Monotropastrum kirishimense (Ericaceae), a new mycoheterotrophic plant from Japan based on multifaceted evidence

Abstract

Due to their reduced morphology, non-photosynthetic plants have been one of the most challenging groups to delimit to species level. The mycoheterotrophic genus Monotropastrum, with the monotypic species M. humile, has been a particularly taxonomically challenging group, owing to its highly reduced vegetative and root morphology. Using integrative species delimitation, we have focused on Japanese Monotropastrum, with a special focus on an unknown taxon with rosy pink petals and sepals. We investigated its flowering phenology, morphology, molecular identity, and associated fungi. Detailed morphological investigation has indicated that it can be distinguished from M. humile by its rosy pink tepals and sepals that are generally more numerous, elliptic, and constantly appressed to the petals throughout its flowering period, and by its obscure root balls that are unified with the surrounding soil, with root tips that hardly protrude. Based on genome-wide single-nucleotide polymorphisms, molecular data has provided clear genetic differentiation between this unknown taxon and M. humile. Monotropastrum humile and this taxon are associated with different Russula lineages, even when they are sympatric. Based on this multifaceted evidence, we describe this unknown taxon as the new species M. kirishimense. Assortative mating resulting from phenological differences has likely contributed to the persistent sympatry between these two species, with distinct mycorrhizal specificity.

Keywords: Fungal association; Integrative taxonomy; Mycoheterotrophy; Mycorrhizas; Reproductive isolation; SNP data; Speciation; Species delimitation.

Fig. 1

Morphological comparison of the aboveground parts of Monotropastrum kirishimense and M. humile. Monotropastrum kirishimense in a Fujieda-shi, Shizuoka Pref., b Ena-shi, Gifu Pref., and c Kirishima-shi, Kagoshima Pref. Monotropastrum humile in d Waga-gun, Iwate Pref. and e Tarumizu-shi, Kagoshima Pref., and f M. humile f. roseum in Sakyo-ku, Kyoto Pref. Arrowheads indicate spreading sepals. Scale bars: 3 cm. Photographed by Masayuki Sato (a), Katsumi Iwahori (b), Shuichi Kurogi (c), Shin Terui (d), Kazushige Uemori (e), and Kenji Suetsugu (f)

Fig. 2

Monotropastrum humile and its monotropoid association found at the M. kirishimense type locality (on June 25, 2019, except for the flowering plants). a Flowering plants (on May 17, 2019). Arrowheads indicate spreading sepals. b Fruiting plant. c Fruiting scapes with root ball. d, e Magnification of the root ball. Root tips and branching are easily recognizable. Arrowheads indicate root tip apices. Scale bars: 3 cm (a–c), 1 cm (d), and 5 mm (e). Photographed by Hideo Shimada (a) and Kenji Suetsugu (b–e)

Fig. 3

Monotropastrum kirishimense and its monotropoid association (holotype). a Flowering scape with root ball. b, c Flowering plants. d Flowers, top view. e, f Magnification of the root ball. Root tips are not apparent, but white fungal hyphae are visible. Arrowheads indicate the root tip apices. Scale bars: 3 cm (a–‍c), 1 cm (d, e), and 5 mm (f). Photographed by Kenji Suetsugu

Fig. 4

Monotropastrum kirishimense (holotype). a Flower. b Root ball with the interpenetrating Pinus densiflora root system. c Flower with perianth removed. d Floral discs with the basal part of the filaments and protrusions from the floral disc. e Petal, adaxial view. f Petal, abaxial view. g Anther. Scale bars: 1 cm (a, b), 5 mm (c, e, f), and 2 mm (d, g). Photographed by Kumi Hamasaki

Fig. 5

Monotropastrum kirishimense (drawn from the holotype). a Flowering scapes with root ball, with the interpenetrating Pinus densiflora root system. b Flower, side view. c Flower, top view. d Flower after the removal of perianth. e Flower after the removal of perianth and filaments. f Sepals, adaxial view. g Petal, adaxial view. h Scale leaf, adaxial view. i Floral discs and basal part of ovary. j Filaments. Scale bars: 3 cm (a), 1 cm (b, c), 5 mm (d–i), and 2 mm (j). Drawn by Kumi Hamasaki

Fig. 6

Flowering phenology of Monotropastrum kirishimense (a, b) and M. humile (c, d) in the Onami population (31°55’N 130°50’E), where both species occur sympatrically

Fig. 7

Phylogenetic tree of Monotropastrum kirishimense and M. humile reconstructed from MIG-seq data. Nodes supported by bootstrap values < 50% are not shown. Branch length represents the average number of substitutions per site

Fig. 8

Relative abundance of ectomycorrhizal communities associated with Monotropastrum kirishimense and M. humile, at the operational taxonomic unit (OTU) level

Fig. 9

Phylogenetic tree of partial internal transcribed spacer rDNA sequences from mycorrhizal fungi of the Monotropastrum kirishimense and M. humile plants investigated in this study (bold type) and from the International Nucleotide Sequence Database Collaboration (INSDC) database. Nodes supported by bootstrap values < 50% are not shown