Morphological and anatomical adaptations to dry, shady environments in Adiantum reniforme var. sinense (Pteridaceae)

The natural distribution of the rare perennial fern Adiantum reniforme var. sinense (Pteridaceae), which is endemic to shady cliff environments, is limited to small areas of Wanzhou County, Chongqing, China. In this study, we used brightfield and epifluorescence microscopy to investigate the anatomical structures and histochemical features that may allow this species to thrive in shady, dry cliff environments. The A. reniforme var. sinense sporophyte had a primary structure and a dictyostele. The plants of this species had an endodermis, sclerenchyma layers and hypodermal sterome, reflecting an adaption to dry cliff environments. Blades had a thin cuticle and isolateral mesophyll, suggesting a tolerance of shady environments. These characteristics are similar to many sciophyte ferns such as Lygodium japonicum and Pteris multifida. Thus, the morphological and anatomical characteristics of A. reniforme var. sinense identified in this study are consistent with adaptations to shady, dry cliff environments.


INTRODUCTION
Adiantum reniforme var. sinense (Pteridaceae, subfamily Vittarioideae) is a rare cliff-dwelling perennial pteridophyte, with a natural distribution limited to small areas of Wanzhou County, Chongqing, China. This plant has been used in Chinese medicine for more than 100 years (Lin, 1980;Rothfels & Schuettpelz, 2014;PPG, 2016;Pryer et al., 2016). A. reniforme is of immense botanical interest due to its intercontinental distribution; A. reniforme is found in the Azores, A. reniforme var. sinense in China and A. reniforme var. asarifolium in south-central Africa (Lin, 1980;Wang et al., 2015). The Chinese variants of this species have low genetic diversity (Pan, Ji & Chen, 2005;Liu, Gituru & Chen, 2007;Wang et al., 2015). In China, the natural habitat of this plant was lost due the construction of Three Gorges Dam, which was completed in 2012. Since this time, A. reniforme var. sinense has been conserved ex situ in the germplasm resource nursery managed by the China Three Gorges Corporation and the Wuhan Botanical Garden (Pan, Ji & Chen, 2005;Liao et al., 2007;Zhou, Jiang & Huang, 2008;Wu, 2012). Due to its endangered status, narrow distribution, ex situ conservation, and low genetic diversity, A. reniforme var. sinense is listed as a class II protected fern in China (Lin, 1980;Xu, Zhen & Jin, 1987;Fu & Jin, 1992;Pan, Ji & Chen, 2005;Liu, Gituru & Chen, 2007;Wang et al., 2015).
Phylogenetic and ontogenetic relationships within Adiantum have historically been based on anatomical characteristics, particularly the presence of dictyostele in the leaves (Wylie, 1949;Imaichi, 1988;Huiet et al., 2018). However, recent molecular genetic analyses have suggested that A. reniforme var. sinense is synonymous with A. nelumboides, in opposition to classical morphological taxonomy (Lin, 1980;Wang et al., 2015). In addition, although A. reniforme var. sinense has been well studied with respect to optimal spore culture conditions, structural sporophyte anatomy and photosynthetic capacity have received little attention (Wu et al., 2010(Wu et al., , 2011Liao et al., 2007).
Here, we aimed to investigate the structural and histochemical features of the A. reniforme var. sinense sporophyte to determine whether these features were adaptations to dry cliff environments. We also sampled the leaves of A. reniforme var. sinense sporophytes in the sun and shade to identify the morphological and anatomical traits that indicated adaptations to shady environments; the leaves exhibited the same traits observed in other species. Evidence of such adaptive characteristics might help to explain the ability of A. reniforme var. sinense to grow in shady, dry cliff environments. Our results may also inform future studies of the ex situ conservation, taxonomy, evolution and phylogeny of this rare plant and its relatives.

Plant sourcing and collection
Adiantum reniforme var. sinense specimens were cultivated at the Chinese Germplasm Resource Nursery of the Three Gorges Corporation (Ichang, Hubei, China). Several sporophytes were collected in October 2019. From each plant, we collected approximately 10 roots, eight rhizomes, five leaves growing in the sun (intensity of illumination about 4,840 lux; humidity 51.6%) and five leaves growing in the shade (intensity of illumination about 805.5 lux; humidity 49.5%). Freshly collected samples were immediately fixed in formaldehyde-alcohol-acetic acid (FAA) (Ruzin, 1999).

Microstructure and histochemistry
Root tissues were then sectioned freehand, using a two-sided razor blade, under a stereoscope (JNOEC JSZ6, China). Root sections were cut at 5, 10 and 20 mm from the root tip. Rhizomes were cut into two sections: young (~10 mm from the tip; white surface coloration) and mature (~30 mm from the tip; brown surface). Petioles were also cut into young (white surface) and mature (black surface) sections. Blades were cut in the center to determine tissue thickness. Sections were divided into three sets such that each set contained 3-6 sections (obtained from different specimens) representing each distance from the root tip; the young and mature rhizomes; the young and mature petioles; and the blade centers and margins.

Blade data collection and statistical analyses
The length and width of leaf area was measured with a centimeter ruler. Tissue thickness data was collected from sections stained by SR7B, BAB and TBO as discussed in the above section. We sectioned the leaflet at the blade margin to measure the density of the fine veins. We also sectioned the leaflet at the blade center (not along the blade margin) to measure the stoma and cell number and size of tissue density and epidermal features. All sections included five blade samples that remained unstained and were mounted with sterile water. Specimens were observed under a Leica DME microscope with a micrometer. Differences between the morphological and anatomical traits from sunny and shady blades were analyzed with the paired-samples T-test using SPSS (version 13.0; SPSS Inc., Chicago, IL, USA).

RESULTS
The stele within the adventitious roots had diarch symmetry with protoxylem poles (Figs. 1A-1I). At 5 mm from the root tip, we observed faint Casparian bands in the endodermis of the inner root cortex, a thin-walled sclerenchyma layer around the endodermis, and a rhizodermis on the root surface (Figs. 1A and 1B). At 10 mm from the root tip, lateral roots emerged from the stele, and the stele had prominent protoxylem and protophloem (Figs. 1C-1F). In addition, the endodermis had complete suberin lamellae with a few passage cells, and the sclerenchyma layer had thicker walls (except for the idioblasts) opposite the passage cells and the protoxylem (Figs. 1C-1F). At 20 mm from the root base, the stele had primary xylem and phloem tissues, as well as deep suberin lamellae in the endodermis; the sclerenchyma layer was thick-walled (Figs. 1G-1I).
Both young and mature rhizomes had a dictyostele surrounded by sclerenchyma layers. The dictyostele included petiole vascular bundles with a prominent central protoxylem ( Figs Young and mature petioles had a single vascular bundle with a central endodermis, a cortex, a peripheral sclerenchyma layer and an epidermis (Figs. 3A-3C). At maturity, the endodermis had Casparian bands and suberized lamellae (Fig. 3B). The petiole surface had a thin cuticle (Fig. 3C). Table 1 shows the morpho-anatomical characteristics of the blades: leaf area, tissue thicknesses, tissue densities, and epidermal features. The leaf blades from both sunny and shady environments had an epidermis, isolateral mesophyll tissue, and a dichotomous vein with a sclerenchyma layer (Figs. 3D-3I; Table 1); the endodermis of the dichotomous vein had Casparian bands and suberized lamellae (Figs. 3E, 3F, 3H and 3I). The stoma was only present on the abaxial epidermis (Figs. 3J and 3K); the shady blades had fewer and larger stoma compared to the sunny blades ( Table 1). The thin cuticle was slightly thicker on the adaxial and abaxial on the sunny blades compared to shady blades (Figs. 3D-3I; Table 1). The surface of the leaf blade had a hypodermal sterome (Figs. 3D-3I). Lastly, the mesophyll was thicker in the middle of the sunny blades than the shady blades (Figs. 3D-3I; Table 1).

DISCUSSION
We observed various morphological and anatomical characteristics that were likely to support the successful colonization of dry and shady environments by A. reniforme var.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This work was supported by the China Three Gorges Corporation (2019H210). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: China Three Gorges Corporation: 2019H210.

Competing Interests
Di Wu, Linbao Li, Xiaobo Ma and Guiyun Huang are employed by Three Gorges Corporation that have no conflicts of interest. The authors have no conflicts of interest to declare.

Author Contributions
Di Wu conceived and designed the experiments, performed the experiments, prepared figures and/or tables, and approved the final draft. LinBao Li performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.
Xiaobo Ma performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft. Guiyun Huang conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft. Chaodong Yang conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Data Availability
The following information was supplied regarding data availability: The raw data is in Figs. 1-3 and a Supplemental File.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.9937#supplemental-information.