Materials & Methods
The research was conducted on Navarino Island (54°S, 67°W), Magellanic region in sub-Antarctic Chile, at the southern end of the Americas. The island has a rugged topography with marked, but still moderate seasons. At the lower ranges, the forest is covered by a mix of southern beech species (Nothofagus betuloides and Nothofagus pumilio ) growing in krummholz formation at the tree line. Above the tree line, a rich community of small plants, including cushion plants, some graminoids, lichens and mosses, thrive in a Magellanic tundra environment (Méndez et al., 2013). We sampled C. pictafeces at sea level and C. picta and A. malouinus feces at ca. 700 masl, in open meadows some 100 m above the tree line. Field and laboratory work were conducted over the course of five weeks and the growth of recovered fragments was monitored over an additional 13 weeks in a growth chamber in Navarino Island, from December 2018 to April 2019.
In the field, fresh C. picta and A. malouinus fecal samples were collected from six locations, focusing on upland and lowland sites. We sampled opportunistically two sites near sea level, Lake Zañartu (19F 587064.87E 3911888.71S) and Robalo Bay (19F 585969.41E 3911044.12S), and four sites above the tree line, near streams and large ponds on the Cerro Bandera summit (19F 587490.15E 3908430.45S,19F 586779.97E 3907237.18S, 19F 586790.75E 3907270.36S). Fecal samples of both birds were identified in the field through our personal experience observing these birds in situ . Multiple fecal samples that visibly contained bryophyte fragments were collected from each site and later only one was selected from each site for processing (n =6), three from each bird species. Of those six samples, there were twoC. picta feces from lowland sites, one C. picta feces from an upland site, and three A. malouinus feces from upland sites. Additionally, mosses from three families (Polytrichum strictum , Brid., family Polytrichaceae, also known as “pigeon wheat”;Syntrichia robusta (Hook. & Grev.) R.H. Zander, family Pottiaceae; and Conostomum tetragonum (Hedw.) Lindb., family Bartramiaceae), were sampled near fecal sample sites to test for regeneration capabilities compared to fragments from fecal samples under the same growing conditions. Bryophytes collected in the field were identified by RM using the preliminary key to mosses of Isla Navarino (Buck & Goffinet, 2010).
In the laboratory, C. picta and A. malouinus fecal samples were stored in paper bags at room temperature before processing. We used a disinfected precision knife to remove the outer layer of the feces to eliminate the possibility of contamination by wind- or soil-borne spores and fragments. Two thin discs were then sliced from each fecal sample (approximately 1 mm in thickness). Each disc was dissolved separately in clear dishes using filtered water collected from the Robalo River (0.22-μm PVDF sterile syringe filters, Millipore, Cork, Ireland). The disc contents were observed under a Leica ICC50 HD compound microscope at 40x and 100x magnification. As many bryophyte fragments as possible were picked out from the dishes using a probe and forceps, placed into Eppendorf tubes corresponding to each disc sample and partially filled with filtered river water, following Russo et al.’s (2020) protocol. These samples were stored for a few days at 4° C until inoculation. Additionally, the wild collected mosses sampled were also cut into fragments.
The six fecal samples (three from each bird species) resulted in 11 disc samples analyzed, and 138 bryophyte fragments recovered and inoculated (106 belonging to C. picta and 32 to A. malouinussamples). The treatments tested were either a culture container, two plastic cups sealed together at the openings with parafilm, with commercial sterile peat soil (Kekkilä Professional Substrate) or a microplate for in vitro growth. The in vitro treatment was made using an agar-Gamborg Mixture Basal Salt (B5 salts) medium (G768, Phytotechnology Laboratories) with a pH of 5.8, a specific medium for moss growth (González et al., 2006). Once all fecal samples were processed, we randomly assigned the fragments from each fecal disc sample and from each moss species sample to either treatment to be propagated, with the exception of one A. malouinus sample, which only had one disc that contained one bryophyte fragment, and was therefore placed only in the agar-Gamborg treatment. For the peat soil treatment, the fragments were placed on the soil, thoroughly misted with filtered water and then covered and sealed for the remainder of the experiment with occasional misting throughout the observation period to keep the culture containers humid. High inner humidity and condensation was observed in the sealed containers throughout the experiment. For the agar-Gamborg treatment, the samples were placed in a 28-well agar plate and then covered and sealed with parafilm. A total of 18 culture mediums were placed into a growth chamber, with the temperature fluctuating between a minimum of 5 ºC and maximum of 25 ºC throughout the entirety of the experiment. Bryophyte cultures were grown on red and blue LED lights in a 4:1 ratio, at a day:night cycle of 16:8 h. The wild moss sample fragments inoculated in the peat soil containers were placed in the growth chamber on January 8th, 2019, followed by the fecal sample fragments inoculated in the peat soil containers on January 9th, 2019, and fecal sample and wild moss sample fragments inoculated in the agar-Gamborg on January 11th, 2019. All the samples were left in the growth chamber for a total of 91 days.