!"#$%"$'(")*+$"+,'("+$($%-'#.%/0(%+(12+/'-'#$'3(42./"(5%," ( ( 6/"+$'#( 7 ( Comparing tardigrade abundance and diversity on rock, log, live tree, and canopy in Monteverde, Costa Rica Lily Stander Department of Environmental Studies University of California, Santa Barbara EAP Tropical Biology and Conservation Program Fall 2016 16 December 2016 ABSTRACT This study surveyed the abundance and diversity of Phylum Tardigrada a cross four different substrates: rock, log, live tree, and canopy, in Monteverde, Costa Rica. Tardigrades are understudied micro invertebrates that inhabit every bio me on the planet. I collected t wenty eight samples or 140 subsamples, by hand, soaked them in water for a minimum of four hours, and surveyed for tardigrades. I found and documented forty five tardigrades Ninety one percent of tardigrades fou nd were Class Eutardigrada, while the remaining nine percent were Class Heterotardigrada. Five genera were identified: Echiniscus, Pseudechiniscus, Macrobiotus, Paramacrobiotus, and Minibiotus The canopy and live tree substrates produced equal abundance and diversity levels, each with 18 Eutardigrades and two Heterotardigrades. The log substrate had three tardigrades and th e rock substrate had only two, all Eutardigrada. Although the canopy an d live tree substrates yielded the same results, tardigrades found in t he canopy were more consistently found in all seven of the canopy samples. The tardigrades found on the live tree substrate, however, were pr imarily found all in one samp le. This may indicate that tardigrade abundance is consis tently higher in the canopy, but has a more patchy, random distribution in live trees. In conclusion, tardigrades are abundant and diverse in the canopy of the cloud forest in Monteverde, Costa Rica. Abundanc ia y diversidad de tardÂ’grados (Filo Tardigrada) en cuatro sustratos del bosque nuboso en Monteverde, Costa Rica RESUMEN El presente estudio estimÂ— la abundancia y diversidad del Filo Tardigrada en cuatro sustratos: roca, tronco muerto, base de Â‡rbol vivo y dosel, en el bosque nuboso de Monteverde, Costa Rica. Los tardÂ’grados son un grupo de micro invertebrados que habitan en todos los bromas del planeta. ColectÂŽ 28 muestras a mano (siete por sustrato), las cuales fueron sumergidas en agua por un mÂ’nim o de cuatro horas. Posteriormente dividÂ’ cada muestra en cinco sub muestras, y contÂŽ el nÂœmero de tradÂ’grados presentes en la muestra. ContÂŽ un total de 45 individuos, de los cuales un 91% pertenecen a la Clase Eutardigrada y un 9% a la Clase Heterotardigr ada. Se encontraron cinco gÂŽneros: Echiniscus Pseudechiniscus, Macrobiotus, Paramacrobiotus, and Minibiotus. Los sustratos con un mayor nÂœmero de tardÂ’grados fueron base de Â‡rbol vivo y dosel, ambos con 18 individuos de la Clase Eutardigrada y
!"#$%"$'(")*+$"+,'("+$($%-'#.%/0(%+(12+/'-'#$'3(42./"(5%," ( ( 6/"+$'#( 8 ( dos individ uos de la Clase Heterotardigrada. Los sustratos de roca y tronco muerto presentaron Âœnicamente individuos de la Clase Eutardigrada, con dos y tres individuos respectivamente. A pesar de que los resultados del dosel y base de Â‡rbol vivo fueron los mismos, e n el dosel cada muestra presentÂ— individuos, mientras que en Â‡rbol vivo solamente dos. Esto parece indicar que la abundancia de tardÂ’grados es constantemente mayor, mientras que en la base le los Â‡rboles presenta una distribuciÂ—n en parches. En conclusiÂ—n encontrÂŽ que los tardÂ’grados en Monteverde son mÂ‡s diversos y abundantes en el dosel del bosque nuboso en Monteverde, Costa Rica. Tardigrades are microscopic, aquatic animals more commonly referred to as "water bears" or "moss piglets". These bilaterally symmetrical micro invertebrates have five body segments; one cephalic segment and four trunk segments, all somewhat indistinct (Miller 2004). Adults range from 0.2 0.5 mm long and have four pairs of lobopodous legs terminating in claws or sucking disks (Northcote Smith 2012). These tiny organisms have a surprisingly complex anatomy and physiology. Like larger animals, they have a full aliment ary canal and digestive system, a dorsal brain atop a paired ventral nervous system, well developed muscles, a nd separate sexes (Miller 2011). Unlike larger animals, however, they lack respiratory and circulatory systems, and instead use open hemocoel in their body cavity to circulate nutrient s and gas to every cell (Miller 1997). There is relatively little information known about the invertebrate phylum Tardigrada, although they inhabit every biotope on th e planet from the Arctic tundra to the driest of deserts to the high h umidity of rain forests (Miller 1997). Although tardigrades are found everywhere, they are strictly aquatic. The tardigrade species that do not inhabit marine or freshwater environments are limno terrestrial. Limno terrestrial is defined as being or inhabiting a moist terrestrial environment that is subject to immersion and desiccation, as in leaf litter or between moss cushions. In this thin film of water, there is a diverse micro world where tardigrades eat bacteri a, alga e, nem atodes, rotifers, as well as o ther tardigrades (Miller 2004). Tardigrades are best known for how the y have adapted to surviving extreme environmental changes. Limno terrestrial species have three basic sta tes of being: an active state where they eat, grow, and reproduce an anoxybiotic state in response to lack of oxygen, and a cryptobiotic state in response to desiccation (Miller 2011). Terrestrial tardigrades can be found in bryophyte cushions, lichens, bark, leaf litter, and soil, all of which are quite susceptible to desiccation (Northcote Smith 2012). According to William R. Miller (2011) during cr yptobiosis tardigrades lose up to 97% of their body moisture and shrivel up into a structure one third of its original size called a tun In this st ate tardigrades can survive just about anything. F our substrates consisting of rock, fallen log, living tree, and canopy were chosen for their accessibility and because they are often home to moss and lichen. One study done by Sayre and Brunson (1971) determined relative frequency of tardigrades on bryophytes and concluded that 61% were found on trees and 12.5% were found on rock.
!"#$%"$'(")*+$"+,'("+$($%-'#.%/0(%+(12+/'-'#$'3(42./"(5%," ( ( 6/"+$'#( 9 ( Other literature concludes that tardigrades do not have any preference for substrate, but a preference for moisture level of the bryophyte. Glime (2013) speculates that this moisture relationship might explain why mosses on rotten logs seem to have few tar digrades, assuming that they exceed preferred moisture levels. A study done by Miller in 2004 confirms a high abundance of tardigrades in the canopy. Despite the fact th at Costa Rica is known for its biodiversity and scientific research, there have b een very few studies done here about Phylum Tardigrada. I intend to study the abundance and diversity of tardigrades here in Monteverde on four different substrates: rock, fallen log, living tree, and canopy. METHODS AND MATERIALS My study site was th e cloud forest surrounding the EstaciÂ—n BiolÂ—gica in Monteverde, Costa Rica, located at the highest point of the TilarÂ‡n mountain range, between 1400 m and 1800 m above sea level I collected t wenty eight mo ss and lichen samples by hand from four substrates: rock, fallen log, live tree at breast height and canopy branches. I collected between the dates of 21 November 2016 through 2 December 2016. I defined one sample as a handful of moss, approximately ten grams I obtained c anopy samples from freshly fallen branches that fell from the top layer of trees I placed samples in plastic bags where I recorded the substrate type, area, and date of collection. Samples were then placed in mason jars and immersed in water for a minimu m of four hours (Miller 1997) Five subsamples of about two millilit ers each were extracted from each mason jar and placed in petri dishes with blackened bottoms. Each subsample was thoroughly searched using a dissecting microscope at 30x magnification w ith an LED light at a forty five degree angle to t he petri dish. I extracted tardigrades with a thin glass pipette and placed them on a slide to be observed and identified under a compound microscope. I took pictures and videos of each tardigrade. Some pictures were sent to a tardigrade taxonomic specialist, William R. Miller, for genera and specie level identification. RESULTS A total of 45 individual tardigrades were fou nd out of 140 subsamples The rock substrate had the lowest number of water bea rs yielding only five percent of t otal tardigrades found Only two tardigrades of Class Eutardigrada were found from the rock samples ( Fig 1 ) The log sam ples produced three total tardi gr ades seven percent of the total, all Class Eutardigrada (Fig 1 ) The live tree and canopy samples surprisingly produced the exact same results each with 44% of the total number of tardigrades found Both had 20 tardigrades, 18 of which were Eutardigrada, two of which were Heterotardigrada (Fig 1 ) Therefore, of the 45 total tardigrades, 41 were Eutardigrada and only four we re Heterotardigrada. Five genera were identified. The two Heterotardigrada genera identified were Echiniscus and Pseu dechiniscus (Table 1, Fig. 3 ) Macrobiotus, Para macrobiotus, and Minibiotu s were the three Eutartigrada genera described (Table 1, Fig 2 ) All five genera were found in bo th canopy and live tree
!"#$%"$'(")*+$"+,'("+$($%-'#.%/0(%+(12+/'-'#$'3(42./"(5%," ( ( 6/"+$'#( : ( samples, while only Macrobiotus and Paramacrobiotus were found on rock and log substrates. Figure 1 Tardigrade abundance found in 28 samples across four different substrates. Also compares tardigrade diversity between two classes: Eutardigrada and Heterotardigrada (
!"#$%"$'(")*+$"+,'("+$($%-'#.%/0(%+(12+/'-'#$'3(42./"(5%," ( ( 6/"+$'#( ; ( Figure 2 : Eutardigrades found during N ovember 2016 in the Monteverde cloud f orest A.) Minibiotus sp. B.) Macrobiotus sp. C.) Paramacrobiotus sp. D.) Macrobiotus hufelandi E.) Paramacrobiotus sp. F.) Macrobiotus sp. G.) Macrobiotus sp. H.) Paramacrobiotus sp. I.) Paramacrobiotus sp. < ( = ( ( ( # $ % ( & ( ( ( (
!"#$%"$'(")*+$"+,'("+$($%-'#.%/0(%+(12+/'-'#$'3(42./"(5%," ( ( 6/"+$'#( > ( Table 1 Diversity of tardigrade genera found in all 28 samples. Only 13 tardigrades found could be identified to genera level. !"#"$% & '()*"$&+,"#-./.", & !"#"$"%#&'(&)*+ -(.('(&)*+ # /%0(.(+%*+ $ -"%#&'(&)*+ !+1*21%0(.(+%*+ # DISCUSSION It is evident that tardigrade abundance was remarkably higher on live tree and canopy substrates compar ed to rock and fallen log substrates. Although there is no published literature directly supporting these findings, there have been studies on tardigrade ecology that may offer some insight as to why tardigrades were much more abundant in the canopy and on live trees According to a study done by Chang and collaborators in 2015, there was a s tatistically significant increase in tardigrade density with incr eased height ascending into the canopy Another study done by Miller in 2004 speculates that tardigrades are picked up by wind in their cryptobiotic tun and dispersed across the world, settling wherever wind runs out, often over a forest. These two studi es support the high abundance of the canopy results but provide no insight to the live tree samples' equal abundance and dive rsity. Extrapolating off of the wind dispersal $)*+,-" / ?'/'#2/"#$%"$'.(@2*+$(%+(A2-'B)'#(8C7>(%+(/D'(12+/'-'#$'(,E2*$(@2#'./F(
!"#$%"$'(")*+$"+,'("+$($%-'#.%/0(%+(12+/'-'#$'3(42./"(5%," ( ( 6/"+$'#( H ( hypothesis, it is possible that tardigrades fall and descend from the canopy into l ower strata of the trees presumably living trees However, it is also possible that the relative abundance of tardigrades was not correlated to the type of substrate it was fou nd on, but the individual substrate it was taken from. For example, of the 20 tardigrades found on live trees (Fig. 1 ) 16 came from sample A20. The l ack of equal distribution across all live tree samples could indicate that tardigrades are not necessarily abundant on all live trees, but that that sample was rich in tardigrades by chance In contrast, the canopy samples more consistently contained tardigrades; five out of the seven samples contained at least one tardigrade, and two samples c ontained seven or more. The live tree sample's pattern is supported by a study done by M eyer ( 2006 ) that concludes that tardigrades have a very patchy distribution while the canopy sample's pattern is supported by Miller's 2004 study that corroborates the high abundance of water bears in the canopy. An extensive study by Kaczmarek (2011) on the ecological factors determining tardigrade distribution in Costa Rica discovered that, of all analyzed factors, substrate had the weakest influe nce on tardigrade distribution. Although comprehensive, this study did not investigate local differences in altitude thereby excluding the canopy. Therefore, it can be said that the results of my study reflect a conglomerate of the conclusions reached by aforementioned tardigrade literature. The high abundance and diversity of the canopy is substantiated b y Chang and collaborators (2015) and Miller (2004), while the high abundance and diversity of the live tree samples can be considered somewhat of a fluke, or due to high variation and patchy distribution, discussed in Meyer (2006). In addition, the low ab undance and diversities of the rock and log substrates can be explained by the conclusions reached in Kaczmarek (2011) as well as Meyer (2006) that state tardigrade distribution does not rely on substrate type, and that their distribution is patchy, respec tively. Altho ugh tardigrades are ubiquitous around the world there are some places where they appear to be more prevalent. The canopy of the cloud forest in Monteverde, Costa Rica is abundant and diverse in Phylum Tardigrada. Tardigrades do also inha bit bryophyte cushions on other substrates in the cloud forest, but their distribution is uneven and inconsistent. O ne thing is certain however : there is much more work to be done in understanding Phylum Tardigrada. Despite their overall abundance and worldwide distribution, little is known regarding their ecological requirements and the phylum's contribution to the biodiversity of the world. For example, e ighty percent of described species are Eu tardigrades (Miller, 1997) Is this percentage reflective of the phylum as a whole, or just a result of studies being concentrated in terrestrial mosses and lichens? What is the role of tardigrades in the micro ecosystems they inhabit? How do interspeci fic interactions affect their daily lives? Further research options are endless and it is important that we continue to explore the world of these tiny water bears.
!"#$%"$'(")*+$"+,'("+$($%-'#.%/0(%+(12+/'-'#$'3(42./"(5%," ( ( 6/"+$'#( I ( ACKNOWLEDGEMENTS I would like to thank the EstaciÂ—n BiolÂ—gica Monteverde Costa Rica for graciously allowing me to use their facilities as well as granting me access to collect samples from their surrounding forest. I would also like to thank my advisors Andres Camacho and SofÂ’a Arce for their unwavering support and encouragement in this whole process. I cannot forget to give a shoutout to the tiny squad, Jenna and Taryn for keeping me sane through endless hours of looking at tiny things in the tiny lab. Finally, I would like to profess my eternal gratitude towards William R. Mille r, tardigod, for patiently answering my page long emails helping me with the taxonomic identification process and guiding my methodology techniques LITERATURE CITED Chang L., Powell, D., Miller, W.R., and Lowman, M. (2015, September). Tardigrades of the canopy: Evidence of stratification. Transactions of the Kansas Academy of Science, 118 (3&4), 230 236. doi: 10.1660/062.118.0306 Glime, J. M. ( 2013 ) Tardigrades: Species Relationships. Bryophyte Ecology 2 (4 5) Bryological Interaction Hofmann, I., & Eichelberg, D. (1987). Ecological investigations of the habitat preference of moss inhabiting tardigrades. Zoologische Beitraege, 31 (1), 61 76. Retrieved from http://search.proquest.com/docview/15016902?accountid=14522 Kaczmarek, L., GoAdyn, B., WeAnicz, W., & Michalczyk L. (2011). Ecological factors determining tardigrada distribution in costa rica. Journal of Zoological Systematics and Evolutionary Research, 49 78 83. doi:http://dx.doi.org/10.1111/j.1439 0469.2010.00603.x Meyer, H. A. (2006). Small scale spatial distribution variability in terrestrial tardigrade populations. Hydrobiologia, 558 (1), 133 139. doi:http://dx.doi.org/10.1007/s10750 005 1412 x Miller, W. R. (1997) Tardigrades: Bears of the Moss. The Kansas School Naturalist. 43 (3). Retrieved from http://www.emporia.edu/ksn/v43n3 may1997/ Miller, W. R. (2004 ). Tardigrades: Bears of the Canopy. Forest Canopies, 251 258. doi:10.1016/b978 012457553 0/50018 6 Miller, W. R. (2011 ). Tardigrades: These abling, eight legged microscopic "bears of the moss" are cute, ubiquitous, all but indesdructible, and a model organism for education. American Scientist, 384. Northcote Smith, E. (2012). The ecology of tardigrades. The Plymouth Student Scientist,
!"#$%"$'(")*+$"+,'("+$($%-'#.%/0(%+(12+/'-'#$'3(42./"(5%," ( ( 6/"+$'#( J ( 2012, 5, (2), 569 580 Sayre, R.M. and Brunson, L.K. (1971). Microfauna of moss habitats. American Biology Teacher 100 102, 105