI analyses to determine the phylogenetic relationship of the new species. Both analyses generated congruent results and the Bayesian result is shown in Fig 4. The new species is a member of the Chinese sect. Nummularia, and forms a robust monophyletic group (Fig 4), though the phylogenetic position is not resolved.

Discussion
In the classification of Chinese Lysimachia, Chen & Hu [8] divided the genus into five subgenera, i.e., Idophyton, Lysimachia, Palladia, Heterostylandra and Naumburgia on the basis of the classification of Handel-Mazzetti [22]. More than one third of the species are placed in subgen. Lysimachia sensu Handel-Mazzetti [22] and Chen & Hu [8], which is characterized by the yellow flowers that are solitary in the leaf axils or arranged in racemes, panicles or heads, the filaments forming a thin ring adnate to the corolla base, and the anthers opening with slits [5,34]. The recent molecular evidence [34,42,43] reveals that subgen. Lysimachia is paraphyletic and consists of six groups. Sect. Nummularia is the largest section recognized in subgen. Lysimachia by Chen & Hu [8] and was further divided into ten series by them. Molecular studies [34] indicated that sect. Nummularia can be divided into two separate groups and Chinese taxa do not group with the type section. Chinese taxa of sect. Nummularia form a sister group with subgenera Pallada and Lysimachiopsis and differ from the latter by the flowers with oil-producing trichomes and filaments forming a thin ring adnate to the corolla base [5,34]. Here, we treat Chinese taxa of sect. Nummularia as an independent section on the basis of the classification of Chen & Hu [8] and thus the name Lysimachia sect. Hypericoideae Knuth is the correct name for the section. The monophyly of sect. Hypericoideae has been supported by previous studies [34]. According to the subdivision of sect. Hypericoideae (as sect. Nummularia) by Chen & Hu [8] who further divided the section into ten series, the new species L. huangsangensis would be placed in ser. Drimarifoliae Hand.-Mazz. Our combined analysis (rbcL, matK, psbA-trnH, and ITS) using Bayesian Inference (BI) (Fig 4) showed that the new species Lysimachia huangsangensis is nested in sect. Hypericoideae. Results support the placement of L. huangsangensis in sect. Hypericoideae and revealed that species in this section included two main groups, L. longipes representing ser.
Paracladae, and other taxaof other series in this section. Our results do not support the classification by Chen & Hu [8]. Additional analyses using more molecular markers are necessary to clarify relationships within sect. Hypericoideae. We delimited species by implementing the phylogenetic species concept [62][63][64][65]. In our phylogenetic analysis (Fig 4), Lysimachia huangsangensis forms a trichotomy together with other sampled taxa in sect. Hypericoideae except L. longipes. Although the phylogenetic relationships among sect. Hypericoideae are not well resolved, the recognition of Lysimachia huangsangensis is supported because all three accessions clearly cluster together. Morphologically, the new species can be distinguished from the closely related species by the leaf characters.
Surface features of seed coats are surprisingly little affected by the environmental conditions under which a plant grows [74]. Studies of seed morphology with SEM have revealed taxonomically useful microcharacters to support the delimitation of individual or groups of taxa [75]. Shao et al [76] observed microcharacters of seed surface of eleven Chinese Lysimachia species and divided them into two types, Heterogenea-type and Grammica-type, in relation to the subgeneric classification. Seven species of subgen. Pallada show the Heterogenea-type and four species of sect. Hypericoideae (as Nummulariain the sense of Chen & Hu [8]) show the Grammica-type with tuberculate ornamentation. Oh et al. [28] studied seed morphology and character evolution in the genus Lysimachia and its related genera (viz., Anagallis, Ardisiandra, Asterolinon, Glaux, Pelletiera and Trientalis). Seed morphology of 34 species of Lysimachia and 14 species of six related genera were investigated by them [28]. Three major types of seed shapewere identified, i.e., sectoroid (dorsiventrally and/or laterally flattened), polyhedral and coarsely rugose with a concave hilar area. The seed surface pattern of Lysimachia can be divided into six main types, i.e., reticulate, tuberculate, vesiculose, colliculate, undulate and poroid-alveolate. The seed morphological characters imply that the traditional concept of Lysimachia is not monophyletic and this was supported by the molecular studies [34,41]. Oh et al. [28] observed seven species of sect. Hypericoideae (as subgenus Lysimachia F by them) and their results showed that the seed shape in sect. Hypericoideae is sectoroid or polyhedral with tuberculate seed surface pattern that is characterized by cells with convex epidermal facets. The observations of Oh et al [28] are similar to that of Shao et al. [76]. The seed of the new species L. huangsangensis is polyhedral in shape with side-faces slanted towards the ventral face (Fig 2G), outer layer of the surface with convex epidermal cells and the inner layer reticulate ( Fig 2H). The seed morphology also supports the placement of the new species in sect. Hypericoideae.
Supporting Information S1 Table. GenBank accession numbers for all the DNA sequences and voucher information.
(XLS) S1 Text. The specimens of Lysimachia carinata, L. pterantha, L. baoxingensis, L. pteranthoides and L. crista-galli were examined to compare the new species with related species. (PDF)