Mesoamerican Cypripedium: Mycorrhizal Contributions to Promote Their Conservation as Critically Endangered Species

Abstract

In the valuable orchid genus Cypripedium, the section Irapeana consists of a distinctive group of Mesoamerican species that is formed by Cypripedium dickinsonianum Hágsater, C. irapeanum Lex., and C. molle Lindl. All lady slipper orchids exhibit different distributions and abundances. Data analysis that used herbarium accessions and field investigations indicated that the habitats of these three species have been dramatically reduced. Prospecting for suitable habitats based on climatic, vegetation, and soil parameters allows us to predict potential distributions. Conservation strategies, such as ex situ propagation by asymbiotic and symbiotic approaches, have indicated that the culture media used are a determining factor for seedling development. Mycorrhizal isolates play a main role in the compatibility and further development of germinated seeds. The fungi isolated from adult plants belong to two different families, which makes it possible that widely distributed C. irapeanum populations will be fungal-specific as well as restricted for C. molle. Root mycorrhization patterns occur high on the secondary roots. In contrast with other species of the genus, in situ germination can occur over a short period of two months, but we have documented periods as long as ten years. Cypripedium is a highly problematic genus for ex situ conservation because the germination requirements and cultures are poorly documented, and there is great urgency for in situ conservation to develop strategies for identifying hotspot habitats and actualize the protection status to avoid extinction of this genus.

Keywords: habitat destruction; in vitro germination; orchid conservation; orchid mycorrhiza.

Figure 1

Mesoamerican Cypripedium: (a) C. dickinsonianum, (b) C. irapeanum and (c) C. molle, pictures taken by Javier Fortanelli, Mauricio Moreno, and Octavio Gabriel in the years 2014, 2010 and 2019, respectively.

Figure 2

Actual distribution of Mesoamerican Cypripediums from herbarium data. Cypripedium irapeanum = yellow points, C. molle = red points, and C. dickinsonianum = blue points.

Figure 3

Destroyed habitats of Mesoamerican Cypripedium molle in Oaxaca State (a–d) and C. irapeanum in Nayarit State (e–f). (a) Land use change for Agave angustifolia cultivation for mezcal production (white arrow indicates a remaining C. molle plant). (b) Deforestation of a pine forest in the transition from oak to cloud forest habitat. (c) Fire destruction of oak forest habitat in limestone. (d) Habitat destruction by an outdoor dump. (e) Population vulnerability next to the road subject to any perturbation process. (f) Land use change of deciduous forest transitions for mango cultivars (white arrow shows the disappeared population). (a–e), photos taken by Mauricio Moreno in 2019; (f), INEGI [23] and Google Earth ©, 2020.

Figure 4

Distribution of suitable habitats of Mesoamerican Cypripedium based on the most reliable Maxent model. Rasters generated in Maxent [27]. Maps were generated in ArcGIS© 10.5 (ESRI, https://www.esri.com/en-us/home (accessed on 1 April 2022)) (a) C. dickinsonianum, (b) C. irapeanum, and (c) C. molle.

Figure 5

In situ development of Cypripedium molle (a,b) in an oak forest in Oaxaca State and C. irapeanum (c–f) in an oak forest in Mexico State and in a tropical dry forest in Veracruz State. (a) Long, little-branched roots longer than 30 cm under abundant litter (white arrow). (b) Achlorophyllous shoot (red arrow) of an emerging plant sprouting due to the deep litter where roots develop in the humic horizon. (c) Lateral shoot emerging from a near meristematic node (green arrow) and short rhizome development with roots surrounded by mycelial cords (dashed red arrow) with short-distance exploration mycelium (double-line red arrow). (d) Multiple points of emergence of annual plants showing a node with growth of several years (white arrows) and the remainder of a dried shoot from the previous year (blue arrow). (e) Plants usually associated with Bletia orchids (B. purpurea, white arrow). (f) Association with an eight-year-old Bletia punctata plant showing the emerging shoots of the first two submerged bulbs. These belong to the first and second bulbs that developed after germination and the emerging shoots after eight years (white arrow). A dried C. irapeanum shoot from the previous year next to a new emerging shoot (yellow arrow). Photos taken by Mauricio Moreno in the year 2019.

Figure 6

In situ germination of Cypripedium irapeanum from Veracruz State in a tropical dry forest (a,b) and an oak forest in Mexico State (c–h). (a) Seedling less than 3 cm long. (b). Emergence of multiple large roots from seedlings (white arrow). (c) Seed imbibition (white arrow) after the second month of baiting placement (upper right picture), showing that the sand grain sizes are larger than those of the seeds and brown fungal hyphae (scale bar = 500 µm). (d,e) Two-month achlorophyllous protocorms in the baiting showing development of the shoot apical apex (scale bar = 500 µm). (f) Germinated seed after 10 years of baiting in August 2018 with a root that is several times larger than the undeveloped shoot (white arrow) (scale bar = 16 mm). (g) Detail of a root emerging from an undeveloped shoot of the same seedling showing testa remains (white arrow) (scale bar = 500 µm). (h) Comparative size of seedlings regarding baiting dispositive (see (c)) (scale bar = 2 mm). Photos taken by Mauricio Moreno (a,b) in 2020; Jesus Colín (c) in 2012 and M.P. Ortega (d–h) in 2018.

Figure 7

Mycorrhizal root colonization of Mesoamerican Cypripedium. View of one of the main roots (black arrows) and secondary roots (white arrows) of Cypripedium irapeanum (a–e) and C. molle (f–j). Cortical colonization by mycorrhizal fungi occurs in patches on the main roots with long sections without fungal colonization and starch grains (b,c) and some with very dense colonization with digested pelotons (f,g). SR are always densely colonized with undigested and partially digested pelotons (d,e and h,i). Bars in c, e, g, i = 10 µm and bars in b, d, f, h = 100 µm. Bar in a = 10 cm. Photos taken by Mauricio Moreno in year 2019.

Figure 8

Mycorrhizal isolates of Mesoamerican Cypripedium. Isolate from Cypripedium irapeanum (a–d) and from C. molle (e–h). (a) Colony view of Epulorhiza sp. (Tulasnella sp.) from a tropical dry forest in Veracruz State showing a submerged mycelium, yellow-creamy color, and waxy surface. (b) Superficial growth after 5 days of incubation of straight hyphae obtained from a peloton after sowing on GPA medium showing 90° bifurcation of the hyphae (black arrow). Bar = 100 µm. (c) Mycelium from an isolated culture showing less than 4 µm hyphae and straight basal septa (black asterisk). Bar = 10 µm. (d) Monilioid cells. Bar = 10 µm. (e) Colony view of Ceratorhiza sp. (Ceratobasidium sp.) that was isolated from a Quercus forest in Oaxaca State showing an aerial mycelium, brownish-creamy color, and cottony superficial aspect. (f) View of hyphae growing from a submerged peloton with a knobby appearance after 3 days of incubation. Bar = 10 µm. (g) Mycelium from an isolated culture showing hyphae longer than 4 µm and constrained basal septa (black asterisk). Bar = 10 µm. (h) Monilioid cells. Bar = 10 µm. Photos taken by Mauricio Moreno in 2020.

Figure 9

Phylogenetic relationships of endophytes isolated from Mesoamerican Cypripedium. The pink clade represents isolates from Cypripedium irapeanum from a tropical dry forest and Quercus forest. The blue clade contains the isolate from C. molle, which was inferred by using the maximum likelihood method and the Tamura-Nei model log likelihood of –3241.26. The branch length labels represent the bootstrap proportions. Analyses were conducted in Geneious (2021.0.3).

Figure 10

Asymbiotic in vitro germination of Cypripedium irapeanum in Norstog medium. (a) Development stage 0 on day of sowing (black arrows show immature and undeveloped seeds and white arrows show mature seeds). (b) Imbibed seeds two days after sowing (das) (white arrow) (development stage 1). (c) Imbibed seed with rupture of testa 12 das (white arrow) (development stage 2). (d) Protocorm polarization with apical meristem at 45 das (upper structure) (developmental stage 3). (e) Protocorm development with leaf blade at 54 das (developmental stage 4). (f) Pre-seedling showing radical meristem (black dashed arrow) and a long achlorophyllous leaf 96 das (developmental stage 5). Other chlorophyll pre-seedlings with two leaf blades are located at the bottom left. (g) Seedling development with two incipient roots at 122 das (black dashed arrow) (developmental stage 6). (h) Seedling 187 das with four leaves and a greater ratio between root and shoot. (i) Seedling 300 das with lateral shoots and remnant testa (black dashed arrow) from the first shoot and two radical sprouts. The white arrow shows two oxidized pre-seedlings at stage 4. (j) Four seedlings 300 das with long roots and one seedling with two shoots (black dashed arrow). The white arrow shows a protocorm with irregular protuberances, such as rhizoids, or a protocorm-like body (PLB). (k) Seedling at 365 das with three shoots and two elongated and exfoliant rhizodermic roots. Scale bars = 100 µm.

Figure 11

Symbiotic in vitro germination of Cypripedium irapeanum with two Epulorhiza spp. (Tulasnella spp.) isolates obtained from an oak forest in Mexico State and a tropical deciduous forest in Veracruz State with seeds from Puebla State. (a) Seed imbibition (stage 1) before fungal contact at 2 days after sowing (das) (white arrow shows immature and ungerminated seeds with embryos) with a Mexico State isolate. (b) Mycorrhizal contact during seed imbibition at 12 das with the Veracruz isolate. (c) Symbiotic protocorms surrounded by mycelium (not rhizoids) at stages 2 (white arrow) and 3 developing rhizoids (black arrow) at 12 das with a Veracruz isolate. (d) Protocorm out of testa at 12 das stained with acid fuchsine showing pelotons at the micropillar seed region (black arrow) and hyphae growing in medium. (e,f) Symbiotic protocorms at stages 3 (e) and 4 (f) at 22 and 54 das, respectively, with the Veracruz isolate showing early vitrification and fungal incompatibility (black arrows). (g,h) Histological staining evidence of the fungal incompatibility, where pelotons invade apical meristematic tissue cells (black arrows) at stage 3 (g) and stage 4 (h), both at 109 das with an isolate from Mexico State. (i) Symbiotic protocorms at 187 das showing fungal compatibility that are located in a brownish tissue in the micropylar zone (black dashed arrow) with the Veracruz isolate. (j) Histological view of a stained mycorrhized protocorm with dense pink pelotons (black arrow) at the micropylar pole and with intact leaf vascular bundles (white arrow) at 96 das. (k,l) Symbiotic mixotrophic protocorms at stage 4 (with two foliar sheets) at 72 and 109 das with the Veracruz isolate. Scale bar = 100 µm. Photos taken by Mauricio Moreno in 2015.