Selaginelladianzhongensis (Selaginellaceae), a new spikemoss from China

Abstract A new species of spikemoss from Yunnan Province of China, Selaginelladianzhongensis, is described and illustrated based on evidence from gross morphology, micromorphology and molecular phylogeny. S.dianzhongensis is most similar to S.amblyphylla in its habit of creeping stem, leaf size, and obviously dimorphic sporophylls, but is distinct by its ventral leaves ovate-oblong, subcordate at base, basiscopic base entire, axillary leaves ovate and decurrent at base. Molecular phylogeny analysis of three chloroplast gene regions (rbcL, atpI, psbA) shows that S.dianzhongensis forms an independent branch with strong support which is distantly related to S.amblyphlla and S.kurzii, but sister to S.bodinieri which is quite different in habitat of erect or ascending stem and rhizophores restricted to the lower part, and slightly dimorphic sporophyllus.


Introduction
The initial critical taxonomic revision of Chinese Selaginella was published by Alston (1934), who recognized 41 species from China. In the Flora Reipublicae Popularis Sinicae and Flora of China (Zhang 2004, Zhang et al. 2013) the numbers of recognized species increased to 64 and 72, respectively. Recently, several new species and new records have been reported from China (Zhou et al. 2015a, Zhang and Sun 2015, Wu et al. 2017. Yunnan province is one of the species diversity centers of plants in China, with more than 53 species of Selaginella (Chu, 2006).
During field trips in Yunnan, we collected an unknown Selaginella species from Yimen County in central Yunnan. Morphology characters show it is similar to S. amblyphylla Alston, but phylogenetic analysis based on three chloroplast gene regions show it is close to S. bodinieri Hieron. Both molecular and morphology data support the taxon as a new species, which is described and illustrated here.
The new species belongs to subgenus Heterostachys in the classification system of Jermy (1986), and sect. Heterostachys of subgenus Heterostachys in the molecular-phylogeny classification proposed by Zhou and Zhang (2015), but this was rejected in the more robust molecular phylogenetic analysis based on chloroplast and nuclear genes by Weststrand and Korall (2016) that only recognized a broad subgenus Stachygynandrum.

Material and methods
Herbarium specimens, silica gel and living materials were collected from Longquan Forest Park of Yimen (Yunnan province), in evergreen forest at 24°40'86"N, 102°08'27.86"E. Herbarium specimens are preserved in PE, and compared with similar species. The morpho-photographs of the plants were taken with a Nikon DXM 1200F camera connected to a stereomicroscope (Nikon SMZ 1000) and computer, measurements were done by D 3.10 (http:// www.nikoninstruments.com). The application ImageJ (https://imagej.nih.gov/ij/) was used to measure morphological characteristics (such as axillary, dorsal, ventral leaves, stems and strobili).
For study of spore morphology, scanning electron microscopy (SEM) was used. The spores were taken from mature sporangia and fixed on double line tape, and then covered with gold-palladium mixture. Spores were photographed and measured under different magnifications using a Hitachi S-4800 at 10-20 kV.
Phylogenetic tree of combined dataset (rbcL+atpI+psbA) was constructed using maximum likelihood (ML) and Bayesian inferences (BI). jModelTest 0.1.1 (Posada 2008) was used to select the appropriate substitution model for ML and BI analyses. The ML analysis was performed on the XSEDE online computing cluster accessed via the CIPRES Science Gateway (http://www.phylo.org) using RAxML-HPC2 v.8.2.8 (Stamatakis 2014), with 1000 bootstrap replicates under the GTRGAT model. Bayesian analyses and posterior probability (PP BI ) calculation were conducted in Mr-Bayes 3.2.6 (Ronquist et al. 2012) implemented on the CIPRES Science Gateway Portal (Miller et al. 2010). We ran four chains of the Markov chain Monte Carlo (MCMC), sampling one tree every 100 generations for 1,000,000, starting with a random tree. Bayesian posterior probabilities (PP) were calculated as the majority consensus of all sampled trees with the first 25% discarded as burn-in.
Etymology. Dianzhong means central Yunnan in Chinese: the type locality (Yimen) is in the central Yunnan area which is centered on the Provincial capital city Kunming.
Distribution and habitat. Selaginella dianzhongensis is known only from Yimen county, Yunnan, growing on mossy soils in a mixed evergreen forest, at ca. 1576 m a.s.l. (Fig. 3).
Conservation status (VU). Selaginella dianzhongensis is known only from one locality inside the Longquan Forest Park in Yimen county, with more than 300 individuals. This park has a heavy recreational load and human pressure, and there are no specific measures to protect the habitats. Considering the restricted distribution and plausible threats, we tentatively assessed Selaginella dianzhongensis as vulnerable (

Phylogenetic Analysis
The combined data matrix included up to 2045 nucleotides for each of the 37 taxa with 374 parsimony informative sites (374/2045 = 18.29%), consistency index (CI) = 0.66, retention index (RI) = 0.80, when the gaps were treated as missing data. The tree recovered from maximum likelihood (ML) and Bayesian inferences (BI), with bootstrap values (BS) of ML and Bayesian posterior probabilities (PP) for each clade is shown in Fig. 4. The new species sampled from Yimen clustered with Selaginella bodinieri with strong support (BS ML = 99; PP BI = 1.0), but the new species is quite similar to the S. amblyphylla rather than S. bodinieri in morphological characters.

Discussion
Morphologically, the shape and margin of ventral and dorsal leaves of Selaginella dianzhongensis is most similar to S. amblyphylla. But the axillary leaves of the former are ovate, 1.1-2.2 × 0.4-0.8 mm, margin with a few long cilia (vs. ovate or triangular, 2-3 × 0.6-1.2 mm, and denticulate at margin in S. amblyphylla). Ventral leaves of the former are ovate-oblong, apex acute (vs. oblong and obtuse or subacute at apex in S. amblyphylla), basiscopic margin entire and not ciliolate (vs. basiscopic margin sparsely ciliolate at base in S. amblyphylla), acroscopic base of ventral leaf long ciliolate (vs. shortly ciliolate in S. amblyphylla). S. dianzhongensis is indeed similar to S. kurzii, but fertile branches are not erect (vs. erect in S. kurzii), dorsal leaves are ovate to broadly ovate with arista at apex (vs. ovate or ovate-elliptic, acuminate or aristate at apex), and ventral leaves are ovate-oblong, basiscopic margin entire and not ciliolate (vs. ovate-triangular, basiscopic margin entire or with 1 or 2 cilia at base).
Selaginella bodinieri is widely distributed in the limestone areas from central to southwestern China: main differences between this species (and the other ones mentioned above) and S. dianzhongensis are reported in the key below, and in Table 1.
Finally, mega-and microspores of S. dianzhongensis are morphologically different from the spores of similar species studied by Zhou et al. (2015b). Megaspores of S. dianzhongensis have verrucae on proximal and distal side; micro-sculptures of megaspores are densely echinate on both sides (Fig. 2 H-J). Microspores of S. dianzhongensis are verrucate, with blunt spinules (Fig. 2 K-M). Morphological comparison of mega-and microspores between S. dianzhongensis and closely related species is presented in Table 2.
Key to the S. dianzhongensis and related species in Yunnan