A new variety of hornwort from northern Thailand, Phaeoceros perpusillus var. scabrellus is described based on morphological characters and molecular phylogenetic analyses. In this study, phylogenetic analyses supported that the new variety is closely related to P. perpusillus var. perpusillus. Morphologically, it is distinguished from the autonimic variety in nearly smooth spores under light microscope. A taxonomic description, illustrations, and light and scanning electron micrographs are provided. In addition, the new variety is assessed as Endangered (EN), demonstrating its rarity by being currently known from only three subpopulations.

Endangered, hornwort, low-copy nuclear markers, new variety, spore ornamentation

Phaeoceros Prosk. (Notothyladaceae) is the third largest genus of hornwort with about 34 currently accepted species worldwide (Söderström et al. 2016) and widely distributed in both the Northern and Southern Hemispheres (Cargill and Fuhrer 2008). The genus is defined by a smooth solid thallus, single chloroplast per cell, presence of a pyrenoid, antheridial chambers with usually (1–)2–6(–8) antheridia, irregularly arranged jacket cells of the antheridia, and yellow spores (Duff et al. 2007; Cargill and Fuhrer 2008; Chantanaorrapint 2009; Villarreal et al. 2010). In Thailand three species have been reported: P. carolinianus (Michx.) Prosk., P. himalayensis (Kashyap) Prosk. ex Bapna & G.G.Vyas, and P. perpusillus Chantanaorr. (Lai et al. 2008; Chantanaorrapint 2009; Chantanaorrapint et al. 2015).

During the bryological surveys in Chiang Mai Province, northern Thailand, some interesting specimens of the hornwort genus Phaeoceros were collected. These specimens resemble P. perpusillus, an endemic species of northern Thailand, in having small gametophytes, short sporophytes (usually less than 1 cm long), yellow spores, and subquadrate pseudoelater cells. Following a detailed comparison with closely related taxa, we here describe and illustrate these specimens as a new variety of P. perpusillus. We also used for the first time three hornwort specific low-copy nuclear markers. In theory, low-copy nuclear genes tend to have higher mutation rates than organellar genes, resulting in more variable sites that can be used for phylogenetic reconstruction, especially at species-level (Sang 2002; Feliner and Rosselló 2007). However, despite the advantages of biparental inheritance, which provides a more comprehensive view of the genetic history and evolution, low-copy nuclear genes have not been widely used (Zhang et al. 2012). In this study, we combined selected low-copy nuclear sequences with chloroplast sequences to enhance the resolution of our phylogenetic analyses. By using both chloroplast and nuclear markers, our study aims to explore alternative genetic regions for species-level phylogenies, thus providing a greater understanding of hornwort evolution.

A concatenated dataset of the coding region of one plastid and three nuclear markers (rbcL, L138, L178 and L315) contained 2856 characters (892, 549, 781, and 634 characters respectively). Tree topologies generated by Bayesian inference (BI) and maximum likelihood exhibited congruent patterns shown in Fig. 1, with posterior probabilities (PP) and maximum likelihood bootstrap values (MLBS) plotted on the branches. The monophyly of the genus Phaeoceros is well supported by posterior possibility (PP = 1) but weakly supported by maximum likelihood analysis (MLBS = 53). In the tree topology (Fig. 1), Phaeoceros was divided into two major lineages with strong support, clade A including twenty-three terminals, containing the new taxon and other papillate spore Phaeoceros (PP = 1, MLBS = 94), and clade B comprising four terminals of non-papillate spore Phaeoceros including P. himalayensis and P. kashyapii A.K. Asthana & S.C. Srivast. (PP = 1, MLBS = 100).

Figure 1. 

Majority rule consensus tree of phylogenetic relationships of Phaeoceros derived from Bayesian analyses of the combined dataset of rbcL, L138, L178, and L315 genes. Bayesian posterior probability values (PP) and bootstrap percentages values (MLBS) are shown on branches respectively. Nucleotide substitution rates indicated below the tree. Clade A include papillate spore Phaeoceros and the new variety (highlighted), and Clade B include non-papillate spore Phaeoceros.

The inclusion of P. perpusillus var. scabrellus and its autonimic variety in the data matrix resolves this species lineage as monophyletic with good support (PP = 1, MLBS = 90). The new variety is recovered as sister to the autonimic variety with less posterior probability and bootstrap support (PP = 0.56, MLBS = 80).

Taxonomic treatment

 Phaeoceros perpusillus Chantanaorr. var. scabrellus Suwanmala & Chantanaorr., var. nov.

Figs 2, 3, 4

Type

Thailand. Chiang Mai Province: Doi Suthep-Pui, Bhu Bing Palace, 1400 m, 18 October 2020, S. Chantanaorrapint & O. Suwanmala 4077 (holotype: PSU!; isotype: BKF!, NICH!).

Figure 2. 

Phaeoceros perpusillus var. scabrellus A plants in natural habitat B dorsal view of thallus showing marginal tubers (arrow) C ventral view of thallus showing ventral tubers (arrows) D cross-section of thallus showing the large dark lump of Nostoc colony (NC = Nostoc colony) E dorsal epidermal cells of thallus showing a single chloroplast with pyrenoid (arrows) per cell F spores and pseudoelaters. Photos by O. Suwanmala (A from S. Chantanaorrapint & O. Suwanmala 4116 B–F from S. Chantanaorrapint & O. Suwanmala 4077).

Diagnostic

Phaeoceros perpusillus var. scabrellus is similar to the autonimic variety but differs in nearly smooth spores under light microscope (or vermiculate under SEM), whereas the autonimic variety have pluripapillae on the distal surface and vermiculate on the proximal.

Figure 3. 

Phaeoceros perpusillus var. scabrellus A gametophyte forming half-rosettes with sporophyte (arrow indicate tuber) B ensiform thalli and sporophytes C gametophyte showing ventral tuber (arrow) D dorsal epidermal cells of thallus E, F cross sections of thalli G cross section of sporangium (AS = assimilative tissue, EP = epidermal cell of capsule, IN = inner most sporangium wall) H inner most cells of sporangium wall I epidermal cells of capsule with stoma J proximal view of spore K distal view of spore L pseudoelaters. All from holotype and drawings by O. Suwanmala. (All drawing from S. Chantanaorrapint & O. Suwanmala 4116).

Description

Thallus yellowish-green to dark green in fresh material, dull green to blackish- brown in dry material, prostrate or moderately adhering to the substratum, solid, ecostate, orbicular to sub-orbicular, dichotomously branched into several lobes, with a smooth dorsal surface; lobes ensiform or sometimes fan-shaped, up to 0.8 mm long, 1–3 mm wide; margins wavy, nearly entire to shallowly crenulate; apex flat, rarely ascending, occasionally tapering into apical tubers; tubers sometimes present on ventral surface. Thallus in cross section plano-convex to concave-convex, 4–10 cells thick in the middle region, without mucilage cavities. Dorsal epidermal cells rectangular to heptagonal, 28–75 × 25–50 µm, thin-walled, smooth. Chloroplast one per cell, large, occupying almost entire cell, variable in shape; pyrenoid present. Nostoc colonies scattered through the ventral side of thallus, appearing as dark dots. Rhizoids hyaline or pale brown along ventral surface, inner wall smooth or tuberculate. Sexuality monoicous. Androecia scattered and slightly raised over the dorsal surface of thallus, 2–3 antheridia per chamber; antheridia subglobose to globose, exposed at maturity, irregularly arranged jacket cells, shortly stalked, stalk with quadriseriate cells. Archegonia embedded in thallus, connected to the upper surface, scattered near the lobe of thallus. Involucre solitary, conical-cylindrical, up to 2 mm long, 2–4 cells thick, mouth smooth to crenulate. Sporophytes capsule somewhat inclined, stout to narrowly cylindrical, 0.5–1(–1.2) cm long, yellow at apex, dehiscing from top toward base, bivalves rarely twisted when dry; epidermal cells of capsule elongate-rectangular, 68–200 × 12–30 µm, thick-walled, stomata present with two reniform guard cells, surrounded by 5–8 epidermal cells; assimilative layers 3–6 cells thick in cross section; the innermost capsule cells dark brown, subquadrate to rectangular; 27–67 × 22–53 µm; columella well-developed, red-brown, consisting of 16 cells (4 × 4 lines of cells) in cross section. Spores unicellular, yellow, rounded-triangular in polar view, equatorial diameter 32–50 µm in diameter, nearly smooth under light microscope (LM), proximal surface with a distinct trilete mark, bordered by vermiculate strip on each side of trilete mark, each facet covered with fine vermiculate pattern; distal surface with a slightly dome-like region at the center, more densely vermiculate than proximal surface, sometimes with minute granules. Pseudoelaters light brown or yellowish-brown at maturity, thin-walled, occasionally branched; pseudoelater cells subquadrate to short rectangular, 30–45 × 25–30 μm, without helicoidal band.

Figure 4. 

Scanning electron micrographs of spores A–F Phaeoceros perpusillus var. scabrellus A distal view of spore showing a central hump-like projection B proximal view of spore with a distinct triradiate mark C close-up of distal surface showing packed vermiculae D close-up of proximal surface showing loosely arranged vermiculae E proximal surface showing trilete mark and loosely arranged vermiculae F spores and pseudoelaters G–I P. perpusillus var. perpusillus G distal view of spore H proximal view of spore I close-up of distal surface showing pluripapillae. Photos by O. Suwanmala. (A–F images from S. Chantanaorrapint & C. Promma 3129 G–I images from S. Chantanaorrapint & O. Suwanmala 3883).

Etymology

The epithet of the variety refers to scabrate ornamentation observed under light microscope.

Habitat and distribution

Phaeoceros perpusillus var. scabrellus is currently known only from northern Thailand. It grows on disturbed soil and sandstone in open site in grassland, pine-oak mixed montane deciduous forests at elevation of 1390–2100 m. It may grow associated with other bryophytes such as Anthoceros subtilis Steph., Notothylas levieri, N. orbicularis (Schwein.) Sull. ex A.Gray, and P. carolinianus.

Conservation status

This variety is currently known from three subpopulations, which are in protected areas (Chiang Dao Wildlife Sanctuary and Doi Suthep-Pui National Park). One of the subpopulations is located in a camping area, which is a common visiting site for tourists and dominated by Ageratina adenophora (Spreng.) R.M.King & H.Rob. (invasive species). Therefore, habitat quality is threatened by trampling and other destructive activities potentially caused by regular visits by tourists to the area, and invasive plant species. Together, these have the potential to cause a population reduction. The other subpopulation is also somewhat disturbed by human activities such as shifting cultivation. The extent of occurrence (EOO) of P. perpusillus var. scabrellus is estimated to be 262.925 km² and its area of occupancy (AOO) is estimated to be 12 km², which falls within the limits for Endangered status under criterion B1 and B2 of IUCN Red List Categories and Criteria. Conservation efforts should focus on implementing strict regulations to reduce the impact of human activity and controlling invasive species, while also raising awareness among local communities about the importance of protecting the habitat.

Additional specimens examined

Thailand. Chiang Mai Province: Chiang Dao Wildlife Sanctuary, 1700–2000 m, 1 November 2013, S. Chantanaorrapint & C. Promma 3125B, 3129, 3216 (PSU); Doi Suthep-Pui National Park, Doi Mon Long Viewpoint, 1390 m, 4 November 2015, S. Chantanaorrapint & W. Juengprayoon 143B (PSU); 15 November 2020, S. Chantanaorrapint & O. Suwanmala 4089, 4090 (PSU); Bhu Ping Palace, 1400 m, 8 September 2013, S. Rattanamanee 3 (PSU); 18 October 2020, S. Chantanaorrapint & O. Suwanmala 4077 (PSU); 5 October 2021, S. Chantanaorrapint & O. Suwanmala 4116 (PSU).

Phaeoceros perpusillus var. scabrellus is morphologically similar to the autonimic variety which is endemic to northern Thailand (Chantanaorrapint 2009). These two varieties share some common features, viz. small orbicular gametophytes (Fig. 2A, B), monoicous sexual condition, very short capsules (usually less than 1 cm long) (Figs 2B, 3A, B), yellow spores, and pseudoelater cells being subquadrate to short rectangular (Figs 2F, 3L). The new variety also resembles P. exiguus (Steph.) J. Haseg., a species found in Indonesia, New Caledonia and Taiwan (Hasegawa 1986, 1993; Siagian et al. 2021). They are monoicous, and have a small thallus, very short capsules, and small pseudoelaters. However, they can be distinguished by the spore ornamentation. Phaeoceros perpusillus var. scabrellus is distinct from the autonimic variety and P. exiguus in nearly smooth spores under light microscope or vermiculate spores under SEM (Fig. 4A–F). In contrast, spores of P. perpusillus var. perpusillus are pluripapillose on distal face and finely vermiculate on proximal face (Fig. 4G–I), while P. exiguus have button-like papillae on distal face and minutely papillae on proximal face.

In addition, the small plants of P. carolinianus, a common species, can be confused with P. perpusillus or P. exiguus in general appearance. The comparisons of morphological characters between these three monoicous species are summarized in Table 3.

Although both varieties of P. perpusillus have been reported only from the northern part of Thailand, P. perpusillus var. perpusillus seems to have a wider range of distribution and is more abundant than the new variety. The new variety has been found in only three subpopulations, overlapping with the autonimic variety, which is assessed as Endangered (EN) according to IUCN Red List.

The placement of the new variety falls into the papillate spore Phaeoceros lineage (Fig. 1, clade A), despite the absence of spines or papillae on its spore surface which sets the new variety apart from other taxa. Within an assemblage of autonimic variety P. perpusillus and the new variety clade, the two taxa share a sister relationship with low support, and they show only one morphological difference in spore morphology. The vermiculate spore ornamentation observed in P. perpusillus var. scabrellus seems to be an unusual form of the autonimic variety. However, based on careful investigation, it becomes evident that the absence of papillae on the spore surface is consistently observed throughout the entire capsule and reveals a uniform pattern in each population. Spore morphology serves as a key trait to differentiate hornwort species, allowing two distinct spore ornamentations to be considered as separate taxa.

This proposal for the new variety’s classification was made due to its gametophyte and sporophyte morphological similarity to the autonimic variety with the exception of the spore ornamentation, and was also supported by phylogenetic inference, and the shared distribution area.

We would like to thank the curators and staff of EGR, G, HSNU, LWG, LWU, NICH, PSU and TNS for making specimens, including types, available for study through loans or visits. The first author would like to express her sincere appreciation to the Science Achievement Scholarship of Thailand (SAST) and the Canada-ASEAN Scholarships and Educational Exchanges for Development (SEED) program for funding. JCV thanks the Canada Research Chair on Genomics and Metabolomics. The authors thank David Bell (E) and Gabriel Peñaloza‐Bojacá for sharing the raw sequences, and D. Christine Cargill and the anonymous reviewers for their constructive comments on the manuscript.