TRANSACTIONS OF THE KANSAS Vol. 118, no. 3-4 ACADEMY OF SCIENCE p. 230-236 (2015) Tardigrades of the canopy: Evidence of stratification LAUREN CHANG1, DEVON POWEll2, WIllIAM R. MIllER3 AND MARGARET LOWMAN4 1. Department of Biology, Wesleyan University, Middletown, Connecticut 06459 2. Department of Environmental Studies, New College of Florida, Sarasota, Florida 34243 3. Department of Biology, Baker University, Baldwin City, Kansas 66006 4. California Academy of Sciences, San Francisco, California 94118 Corresponding author: [email protected] Mosses and lichens growing on trees and rocks are a common habitat for limno- terrestrial tardigrades all over the world. However, worldwide tardigrade research has been conducted primarily with ground level samples. In order to determine the presence, absence, diversity, distribution and size of tardigrade populations in the canopy, researchers ascended 135 trees of 17 species at nine northeastern Kansas forest sites, and collected tardigrade habitat from varying heights. Analysis of 822 samples clearly evidenced the presence of tardigrade populations in the forest canopy, as well as documented stratification with a statistically significant increase in density of the tardigrades per sample from 6.4 to 11.9 with increased height. This third dimension discovery must now be factored into theories of tardigrade dispersal. Further investigations may hold new, arboreal species. Keywords: Canopy, Stratification, Tardigrades INTRODUCTION their ecology or basic requirements for living (Ramazzotti and Maucci 1983; Miller 1997). Tardigrades are little studied, aquatic, microscopic invertebrates. First viewed Tardigrades are typically 200-700µm long, by Leeuwenhoek in 1674 (Kinchin 1994), with five body segments. The anterior or head tardigrades inhabit a variety of aquatic segment includes the brain, mouth, and eyes. environments, including oceans, rivers, and The four body segments each have a pair of lakes. In addition, limno-terrestrial tardigrades legs ending in claws which allow tardigrades to are able to live in terrestrial habitats that crawl through their aquatic environment. The periodically retain water like a sponge (mosses, limno-terrestrial tardigrades are divided into two lichens, leaf litter, soil, etc.). They exhibit classes. Heterotardigrada have armored plates on cryptobiosis, the process of desiccating with their segments, colored bodies, and four parallel their habitat and reconstituting with the return claws on each foot (Fig. 1). Eutardigrada do not of moisture. This allows them to survive in dry have body plates, are transparent, and have a areas of the world (Kinchin 1994). Recently, pair of claws on each foot (Fig. 2). cryptobiotic tardigrades became the first multi-celled animal to survive exposure to the Forest canopies have been called the “eighth vacuum, temperature, and radiation of space continent of planet Earth” (Wilson 2005) due (Jönsson et al. 2008). Cryptobiotic tardigrades to their complexity and enormous biodiversity. are probably dispersed in the winds (Kinchin With the explosion of canopy exploration 1994; Miller 1997) and thus are rained onto over the last two decades (Lowman 1999, the canopies of the world, where they must Lowman and Rinker 2004), canopy researchers find acceptable habitat to survive (Miller have confirmed that a large portion of global 2004). Despite our knowledge of tardigrade biodiversity inhabits the treetops (Erwin 1982; physiology, we know almost nothing about Lowman and Rinker 2004). Transactions of the Kansas Academy of Science 118(3-4), 2015 231 Figure 1. Class Heterotardigrada, showing Figure 2. Class Eutardigrada showing paired parallel claws, plated dorsal cuticle and claws, smooth dorsal cuticle and elongated rounded body shape. Scale bar = 50 µm. body shape. Scale bar = 50 µm. Historically, limno-terrestrial tardigrades have METHODS been collected within reach at ground level. Kimmel and Meglitsch (1969) presented the Tardigrade habitat samples (moss and lichen) first hint of arboreal tardigrades, finding them were collected from different substrates (tree three meters above the ground in Iowa trees. species) at nine locations in northeastern Voegtlin (1982) reported observing water bears Kansas. Utilizing the double-rope climbing in washings of Douglas-fir canopy needles technique (Haefke et al. 2013), researchers in Oregon. Counts et al. (2001) reported ascended as high as 20 meters (60 ft) into trees tardigrades as “Jewels” in the canopy of Great and collected samples (Fig. 3). Four levels, Smoky Mountains National Park during the increasing in height at three-meter increments, beginnings of the All Taxa Biodiversity Survey were recorded (0-3, 3-6, 6-9, >9 meters). The project (Sharkley 2001). Mitchell et al. (2009) samples from levels above nine meters were documented substrate (tree) selection and pooled because of the limitations of sampling. provided evidence of vertical distribution. Habitat samples were scraped from the bark of Recently, Miller et al. (2013) reported actual the substrate using a pocketknife into a labeled stratification with greater diversity and density paper bag. of tardigrades at higher heights in the canopy (10 meters) of a white pine on the Baker In the lab, habitat samples were soaked in University campus in Kansas. bottled water in small plastic cups for about 12 hours. After soaking, a 1 ml pipette was used This project was designed to demonstrate and to extract water and debris from the soaking document tardigrade canopy usage. It was cup to fill a 25 x 50 mm tray. The tray was hypothesized that tardigrade populations in examined with a dissecting microscope and the upper levels would have the same density, reflected light. Tardigrades were extracted diversity and distribution as the population using an Irwin loop (Schram and Davison found on the base level of the tree. It follows 2012). Tardigrades were mounted on slides that there should be no significant differences in PVA (Salmon 1951) and labeled with a between locations, substrates (tree species), or slide number and sample code. The extraction habitats (epiphytic moss or lichen) relative to process was performed three times per sample: tardigrade populations. twice by the original processor and once by 232 Chang, Powell, Miller and Lowman Figure 3. Lauren Chang using the double-rope climbing technique while collecting moss and lichen habitat at the Kansas State University, Konza Long Term Environmental Research (LTER) site near Manhattan, Kansas. a different researcher. Several days after the RESULTS tardigrades were mounted on slides, the edges of the slide covers were sealed with nail polish During the summer of 2014, a Research to reduce drying. Experiences for Undergraduates (REU) was conducted at Baker University in The density or tardigrades per sample was northeastern Kansas. One hundred thirty-five used as the measure of presence and habitat trees (substrates) of 17 different species were acceptability. Because for 250 years tardigrades climbed at nine different locations within a two- have been collected at the base of trees, the hour radius of Baldwin City. Tardigrade habitats hypotheses of uniformity was tested by the (moss and lichen) were collected at four comparison of the observed tardigrade density different levels at approximately three meters at upper levels to the expected density at apart. Eight hundred twenty-two samples were the base level. The Chi Square test for the taken (250 mosses and 572 lichens) (Table 1). difference between expected and observed Sample distribution by level was reasonably tardigrade density was used to judge significant uniform between 23% and 26% (Table 1). Eight differences. Significance was defined as a thousand sixty-four tardigrades were extracted. calculated X2 value greater than the critical value The two upper levels yielded 51% (419) of the of 3.84 for one degree of freedom or a P value samples but 62% (4,987) of the tardigrades vs. less than 0.05 (X2>3.85, 1df, p<0.05) (Zar 2010). 49% (403) of the samples and only 38% (3,077) of the tardigrades (Table 1). The two upper Transactions of the Kansas Academy of Science 118(3-4), 2015 233 Table 1. Stratification of tardigrades in the canopy. Showing balance of sampling, increasing num- bers of tardigrades at higher levels, and differences between habitats (moss and Lichen). levels (3 and 4) each exhibited significantly mulberry, exhibited at least one level with a (X2>3.85, 1df, p<0.05) more tardigrades per significantly lower species density per sample sample (11.9) when compared to the expected than expected (Table 3). On seven substrate number (6.4) found at the base level (Table 1). species, hackberry, shag bark hickory, honey locust, red oak, Virginia creeper, burr oak, and At eight of the nine collection sites, there was ponderosa pine, all three upper levels exhibited significantly greater tardigrade density in the significantly greater tardigrade density than canopy samples than the base samples (Table expected (Table 3). 2). But at one, the Black Jack Battlefield site, two miles east of the Baker campus, there was DISCUSSION no difference in tardigrade density indicated between the three upper levels and the base at This project aimed to clarify tardigrade canopy this location (Table 2). usage. Based on the data, tardigrades are present in the canopy in great numbers and variety. Seventeen substrates (tree species) were However, it is necessary to reject the hypothesis climbed and sampled. Fourteen substrate