Bolbitis lianhuachihensis (Dryopteridaceae), a new species from Taiwan

Abstract A new species of Bolbitis, B. lianhuachihensissp. nov., was found in central Taiwan. It most resembles B. virens var. compacta and B. hainanensis. A phylogenetic tree of Taiwanese and other Asian species of Bolbitis species supports the recognition of the new species. Morphologically, the combination of anastomosing venation and fewer sterile pinnae are critical characters to discriminate B. lianhuachihensis from other Taiwanese Bolbitis species. Bolbitis lianhuachihensis can be further distinguished from B. virens var. compacta and B. hainanensis by having lanceolate sterile pinnae and absent or fewer free veinlets in small areoles of sterile pinnae. The morphological descriptions, illustration, ecology and distribution of the new species are presented. A key to all Taiwanese Bolbitis is also provided.

In Taiwan, a Bolbitis plant appeared unusual due to its few sterile pinnae (Figs 1 and Suppl. material 1: Figure S1). By this character, it was identified as B. virens var. compacta and resembled B. hainanensis Ching & Chu H. Wang (Knapp 2011), but B.virens var. compacta is found in Indochina (Hennipman 1977), while B. hainanensis is endemic to Hainan and Yunnan, China (Dong and Zhang 2005;Zhang et al. 2013). Taiwanese Bolbitis also differed from these species by venation. This character has been shown to be helpful in distinguishing many species of Bolbitis worldwide (Hennipman 1977). Species of Bolbitis may have either free or anastomosing veins. If the latter, a helpful distinguishing character is the number of areoles between the costae and margins, the presence or absence of included free veinlets in the areoles and whether these veinlets are recurrent or excurrent (Hennipman 1977;Moran et al. 2010a). In this study, we compared the morphological characteristics of the undescribed plant, especially with regards to lamina venation, with other species of Bolbitis in Taiwan. We also examined the phylogenetic relationship of these plants to existing Bolbitis species in Taiwan and to other similar species. Based on the results, we were able to clarify the specific morphological and molecular traits of the Bolbitis plant and describe a new species.

Morphological studies
The undescribed Bolbitis was collected in central Taiwan and herbarium specimens at HAST and TAIF were also studied. The keys, descriptions and illustrations in Hennipman's monograph (1977) were investigated to compare the morphological characteristics of our suspected new species with the known Bolbitis species. Furthermore, the type specimens of two similar species in nearby regions, B. hainanensis (Wallich 1033, K) and B. virens var. compacta (Tagawa et al. 6802,L) were analysed. As the type specimen of B. hainanensis lacks fertile fronds, we also consulted the protologue (Ching and Wang 1983). The venation of the Lienhuachih plants, two similar taxa and the known Bolbitis taxa in Taiwan were depicted. The morphological terminology follows that of Lellinger (2002). Acronyms of herbaria follow Thiers (2019).

Phylogenetic analyses
To clarify the phylogenetic position of the undescribed plant, six more Bolbitis species from Taiwan and B. virens from China were sampled and sequenced in this study. Additional sequences of 19 species were gathered from GenBank, including 17 Bolbitis species and two outgroup species. Elaphoglossum lepervanchii and Teratophyllum koordersii were used as outgroups, based on the phylogenetic trees of Bolbitis and related taxa (Moran et al. 2010a;Chen et al. 2017). A total of 30 samples of Bolbitis species were included and the voucher information is provided in the Appendix 1.
Phylogeny was inferred by Maximum Likelihood (ML) analyses with GARLI v.2.0.1019 (Zwickl 2006). The best tree was created from the ten independent runs with automatic termination following 10,000 generations without a significant (lnL increase of 0.01) change in topology. A majority-rule consensus tree was calculated in PAUP* v. 4.0b10 (Swofford 2002) to obtain bootstrap support based on 1,000 bootstrap replicates with automatic termination at 10,000 generations under one run in GARLI. Genetic data and the accession numbers of the sequences are listed in the Appendix 1.

Results
Based on the morphological study of the undescribed plant and type specimens of Bolbitis hainanensis and B. virens var. compacta (Suppl. material 1: Figure S2 and S3, respectively), specific characteristics were analysed to separate the three taxa; morphological data in the Bolbitis monograph (Hennipman 1977) is also integrated in Table 1. The unknown plant is distinguished from the two morphologically similar species through several characters: smallest fertile pinnae, lanceolate sterile pinna (narrower than others), cuneate bases of sterile pinnae, the smallest angle between veinlets and costae of sterile pinnae and the absence of, or fewer, free veinlets in small areoles of sterile fronds.
The venation in sterile fronds of the unknown plant, Bolbitis hainanensis, B. virens var. compacta and other Bolbitis species in Taiwan is illustrated in Fig. 2, showing one  side of a pinna of each species. We have found that the venation pattern of some taxa vary between small and larger pinnae, which is also reported by Hennipman (1977 We also find the angles of veinlets to costae to vary. Considering the three similar taxa, the unknown plant has smaller angles than B. hainanensis and B. virens var. compacta (65°< 75° or 80°; Fig. 2 J, K, and L); the latter two taxa have sterile pinnae with veinlets more vertical to the costae than the unknown species (also in the type specimens of the three taxa Suppl. material 1: Figures S1, S2, and S3).
Three presumed hybrid taxa present intermediate venation morphology between their putative parents. Bolbitis heteroclita × B. subcordata has more free veinlets included in areoles than B. heteroclita, but fewer free veinlets included in areoles than B. subcordata. Most pinnae of B. laxireticulata, like B. appendiculata, have free veins, but some pinnae have anastomosing veins, similar to those of B. subcordata. Some pinnae of Bolbitis ×nanjenensis have free veins like B. appendiculata, but some have costal areoles like B. heteroclita.
Additional Distribution. Taiwan (Fig. 4). Ecology. Evergreen, broad-leaf forests, often near streams, below 1000 m a.s.l. Etymology. The specific epithet 'lianhuachihensis' refers to the type locality. Note. Bolbitis lianhuachihensis can be delimited by the combination of anastomosing venation and fewer sterile pinnae than any other species of Bolbitis in Taiwan. It was thought to be related to two similar taxa with "thick laminae" in nearby regions, B. virens var. compacta and B. hainanensis (Knapp 2011). However, based on our study, only B. hainanensis has coriaceous laminae, thicker than chartaceous laminar of B. lianhuachihensis and B. virens var. compacta. Moreover, B. lianhuachihensis has lanceolate sterile pinnae and very few free veinlets in areoles of sterile pinnae wider than 3.5 cm, differing from the two similar taxa.
In this study, we revealed the venation diversity in the Taiwanese Bolbitis species and related taxa. Based on the illustration of venation, it is found that every taxon possesses its own venation morphology, supporting the taxonomic value of venation (Hennipman 1977;Moran et al. 2010a). We have applied several characters, including angles of veinlets to costae, free or anastomosing, row number of areoles between the costae and margins in sterile fronds and free veinlets in areoles or not for sterile fronds. We also found that venation patterns are more complicated, composed of multiple areoles, sub-areoles and free veinlets, which is also reported by Hennipman (1977). A character-state change from free venation to anastomosing venation is reported by Moran et al. (2010a). The venation characteristics in Bolbitis species are worthy of further investigation and application to the systematic and evolutionary study. To classify and describe the diverse venation morphology in detail would contribute to further studies of Bolbitis systematics.