﻿Comprehensive molecular and morphological analysis of Brachystemmacalycinum and Stellariaovatifolia in the tribe Alsineae (Caryophyllaceae)

﻿Abstract Over the course of the recent decade, the composition of Alsineae has been drastically changed by means of molecular phylogeny. However, the genus Brachystemma has not been sampled in any of the previous studies, and its phylogenetic position is still pending. In addition, the related species Stellariaovatifolia, which has at times been placed in Brachystemma, Schizotechium, or Stellaria, has also not been sampled. Here, nuclear ribosomal internal transcribed spacer (ITS) and four plastid regions (trnL-F, matK, rbcL, rps16) were used to conduct phylogenetic analyses within Caryophyllaceae and the tribe Alsineae. Ancestral characters (petal margin and number of seeds) were reconstructed in the tribe Alsineae based on the phylogenetic results. Our results indicate that Brachystemma is nested in the tribe Alsineae and forms a monophylum with S.ovatifolia, and apically lobed petals and numerous seeds may be the ancestral characters in the tribe Alsineae. Based on our study, Stellariaovatifolia should be considered within Brachystemma, and Brachystemma is clearly a separate genus and now includes two species.


Introduction
The family Caryophyllaceae has traditionally been divided into three subfamilies . Recently, a new classification system has been proposed based on molecular and morphological evidence in Caryophyllaceae, and eleven tribes were recognized (Harbaugh et al. 2010;Greenberg and Donoghue 2011).
Brachystemma ovatifolium Mizushima was first published in 1955 and is related to Brachystemma calycinum D.Don (Mizushima 1955; Fig. 1 in present paper). Subsequently, Mizushima transferred it to Stellaria as Stellaria ovatifolia (Mizushima) Mizushima due to its two-lobed petals and similar seed morphology (Mizushima 1966), which was also accepted by Flora Reipublicae Popularis Sinicae (Wu and Ke 1996) and Flora of China (Shilong and Rabeler 2001). In the first book, it was incorporated into sect. Schizothecium Fenzl of Stellaria, together with S. delavayi Franch. and S. monosperma Buch.-Ham. ex D.Don (Wu and Ke 1996). Recently, Stellaria sect. Schizotechium has been raised into a separate genus, Schizotechium (Pusalkar and Srivastava 2016), and the new combination Schizotechium monospermum (Buch.-Ham. ex D.Don) Pusalkar & S.K.Srivast. was proposed based on morphological studies (Pusalkar and Srivastava 2016). The molecular studies also indicated that Stellaria monosperma was far from the core Stellaria and nested within Schizotechium (Greenberg and Donoghue 2011;Sharples and Tripp 2019;Arabi et al. 2022). Although Stellaria ovatifolia was hypothesized to be part of Schizotechium (Pusalkar and Srivastava 2016), it has never been sampled and has at times been placed in Brachystemma, Schizotechium, or Stellaria, and its phylogenetic position is still pending.
In this study, we conducted a combined molecular and morphological analysis in order to (1) confirm the phylogenetic position of Brachystemma; (2) clarify the relationship of Stellaria ovatifolia among Stellaria, Schizotechium, and Brachystemma; (3) estimate the character evolution of seed number and petal margin in the tribe Alsineae.

Taxon sampling and DNA sequencing
The samples of Brachystemma calycinum and Stellaria ovatifolia were collected from silica-dried leaves tissue, and the vouchers were deposited in the herbarium of the College of Agriculture, Guangxi University (GAUA) and the detailed information is shown in Suppl. material 1. The total DNA of the samples were extracted by the CTAB protocol (Maddison and Maddison 2014). The PCR amplification of ITS [5F (White et al. 1990), 4R (White et al. 1990)], matK [390F (Smissen et al. 2002), 1440R (Smissen et al. 2002)], rbcL [1F (Kress and Erickson 2007), 724R (Kress and Erickson 2007)], rps16 [F (Popp and Oxelman 2001), R (Popp and Oxelman 2001)], trnL-F [C (Taberlet et al. 1991), F (Taberlet et al. 1991)] were performed as above cited. The sequencing of PCR products was performed by the Beijing Genomics Institute (BGI). Newly generated sequences are available in GenBank (https://www.ncbi.nlm.nih.gov/), and their accession numbers (in bold) and the sequences of Caryophyllaceae members downloaded from GenBank are listed in Table 1. The absent sequences were coded as missing data.

Phylogenetic analyses
Sequences alignment were performed with MAFFT v.7.313 (Katoh and Standley 2013). Phylogenetic analyses were conducted separately on the nuclear ribosomal internal transcribed spacer (ITS) and plastid regions (matK, rbcL, trnL-F, and rps16) and then combined; no notable incongruence was found (Fig. 2). The Bayesian Inference (BI) trees were constructed using MRBAYES 3.2.6 (Ronquist and Huelsenbeck 2003), and the maximum likelihood (ML) trees were constructed by RAXML-HPC2 (Stamatakis 2006). ML trees were constructed on CIPRES Science Gateway (Miller et al. 2010) under the GTRGAMMA model with 1,000 bootstrap replicates and default values for the remaining parameters. In Bayesian inference analysis, PARTITIONFINDER v.2.1.1 (Lanfear et al. 2016) was applied to selected models of nucleotide substitution under the Akaike Information Criterion (AIC). Selected models consisted of SYM+I+G for ITS, GTR+G for matK, trnL-F, and rps16, HKY+I+G for rbcL. Each Markov chain Monte Carlo (MCMC) analysis was run for 2,000,000 generations with the tree sampled every 100 generations. The first 25% trees of each run as burn-in were discarded.

Ancestral characters
Two morphological characters (petal margin and number of seeds) which were diagnostic characters in Brachystemma were selected to reconstruct the ancestral characters in the tribe Alsineae. MESQUITE v.3.6 (Maddison and Maddison 2014) was used to reconstruct the ancestral characters with default parameters, using the ML tree from the combined tree. Morphological characters were coded as the following: (a) the petals are entire or emarginate (coded as 0), apex lobed (less than 1/2 the Table 1. List of sampled taxa and their GenBank accession numbers of sequences. The arrangement of sequences in the table shows sequences used to generate the trees shown in Fig. 3A, B. Sequences in bold were generated in this study.

Ancestral character
The results of the ancestral character reconstruction indicated that petals with a lobed apex and numerous seeds may be the ancestral characters of the tribe Alsineae (Fig. 4). The presence of entire petals and 1-3 seeds became the diagnostic characters between Brachystemma and related genera. In addition, B. calycinum and S. ovatifolia shared the characters of 1-3 seeds and neither taxa has deeply bifid petals. It suggested a close relationship between B. calycinum and S. ovatifolia.

Phylogenetic position and distinction of Brachystemma
As currently defined, Brachystemma is a monotypic genus in the tribe Alsineae, which is characterized by annual subscandent life form, lax thyrse with many flowers, petals shorter than 1/2 the length of the sepals with entire margins, two styles, four-valved capsules, and one mature seed ( Fig. 1) . Our phylogenetic results also revealed that Brachystemma formed a single branch with S. ovatifolia ( Fig. 2 and  Fig. 3) and demonstrated that Brachystemma is an independent genus (S. ovatifolia will be discussed in the following paragraphs), which is consistent with traditional morphological studies (Fenzl 1840; Bentham and Hooker 1862;Pax and Hoffmann 1934;Bittrich 1993;Takhtajan 2009). Furthermore, the phylogenetic position of Brachystemma was nested in the tribe Alsineae and sister to the clade composed of Schizotechium, Mesostemma, Lepyrodiclis, Shivparvatia, Odontostemma, and Pseudostellaria (Fig. 3). Nevertheless, Brachystemma can be morphologically distinguished from the related genera of this clade. Brachystemma and Lepyrodiclis share characters such as annual life form, lax thyrse, and two styles, but Brachystemma differs from the latter by subscandent life form and four-valved capsules . It also can be distinguished from Mesostemma, Pseudostellaria, and Schizotechium by annual life form, petals with entire margins, lax thyrse, and two styles Arabi et al. 2022). It can be clearly distinguished from Shivparvatia by annual habit, lax thyrse, and two styles Keshav and Kumar 2015). Finally, it can be segregated from Odontostemma by lax thyrse, petals with entire margin and wingless seeds Sadeghian et al. 2015).

Character evolution
Our results indicated that petals with a lobed apex and numerous seeds may be the ancestral characters of the tribe Alsineae, which was consistent with previous studies (Greenberg and Donoghue 2011; Zhang et al. 2017). Brachystemma has entire petal margins, but it is sister to the clade composed of genera having lobed petals Schizotechium, Mesostemma, Odontostemma, and Pseudostellaria (except Pseudostellaria maximowicziana and Pseudostellaria tibetica) (Fig. 4). Moreover, the tribe Alsineae is defined by a many- seeded (rarely few-or one-seeded) capsule or a rarely indehiscent nutlet (Harbaugh et al. 2010;Greenberg and Donoghue 2011;Arabi et al. 2022), but above genera having lobed petals share the character of fewer seeds (a capsule) (Fig. 4). The tribe Alsineae may have developed in an evolutionary direction toward fewer seeds. In addition, B. calycinum may be a species with diverse petals based on our field observations. B. calycinum may also include long (longer than sepals) and apically lobed petals (Fig. 1H), instead of only short (shorter than 1/2 the sepal length) and entire petals in the protologue (Fig. 1I). While additional observations in the field and specimens are required to confirm the petal condition, the petal condition in Brachystemma is coded here in accordance with the protologue.

Classification of Stellaria ovatifolia
Although the placement of Stellaria ovatifolia among Brachystemma, Schizotechium and Stellaria has been uncertain for a long time, S. ovatifolia was considered more similar to B. calycinum in general appearance (Mizushima 1955;Wu and Ke 1996). It was clearly distinguished from the core Stellaria by subscandent life form (vs. non-scandent), lax thyrse (vs. cymes, rarely solitary), two styles (vs. three, rarely four or five), two-lobed (nearly to half of petal length) petals (vs. deeply-bifid petals), four-valved capsules (vs. six-valved capsules), and one mature seed (vs. many mature seeds) (Wu and Ke 1996;Shilong and Rabeler 2001;Sharples and Tripp 2019). Despite being hypothesized to belong to Schizotechium (Pusalkar and Srivastava 2016), S. ovatifolia shows noticeable differences with Schizotechium, including a lax thyrse (vs. many-flowered compound cymes), two styles (vs. three styles), four-valved capsules (vs. six-valved capsules), and one mature seed (vs. one or two mature seeds) (Wu and Ke 1996;Shilong and Rabeler 2001;Pusalkar and Srivastava 2016). What is more, S. ovatifolia differs from Brachystemma by having two-lobed petals (nearly to half of petal length) and Stellaria type seeds, but they both share the following characters: subscandent life form, lax thyrse, two styles, four-valved capsules, and one mature seed ( Fig. 1) (Wu and Ke 1996;Shilong and Rabeler 2001). Hence, S. ovatifolia is highly similar to Brachystemma, instead of either Stellaria or Schizotechium. In terms of our molecular phylogeny, Stellaria ovatifolia is nested with Brachystemma calycinum in a clade with strong support (PP = 1.00, BS = 100) and not closely related to either Stellaria or Schizotechium in the nrDNA tree, cpDNA tree, and combined tree ( Fig. 2 and Fig. 3). We believe that S. ovatifolia should be reclassified as a species of Brachystemma combining the evidence of similar general appearance and close phylogenetic relationship. As a result, the scientific name Brachystemma ovatifolium Mizushima is reinstated here. The main characters of Brachystemma now are: herbs annual or perennial; stems subscandent, branched; leaves opposite, petiolate; leaf ovate-lanceolate to lanceolate; stipules absent; inflorescence a thyrse or numerous in dichotomous, nearly subglobose cymes, terminal or axillary; flowers numerous, 5-merous, pedicellate; sepals free, subscarious, persisting in fruit; petals lanceolate or minute, much shorter than sepals, margin entire or bifid; stamens 5 or 10; styles 2; fruit a capsule, oblate, 4-valved, 1-seeded; seed reniform or globose.  Mizushima, Acta Phytotax. Geobot. 16: 42. 1955.

Conclusion
Based on our study, Brachystemma is clearly a separate genus nested in the tribe Alsineae and now includes two Asiatic species B. calycinum and B. ovatifolium. The native range of B. calycinum is Assam (India), Cambodia, South-West (Tibet, Xizang province) and South-Central China, East Himalaya, Laos, Myanmar, Nepal, Thailand, Vietnam (Wu and Ke 1996;Shilong and Rabeler 2001). The native range of B. ovatifolium is Nepal and China (Tibet) (Wu and Ke 1996;Shilong and Rabeler 2001).