An Extraordinary Rosette and Resurrection New Spikemoss, Selaginella iridescens (Selaginellaceae) from Hainan Island, China

: Selaginella iridescens (Selaginellaceae), a new species of spikemoss from Hainan Island, China, is described based on morphological and molecular evidence. The new species morphologically resembles S. pseudotamariscina from Vietnam which was recently recognized and segregated from S. tamariscina and S. pulvinata . Some characters of S. iridescens , including the iridescent leaves and long arista (0.35–1.20 mm long), the widely membranaceous margin of axillary leaves (ca. 2/3), dorsal leaves sulcate extending to the top, and slender main stem, dorsoventrally prostrate, distin-guish it from S. pseudotamariscina . Phylogenetic results based on rbcL of 28 Selaginella species indicate that S. iridescens is sister to S. pseudotamariscina , and distant from the S. tamariscina - S. pulvinata clade. The ancestral character reconstruction result reveals that the rosette is apomorphic and has evolved independently at least six times in Selaginella .


Introduction
Selaginella P. Beauv., the single genus in Selaginellaceae, is the largest extant lycophyte genus that comprises approximately 700-800 taxonomically difficult species [1][2][3][4]. The monophyly of Selaginella was well-supported by recent phylogenetic studies, however, the infrageneric classification is still uncertain [1,[5][6][7]. Based on the morphological characters, Jermy [1] divided the genus Selaginella into five subgenera, whose classification was the most widely accepted before molecular phylogenetic studies. Recently, Weststrand and Korall [3,4] divided the genus Selaginella into seven subgenera based on DNA sequences (rbcL, pgiC, and SQD1) and morphological data. This classification system was supported by the phylogenomic study based on plastome datasets, which further resolved the uncertainty of the S. sanguinolenta group as the most basal clade of S. subg. Stachygynandrum [7].
Species of Selaginella have various growth forms, including rosette, creeping, climbing, prostrate, erect, and suberect [2,8,9]. The rosette is a special morphological feature in Selaginella, but only few species are known to be rosette-forming [4,6,10]. In China, S. tamariscina (P. Beauv.) Spring and S. pulvinata (Hook. et Grev.) Maxim are two famous rosette-forming species of Selaginella [2,9]. In Hainan Island, about 14 Selaginella species are recorded in Flora of China (FOC), including two endemic species (S. hainanensis X. C. Zhang and Nooteboom and S. scarbrifolia Ching and Chu H. Wang) and one rosette-forming species (S. tamariscina) [2,11]. During a field trip in Hainan Island of China, a new rosette-forming Selaginella species was collected. A detailed examination on morphology of our newly collected materials and potential similar species revealed that these specimens were morphologically distinct from the two famous rosette-forming species in China, S. tamariscina and S. pulvinata, but morphologically similar to S. pseudotamariscina X.C. Zhang and C.W. Chen, a new species recently described from Vietnam [12]. Then, we conducted phylogenetic analyses using rbcL sequences, including this new species and other rosette species within Selaginella. In this paper, we present evidence from morphology and molecular phylogenetic analysis to support the new species and conduct ancestral character reconstruction to reveal the evolutionary history of rosette character in Selaginella.

Morphological Observations
Morphology of the newly collected specimens was examined and photographed under a Leica S9D stereo microscope, and compared with S. pseudotamariscina, S. pulvinata and S. tamariscina. The sterile leaves, strobili, and sporophylls were observed and measured.

Taxon Sampling and Sequencing
We sampled three individuals of the new species from one site on Hainan Island ( Figure 1). Total genomic DNA was extracted from silica gel dried materials using the Plant Genomic DNA Kit DP305 (TianGen Biotech, Beijing, China) following the manufacturer's protocol. Libraries for pair-end 150 bp sequencing with a 350 bp insert size were conducted using an Illumina NovaSeq 6000 platform at BioMarker Co (Beijing, China). Raw reads were filtered based on the following criteria: pair-end reads with >10% 'N' bases; reads, on which more than 50% of the bases have a quality score less than 10 (Phred-like score). Finally, approximate 6 Gb high-quality sequences were obtained for each sample (only rbcL sequences were extracted for this study, this high-throughput data will be used for further study). and Nooteboom and S. scarbrifolia Ching and Chu H. Wang) and one rosette-forming species (S. tamariscina) [2,11]. During a field trip in Hainan Island of China, a new rosetteforming Selaginella species was collected. A detailed examination on morphology of our newly collected materials and potential similar species revealed that these specimens were morphologically distinct from the two famous rosette-forming species in China, S. tamariscina and S. pulvinata, but morphologically similar to S. pseudotamariscina X.C. Zhang and C.W. Chen, a new species recently described from Vietnam [12]. Then, we conducted phylogenetic analyses using rbcL sequences, including this new species and other rosette species within Selaginella. In this paper, we present evidence from morphology and molecular phylogenetic analysis to support the new species and conduct ancestral character reconstruction to reveal the evolutionary history of rosette character in Selaginella.

Morphological Observations
Morphology of the newly collected specimens was examined and photographed under a Leica S9D stereo microscope, and compared with S. pseudotamariscina, S. pulvinata and S. tamariscina. The sterile leaves, strobili, and sporophylls were observed and measured.

Taxon Sampling and Sequencing
We sampled three individuals of the new species from one site on Hainan Island ( Figure 1). Total genomic DNA was extracted from silica gel dried materials using the Plant Genomic DNA Kit DP305 (TianGen Biotech, Beijing, China) following the manufacturer's protocol. Libraries for pair-end 150 bp sequencing with a 350 bp insert size were conducted using an Illumina NovaSeq 6000 platform at BioMarker Co (Beijing, China). Raw reads were filtered based on the following criteria: pair-end reads with >10% 'N' bases; reads, on which more than 50% of the bases have a quality score less than 10 (Phredlike score). Finally, approximate 6 Gb high-quality sequences were obtained for each sample (only rbcL sequences were extracted for this study, this high-throughput data will be used for further study).

Phylogenetic Analysis
The chloroplast rbcL gene was the most commonly used marker in Selaginella [3,5,6,10]. Therefore, rbcL was selected to reconstruct a phylogeny with an extensive species sampling to clarify the relationships between the new Selaginella species and other rosette species. In total, 32 individuals of 28 ingroup species representing all the seven subgenera of Selaginella according to Weststrand and Korall [3,4] were included, while Isoetes histrix Bory and Durand and I. sinensis Palmer were selected as outgroups. The rbcL of new species were obtained by Geneious mapping using Illumina short reads in Geneious v. 11.1.4 [13] and rbcL sequences for other species were downloaded from GenBank (voucher information and GenBank accession numbers listed in the Table 1). All the sequences were aligned using MAFFTT v7.313 [14,15]. Both maximum likelihood (ML) analysis and Bayesian inference (BI) were carried out in this study. ML analysis was performed using RAxML 7.2.6 [16], with 1000 bootstrap replicates under  [17]. BI analysis was performed using MrBayes v. 3.2.6. [18], under the SYM + G4 model selected according to the BIC by ModelFinder [17]. For each Bayesian analysis, four MCMC chains were run simultaneously for 1 million generations and sampled every 1000 generations. The average standard deviation of split frequencies (<0.01) was used to assess the convergence. ML and BI trees and the branch support values were visualized using FigTree v.1.4.2 [19].

Character Evolution Analysis
We used Mesquite v.3.61 [20] to infer the ancestral states of rosette character. The ancestral character reconstruction was performed under ML models "Mk1" [21], with the phylograms of 1000 RAxML bootstrap trees based on rbcL sequences as input phylogenies in order to consider any phylogenetic uncertainties. The results were finally summarized as percentage of changes of character states on a given branch across all 1000 trees using the "Average-frequencies-across-trees" option.
investigation conducted both in 2020 and 2021. Moreover, detailed examination of relevant specimens collected from Hainan and adjacent regions of rosette Selaginella species in virtual herbaria (CVH and GBIF) revealed no additional specimens of this species. Even if the assignment of a conservation status of this new species could be premature, it is most likely an endemic to Hainan Island and could be temporarily considered as Vulnerable (VU) according to the IUCN Red List guidelines criterion D1D2 based on current data [22].   investigation conducted both in 2020 and 2021. Moreover, detailed examination of relevant specimens collected from Hainan and adjacent regions of rosette Selaginella species in virtual herbaria (CVH and GBIF) revealed no additional specimens of this species. Even if the assignment of a conservation status of this new species could be premature, it is most likely an endemic to Hainan Island and could be temporarily considered as Vulnerable (VU) according to the IUCN Red List guidelines criterion D1D2 based on current data [22].        Figure 1).
Etymology. The specific epithet 'iridescens' refers to its iridescent leaves. Conservation status. Selaginella iridescens is known only from one population with about 300 individuals in the type locality. No other localities were discovered during our investigation conducted both in 2020 and 2021. Moreover, detailed examination of relevant specimens collected from Hainan and adjacent regions of rosette Selaginella species in virtual herbaria (CVH and GBIF) revealed no additional specimens of this species. Even if the assignment of a conservation status of this new species could be premature, it is most likely an endemic to Hainan Island and could be temporarily considered as Vulnerable (VU) according to the IUCN Red List guidelines criterion D1D2 based on current data [22].

Phylogenetic Analysis
The total length of rbcL alignment is 1428 bp, with 475 parsimony informative sites. The sequences of three Sealginella iridescens individuals are identical and have 14 nucleotide variations when compared with S. pseudotamariscina, the most related species to S. iridescens.
Our phylogenetic analyses of 28 species of Selaginella based on rbcL gene are generally similar to the former phylogeny studies and consistent with the seven subgenera classification [3][4][5]. Three individuals of S. iridescens are clustered together and sister to S. pseudotamariscina, and formed a monophyletic clade belonging to the Subg. Stachygynandrum ( Figure 5). This clade is sister to the S. helicoclada-S. imbricata clade with strong support (BS = 100/PP = 100). The clade grouping of these two latter clades (S. iridescens-S. pseudotamariscina clade and S. helicoclada-S. imbricata clade) is sister to the S. pulvinata-S. stauntoniana clade (BS = 99/PP = 100).

Ancestral Reconstruction of Rosette Character
Ancestral character reconstruction results reveal that rosette is apomorphic in Selaginella. This character has experienced a complicated evolutionary history and has evolved independently at least six times in Selaginella. There were five times in subg. Stachygynandrum and once in subg. Lepidophyllae. The five times in subg. Stachygynandrum appeared in S. nothohybrida-S. pallescens clade, S. convoluta-S. nubigena clade, S. pulvinata-S. tamariscina clade, S. irisdescens-S. pseudotamariscina clade, and S. pilifera clade, respectively ( Figure  6).

Ancestral Reconstruction of Rosette Character
Ancestral character reconstruction results reveal that rosette is apomorphic in Selaginella. This character has experienced a complicated evolutionary history and has evolved independently at least six times in Selaginella. There were five times in subg. Stachygynandrum and once in subg. Lepidophyllae. The five times in subg. Stachygynandrum appeared in S. nothohybrida-S. pallescens clade, S. convoluta-S. nubigena clade, S. pulvinata-S. tamariscina clade, S. irisdescens-S. pseudotamariscina clade, and S. pilifera clade, respectively ( Figure 6).
Ancestral character reconstruction results reveal that rosette is apomorphic in Selaginella. This character has experienced a complicated evolutionary history and has evolved independently at least six times in Selaginella. There were five times in subg. Stachygynandrum and once in subg. Lepidophyllae. The five times in subg. Stachygynandrum appeared in S. nothohybrida-S. pallescens clade, S. convoluta-S. nubigena clade, S. pulvinata-S. tamariscina clade, S. irisdescens-S. pseudotamariscina clade, and S. pilifera clade, respectively ( Figure  6).

Morphological Comparison with Three Related Rosette Species
Morphological comparison with S. pulvinata and S. tamariscina shows that S. iridescens is distinguished by its fresh leaves iridescent; dorsal leaves symmetrical, lanceate, adaxially sulcate, margin ciliolate or denticulate; ventral leaves adaxially shallow sulcate; strobili dorsoventrally complanate, similar shape of dorsal sporophylls and dorsal sterile leaves; dorsal sporophylls smaller than the ventral ones, and sporangia borne only on the ventral sporophylls ( Figure 3 and Table 2). The leaves of S. pulvinata and S. tamariscina are not iridescent, dorsal leaves asymmetrical, ventral leaves adaxially not sulcate ( Figure 4). The strobili of S. pulvinata and S. tamariscina are tetragonal, sporophylls isophyllous, margin denticulate, sporangia borne on both dorsal and ventral sporophylls ( Table 2).
Morphological comparison with S. pseudotamariscina shows that S. iridescens is similar to S. pseudotamariscina by its rosette-forming habit and lanceate symmetrical dorsal leaves. However, S. iridescens is further characterized by the following unique features: the leaves iridescent with long arista apex (0.35-1.20 mm), dorsal leaves sulcate extending to the top, margins of axillary leaves widely membranaceous (ca. 2/3), and main stem slender, dorsiventrally prostrate (Figures 2-4 and Table 2). The leaves of S. pseudotamariscina are not iridescent and the apex is short arista (0.02-0.40 mm), dorsal leaves sulcate to the middle of leaves, margins of axillary leaves widely membranaceous (ca. 1/2) ( Figure 4 and Table 2). The main stem of S. pseudotamariscina is thick, radial, and erect. It is obvious that these two similar species are very distinct in detail.

How Many Rosette Species in Selaginella?
Rosette is a special morphological feature, which has evolved independently at least six times in Selaginella ( Figure 6). However, there are only about 12 Selaginella species known to be rosette-forming according to our new finding and former studies [3,6,12]. These 12 rosette species only disperse in two subgenera of Selaginella, S. subg. Lepidophyllae, and subg. Stachygynandrum. Subg. Lepidophyllae contains four rosette species mainly distributed in North America, but only two species (S. lepidophylla and S. novoleonensis) have been sequenced and obtained a confirmed position [3,4]. There are still two rosette-forming resurrection species, S. gypsophila A. R. Sm. and T. Reeves and S. ribae Valdespino with unknown systematic position, that may still be members of subg. Lepidophyllae [4,23,24]. There are eight rosette-forming species in subg. Stachygynandrum, mainly distributed in America (S. nothohybrida, S. pallescens, S. convoluta, and S. pilifera) and East Asia (S. iridescens, S. pseudotamariscina, S. pulvinata, and S. tamariscina) [3,10,12]. These eight rosette-forming species are not monophyletic, they fell into four small groups and occupied different positions both in present and previous phylogenetic analyses [3,10,12]