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

Abstract

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, distinguish 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.

1. 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.

2. Materials and Methods

2.1. 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.

2.2. 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).

2.3. 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. Species names and GenBank accession numbers of rbcL sequences used in this study.

No.	Species	Collector and Voucher No.	Locality	Herbarium	Accession No.
1	Selaginella bryopteris (L.) Baker	C. R. Fraser-Jenkins 4370	Nepal	L	KY022983
2	S. convoluta (Arn.) Spring	R. M. Harley 16181	Brazil: Bahia	U	KY023003
3	S. deflexa Brack.	D. Pamer 2651	USA: Hawaii	KANU	AF093253
4	S. digitata Spring	N. Wikström et al. 110319-2	Madagascar	S	KY023013
5	S. digitata Spring	P. Phillipson 1826	Madagascar	L	KY023012
6	S. exaltata (Kunze) Spring	Korall 1996-1	Sweden	S	AJ010849
7	S. helicoclada Alston	Rakotondrainibe 3262	Madagascar	P	AJ295896
8	S. heterostachys Baker	X. C. Zhang 7088	China: Guizhou	PE	MH814896
9	S. imbricata (Forssk.) Spring ex Decne.	Rothfels et al. 4275	Oman	DUKE	KT161486
10	S. iridescens X. C. Zhang and Y. R. Wang	Y. R. Wang and L. X. Yuan wyr20200110-1	China: Hainan	PE	OK181884
11	S. iridescens X. C. Zhang and Y. R. Wang	Y. R. Wang and L. X. Yuan wyr20200110-2	China: Hainan	PE	OK181885
12	S. iridescens X. C. Zhang and Y. R. Wang	Y. R. Wang and L. X. Yuan wyr20200110-3	China: Hainan	PE	OK181886
13	S. kraussiana A. Braun	M. Mokoso 3098	Democratic Republic of the Congo	BR	KY023057
14	S. lepidophylla (Hook. and Grev.) Spring	Worthington s.n.	USA: Texas	US	AF419051
15	S. moellendorffii Hieron.	Ju and Deng HGX12295	China: Sichuan	CDBI	KT161531
16	S. nipponica Franch. and Sav.	X. C. Zhang et al. 7066	China: Guizhou	PE	MW407367
17	S. nothohybrida Valdespino	Rothfels et al. 3069	Mexico: San Luis Potosí	DUKE	KT161545
18	S. novoleonensis Hieron.	F. Drouet and D. Richards 3942	Mexico: Sonora	S	KY023097
19	S. nubigena J.P.Roux	A. Larsson AL810	South Africa	UPS	KY023098
20	S. pallescens (C. Presl) Spring	Beck 1120	Mexico: Hidalgo	DUKE	KT161555
21	S. pilifera A. Braun	Pringle 13959	N/A 1	S	AJ295862
22	S. pseudotamariscina X.C. Zhang and C.W. Chen	C.W. Chen Wade 5314	Vietnam: Khanh Hoa Province	PE	MZ159980
23	S. pulvinata (Hook. et Grev.) Maxim	D. E. Boufford et al. 37879	China: Sichuan	A	KY023124
24	S. pulvinata	D. E. Boufford et al. 35254	China: Yunnan	A	KY023125
25	S. remotifolia Spring	Gaoligong Shan Biodiversity Survey; 21081	China: Yunnan	GH	KY023130
26	S. selaginoides (L.) P. Beauv. ex Mart. and Schrank	S. Weststrand 104	Sweden	UPS	KY023148
27	S. sibirica (Milde) Hieron.	L. A. Viereck and K. Jones 5667	USA: Alaska	S	KY023153
28	S. stauntoniana Spring	Zhao 169	China: Beijing	CDBI	KT161614
29	S. tamariscina (P.Beauv.) Spring	N/A TNS759348	Japan: Okinawa	TNS	AB574655
30	S. uliginosa Spring	Holmgren and Wanntorp 253	Sweden	S	AJ010843
31	S. uncinata (Desv. ex Poir.) Spring	Zhang and Zhou DJY04101	China: Sichuan	CDBI	KT161626
32	S. vardei H. Lév.	D. E. Boufford et al. 32425	China: Tibet	A	KY023169
33	Isoetes sinensis Palmer	N/A 743727	Japan	TNS	AB574660
34	I. histrix Bory and Durand	Wanntorp N/A	Sweden	S	AF404497

¹ N/A = not available.

).

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 the GTRGAMMA model selected according to the Bayesian Information Criterion (BIC) by ModelFinder [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].

2.4. 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.

3. Results

3.1. Taxonomy Treatment

Selaginella iridescens X. C. Zhang and Y. R. Wang, sp. nov.

Type. CHINA, Hainan, Changjiang, Changhua, at foot of Mt. Daling, on quartzite rocky slope, in open grass, 108.700° E, 19.328° N, altitude 31 m, 10 January 2020, Y. R. Wang and L. X. Yuan wyr20200110 (holotype, PE!); ibid. 11 June 2021, X. C. Zhang and E. F. Huang 11,594 (paratype, PE!).

Diagnosis. Selaginella iridescens differs from S. pseudotamariscina by its iridescent leaves and apex with long arista (0.35–1.2 mm), dorsal leaves adaxially sulcate, margins of axillary leaves widely membranaceous (ca. 2/3), and main stem slender, dorsiventrally prostrate (Figure 2, Figure 3 and Figure 4;

Table 2. Character comparison of S. iridescens, S. pseudotamariscina, S. pulvinata, and S. tamariscina.

Characters/Species	S. iridescens	S. pseudotamariscina	S. pulvinata	S. tamariscina
Distribution	Hainan of China	Khanh Hoa and Ninh Thuan of Vietnam	Southwestern and central China, India, Thailand, Vietnam	Eastern and Southeastern China (including Hainan), Japan, Korean Peninsula, Philippines, Thailand
Habit	Stems and roots entangled forming slender, dorsiventrally prostrate trunk	Stems and roots entangled forming thick, radial erect treelike trunk	Stems and roots entangled not forming treelike trunk	Stems and roots entangled forming treelike trunk
Leaves	Iridescent	Not iridescent	Not iridescent	Not iridescent
Axillary leaves	Ovate-oblong, margin membranaceous (about 2/3 of the entire leaf), lacerate or subentire, base ciliolate or denticulate	Lanceate, or ovate-lanceate, margin membranaceous (about 1/2 of the entire leaf), acerate-ciliolate	Ovate to triangular, margin membranaceous (about 1/2 of the entire leaf), lacerate-ciliolate, base ciliolate or lacerate	Ovate, ovate-triangular, or elliptic, margin membranaceous (about 1/3 of the entire leaf), denticulate-lacerate, base ciliolate or denticulate
Dorsal leaves	Symmetrical, lanceate, adaxially sulcate extends to the top, apex with long arista (ca. 0.76 mm long), margin ciliolate or denticulate	Symmetrical, lanceate, adaxially sulcate extends to half of the leaves, apex with short arista (ca. 0.2 mm long), margin ciliolate or denticulate	Asymmetrical, obliquely ovate or triangular, adaxially not sulcate, margin lacerate or entire	Asymmetrical, elliptic, adaxially not sulcate, margin denticulate (shortly ciliolate at base)
Ventral leaves	Ovate-lanceate to ovate-triangular, adaxially sulcate, apex with long arista (ca. 0.35–1.20 mm long)	Ovate-lanceate to ovate-triangular, adaxially sulcate, apex with short arista (0.02–0.40 mm long)	Oblong, adaxially not sulcate; apex with long arista (ca. 0.35–1.20 mm long)	Ovate to triangular or oblong-ovate, adaxially not sulcate; apex arista
Strobili	Slightly dorsoventrallycomplanate	Slightly dorsoventrally complanate	Tetragonal	Tetragonal
Sporophylls	Slightly anisophyllous; sporangia borne only on the ventral sporophylls	Slightly anisophyllous; sporangia borne only on the ventral sporophylls	Isophyllous; sporangia borne on both dorsal and ventral sporophylls	Isophyllous; sporangia borne on both dorsal and ventral sporophylls

).

Description. Rosettes, xerophytic. Rhizophores restricted to base of stem; rhizophores much forked, forming thick massive rootstock. Stems and roots entangled forming prostrate dorsiventral trunks; main stems branched near or above the base, pinnately or anisotomously branched, stramineous or brown; primary leafy 2–3 pinnately branched, branchlets compact and regular. Leaves thick and surface smooth, fresh leaves iridescent. Axillary leaves on branches symmetrical, ovate-oblong, ca. 2.02 × 0.87 mm, margin widely membranaceous (about 2/3 of the entire leaf), lacerate or subentire, base obtuse, ciliolate or denticulate, apex arista (ca. 0.52 mm long). Dorsal leaves strongly ascending, imbricate, symmetrical, lanceolate, 2.27 × 0.70 mm, lower side sulcate extends to the top, upper side carinate, base obtuse, margin ciliolate or denticulate, not membranaceous, apex arista (ca. 0.76 mm long). Ventral leaves slightly ascending, overlapping, asymmetrical, ovate-lanceolate to ovate-triangular, ca. 2.6 × 1.5 mm, adaxially sulcate extends to the top, apex arista (ca. 0.35–1.20 mm long); basiscopic margin ciliolate or denticulate; acroscopic base enlarged, broader, widely membranaceous, lacerate or subentire, ciliolate or lacerate at base. Strobili solitary, terminal, compact, slightly dorsoventrally complanate, very short (ca. 1–5 mm long); sporophylls anisophyllous; dorsal sporophylls smaller than the ventral ones, symmetrical, lanceolate, ca. 2.0 × 0.8 mm, margin slightly membranaceous, shortly ciliolate, apex arista; ventral sporophylls triangular, ca. 2.21 × 1.30 mm, margin widely membranaceous, denticulate, ciliolate or lacerate, apex arista; sporangia borne only on the ventral sporophylls; microspores yellowish orange, megaspores pale yellow to white (Figure 2 and Figure 3).

Distribution and habitat. Selaginella iridescens is only known from Western Hainan, Changjiang County, on quartzite rocky slope, open area (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].

3.2. 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).

3.3. 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).

4. Discussion

4.1. 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 (Figure 2, Figure 3 and Figure 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.

4.2. 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].

4.3. Key to S. iridescens, S. pseudotamariscina, S. pulvinata, and S. tamariscina

1. Strobili dorsoventrally complanate; sporangia borne only on the ventral sporophylls; dorsal leaves symmetrical, lanceolate, adaxially sulcate; ventral leaves abaxially shallowly sulcate………………….…………………………..………………………………… 2.

2. Main stem slender, dorsiventrally prostrate; leaves iridescent, apex long arista (0.35–1.20 mm long); margins of axillary leaves widely membranaceous (ca. 2/3); dorsal leaves adaxially sulcate to the apex………………………….……….……….…. S. iridescens.

2. Main stem thick, radially erect; leaves not iridescent, apex shortly arista (0.02–0.40 mm long); margins of axillary leaves membranaceous (ca. 1/2); dorsal leaves adaxially sulcate not to the apex ………………………………………………….. S. pseudotamariscina.

1. Strobili tetragonal; sporangia borne on both ventral and dorsal sporophylls; dorsal leaves asymmetrical, obliquely ovate, triangular, or elliptic, not sulcate………………………………..………………..……………………………………………. 3.

3. Stems and roots entangled not forming treelike trunk; inner margins of dorsal leaves lacerate or entire, outer margins revolute and entire………………..… S. pulvinata.

3. Stems and roots entangled often forming treelike trunk; inner margins of dorsal leaves denticulate, outer margins denticulate or shortly ciliolate…………… S. tamariscina.