Protocorms of an epiphytic orchid (Epidendrum amphistomum A. Richard) recovered in situ, and subsequent identification of associated mycorrhizal fungi using molecular markers

Abstract

ProtocormS of an EPiPhytic orchid (EpidEndrum amphistomum a. richard) rEcovErEd in situ, and SubSEquEnt idEntification of aSSociatEd mycorrhizal fungi uSing molEcular markErS L aw r ence w. Z et t L er 1, * , L au r a L . cor ey 1 , L a r ry w. r ich a r dson 2 , a pr i L y. ros s 1, 3 , and L i L L i a n MoL L e r-Jacob s 1, 4 1 department of biology, illinois college, 1101 west college avenue, Jacksonville, iL 62650 usa 2 Florida panther national wildlife refuge, u.s. Fish and wildlife service, 3860 tollgate boulevard, suite 300, naples, FL 34114, usa 3 current address: department of public heath, university of illinois at springfield, springfield iL, 62703 usa 4 current address: department of entomology, 501 asi building, pennsylvania state university, university park, pa 16802 usa * corresponding author: lwzettle@ic.edu; tel.: (217) 245-3479; Fax: (217) 245-3358 AbstrActEpiphytic orchids have received considerable study, yet little has been published on their germination requirements in situ involving mycorrhizal fungi. Such research has been hampered by the small, dust-like size of seeds and leafless seedlings (protocorms) which are difficult to pinpoint on natural substrates, especially those on arboreal substrates (tree limbs). We report a novel seed sowing and retrieval method, modified from one applied to terrestrial orchids, used in the acquisition of epiphytic orchid protocorms from the Florida Panther National Wildlife Refuge. Seeds from two epiphytic orchid species (Epidendrum amphistomum A. Richard, E. nocturnum Jacquin) were placed in separate nylon mesh packets secured within 35 mm plastic slide mounts, and affixed to tree bark using gutter mesh and a staple gun. To confirm that the embryos were viable, some seeds were also sown on asymbiotic media in the laboratory which subsequently germinated after 52 days incubation. Of 60 packets distributed among 18 tree limb sites, one packet -harboring seeds of E. amphistomum affixed to pop ash (Fraxinus caroliniana Mill.) on a moss substrate -harbored protocorms after 267 days. Using molecular markers, a fungus assignable to the Ceratobasidiaceae, appears to be the mycorrhizal associate of these protocorms suggesting that this fungus may be associated with the germination process in situ.

What constrains the distribution of orchid populations?

“…The distribution of mycorrhizal fungi associated with epiphytic orchids is less well studied, perhaps because early seed packet techniques using slide mounts were less successful on branches than in the ground (Zettler et al ., ). Otero et al .…”

“…Additional experimental manipulation of environments and mycorrhizal fungi will provide further insight into the mechanisms behind observed patterns of orchid and fungus distribution. The distribution of mycorrhizal fungi associated with epiphytic orchids is less well studied, perhaps because early seed packet techniques using slide mounts were less successful on branches than in the ground (Zettler et al, 2011). Otero et al (2002) suggested that epiphytic orchids may also be affected by the distribution and abundance of their mycorrhizal fungi, but research is only beginning on mycorrhizal fungus distribution in epiphytes (Kartzinel et al, 2013).…”

Seedling mycorrhiza: a discussion of origin and evolution in Orchidaceae

“…(), Zettler et al . (, , , ), and Zettler & P iskin (). The illustration of orchid phylogeny is based on data from C hase et al .…”

A novel seed baiting technique for the epiphytic orchid Rhynchostele cervantesii, a means to acquire mycorrhizal fungi from protocorms

“…The seed baiting technique initially developed by Rasmussen and Whigham (1993) has helped considerably with the former, resulting in more than a hundred studies carried out in situ during the past 20 years, mostly involving terrestrial orchids (Rasmussen, 2002). For epiphytic orchids, a modified seed baiting technique has since been developed that emphasizes affixing seed packets to arboreal substrates (Zettler et al 2011;Zi et al 2014) which may have potentials for use in a wide range of taxa. Nevertheless, seeds in packets that are attached to aerial substrates are highly vulnerable to desiccation, even in packets placed in direct contact with (moist) moss in continuous shade.…”

“…Nevertheless, seeds in packets that are attached to aerial substrates are highly vulnerable to desiccation, even in packets placed in direct contact with (moist) moss in continuous shade. Zettler et al (2011) reported that only one of 60 seed packets affixed to trees harbored protocorms of Epidendrum amphistomum in south Florida, for example, and they proposed that moisture-rich substrates were needed for fungal growth and proliferation to trigger epiphytic orchid seed germination. If true, it is conceivable that placing a water absorbing/retaining material within the seed packet itself may raise moisture to levels conducive to germination.…”