Ecology and evolution in changing environments: Mechanisms to Responses

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2013 REU Student Projects and Abstracts

Steven Rosenzweig - SUNY Geneseo - Reestablishment of ecosystem services following restoration of tallgrass prairie. (Mentor: John Blair)

Steffanie Munguía - University of South Florida - Why do territorial male Grasshopper Sparrows aggregate? (Mentor: Alice Boyle)

Emily Pavlovic - Earlham College - The development of microRNA genes as a barcode to characterize nematode community composition. (Mentor: Michael Herman)

Nicole Richardson - University of Iowa- The Evolution of Multicellularity: Driven by Predation? (Mentor: Bradley J.S.C. Olson)

Elizabeth S. Mays - Colorado College - The role of sterols in cellulose synthesis: genetic and evolutionary approaches (Mentor: Janet M. Paper and Kathrin Schrick).

Bonnie Bernard - Centenary College of Louisiana - Cytotypic and cryptic morphological variation in and among Phlox hoodii populations of the Black Hills. (Mentors: Carolyn Ferguson and Mark Mayfield)

Casie Lee - University of California, Berkeley -The Thrill of Victory, the Agony of Defeat: Developing and Testing a Standard Protocol for Field Estimates of Short Term Growth in Fish Predators. (Mentor: Martha Mather)

Benjamin Ketter - Kansas State University - Natural abundance of stable isotopes as an indicator of hydraulic lift under field conditions. (Mentor: Kim O’Keefe and Jesse Nippert)

Liliana N. Calderon - University of Illinois - Effects of Fire Treatments and Slope Position on Macroinvertebrates in North American Tallgrass Prairie. (Mentor: Ellen Welti and Bruce A. Snyder)

Armand Cann -  Xavier University - Small mammal responses in relation to vegetation recovery in patch-burn grazing pastures.  (Mentors: Drew Ricketts and Brett Sandercock)

Alessandro Bartolo -  Hampshire College - Effects of Patch-Burn Grazing on Dickcissel Territory Size. (Mentors: Bram Verheijen and Brett Sandercock)

Jenny Lohmiller - Bethany Lutheran College - Vegetation architecture effects on the diversity and abundance of web-building spiders (Mentors: Jesús Gómez and Anthony Joern)

Project Abstracts - 2013

(listed in order of presentation)

Steven Rosenzweig - Reestablishment of ecosystem services following restoration of tallgrass prairie (Mentor: John Blair).

Conversion of tallgrass prairie to cropland has depleted soil carbon (C) and nitrogen (N) pools on a large spatial scale, resulting in widespread damage to environmental health, and the loss of many ecosystem services such as C sequestration, nutrient retention and maintenance of soil fertility. Restoration of perennial grassland species on formerly cultivated fields can re-establish critical soil processes and rebuild soil organic matter (SOM) pools, but the extent to which restored grasslands can reestablish ecosystem services is unknown, and predicted time scales for a successful restoration are highly variable. We examined changes in soil physical, biological, and chemical characteristics over a chronosequence consisting of grasslands restored for 1, 3, 7, 15, 26, and 35 years, in addition to a currently cultivated wheat field and unplowed, native prairie. Initial concentrations of total C and N in the agricultural soil were 45.5% and 48.9% of those in native prairie, and had increased to 68.8% and 70.5% in the 35-year restored field, with the greatest increase occurring between years 26 and 35. Soil bulk density was highest in the agricultural site and decreased with grassland age. Although we detected no statistically significant change in standing stock of soil C and N in the first 26 years of restoration, differences between the 35- and 26-year restorations indicate C and N accrual rates of 77.7 g Cm-2yr-1 and 3.1 g Nm-2yr-1 in the surface 10cm of soil. Microbial biomass C and N increased linearly over the chronosequence, and are expected to reach native prairie levels in the 53rd and 52nd year of restoration, respectively. Potentially mineralizable soil C and in situ measurements of soil CO2 efflux both increased linearly with time since restoration, and resembled native prairie by the 26th year of restoration. Available soil P and total inorganic N both decreased with time, reflecting increasing nutrient limitation in reestablishing grasslands. We conclude that the smaller, more labile SOM pools such as plant-available nutrients and microbial biomass C and N return to native prairie levels more quickly than the total C and N pools. This suggests that ecosystem services such as nutrient retention and nutrient cycle regulation can be reestablished within the first few decades of restoration, while C sequestration occurs on a longer time scale.

Steffanie Munguía - Why do territorial male Grasshopper Sparrows aggregate? (Mentor: Alice Boyle)

Aggregation of animals can occur due to spatial variation of resources or can function in multiple social contexts ranging from mate attraction to group defense.  In territorial species, group defense is often an important driver of aggregation.  One such territorial species is the Grasshopper Sparrow (Ammodramus savannarum), a small grassland songbird, in which preliminary observations suggested that the distribution of territories ranged from widely separated to highly aggregated.  The objectives of this study were to 1) document the spatial distribution of Grasshopper Sparrow territories on the Konza Prairie in northeast Kansas, 2) examine the relationship between habitat characteristics and sparrow densities, and 3) test nest predator and nest parasite defense hypotheses for the function of territorial aggregations.  We mapped territories on randomly-located 10 Ha plots in 18 watersheds differing in grazing and fire regimes from June 17 – July 8, 2013.  We examined how territory density varied as a function of landuse as an indirect test of the habitat quality hypothesis.  We then experimentally tested the group defense hypothesis by presenting models of a Black Rat Snake (nest predator), Brown-headed Cowbird (nest parasite), and House Sparrow (control) in paired aggregated and non-aggregated territories and assessing behavioral responses.  Grasshopper Sparrow territories on the Konza ranged from widely spaced to highly aggregated with nearest neighbor distance ranging from 12 – 178 m.  Densities of sparrows varied in response to grazing treatment but not fire treatment with watersheds grazed by cattle having higher densities of territories (0.4+ 0.1 territories/Ha) than those grazed by bison (0.2 + .1 territories/Ha) or not grazed (0.2 + .1 territories/Ha).  Behavioral assays of response times, time until nearest approach to mounts, and the nearest approach distance revealed no differences between aggregated and non-aggregated males.  We found no support for group defense hypotheses but some results were consistent with the habitat quality hypothesis.  Our findings suggest that spatial aggregation of territories may not function in the context of nest defense in grassland birds, and that habitat quality is likely a more important factor shaping the distribution of individuals on the landscape.  

Emily Pavlovic - The development of microRNA genes as a barcode to characterize nematode community composition (Mentor: Michael Herman).

Nematodes play important roles in soil ecosystems and respond to environmental perturbations through changes in community composition.  In order to understand the role nematodes play in nutrient cycling and their interactions with other microorganisms, we require accurate methods of enumeration.  Traditionally, this has involved sampling and isolation of individuals that were identified based on morphology.  Recently, amplicon sequencing of the 18S rRNA gene has been used as a replacement for morphological identification due to its challenging and time-consuming nature.  However, we recently found that certain nematode species contain variable numbers of copies of this gene, causing the results to differ greatly from those based on morphology.  In order to find a more viable method, we looked for genes that were present in a single copy, were encoded in the nuclear genome, were noncoding, and were highly conserved across nematode species.  MicroRNA (miRNA) genes fit these parameters.  The aim of our study was to develop a method to investigate the feasibility of using miRNA genes as a barcode for species identification through amplicon sequencing.  For this to be useful, we will need a sequence database of diagnostic nematode miRNA genes, as the presence of miRNA genes in current databases are limited to a few species.  Thus, we also wanted to begin development of such a miRNA gene database.  Using sequence databases, two miRNA genes, mir-1 and mir-236, were selected as the best candidates based on the degree of sequence conservation and potential for species discrimination.  We adapted our method from a genome walking technique that involves digesting genomic DNA with restriction enzymes, ligating adaptors, and using adaptor and gene specific primers to amplify the desired sequence.  Due to differences in the gene sequences, mir-236 produced better results than mir-1.  We hypothesize that the reason for this was that the Tm for the mir-1 primer is too low to be compatible with the adaptor primers.  Using the restriction enzyme TaqI we successfully amplified mir-236 from Caenorhabditis elegans and confirmed the results through sequencing.  Amplicon sequencing by genome walking of mir-236 was also attempted for four other nematode species: Oscheius tipulae, two Mesorhabditis sp., and Panagrellus sp.  We are currently analyzing these results and will report our findings.  Based upon our current data, it appears that this method shows promise, but further refinements still need to be made in order to make this a viable technique for use on complex environmental samples.

Nicole Richardson - The Evolution of Multicellularity: Driven by Predation? (Mentor: Bradley J.S.C. Olson)

The evolution of multicellular organisms is a major evolutionary transition, having happened at least twenty-five times in a wide array of taxa. However, our understanding of the genetic and ecological basis of this transition is largely unknown. To understand how multicellular organisms evolved, we use the Volvocine algae as the model system because they have organisms that with morphologies ranging from unicellular (e.g. Chlamydomonas) to colonial multicellular (e.g. Gonium), to organisms with differentiated tissues (e.g. Volvox). More importantly, the Volvocine algae have recently evolved multicellularity, about 200 million years ago, compared to >1 billion years ago in most other lineages. One hypothesis about how multicellularity evolved is predation selected for larger organisms that was accomplished by having multiple cells. Interestingly, the Olson lab has found that unicellular Chlamydomonas cells cooperate by forming aggregates in response to predation. This suggests that the genetic permanence of the predator response might be a mechanism by which multicellular organisms evolved. For this to be true, the Chlamydomonas predator response should provide a fitness benefit and thus we predict that Chlamydomonas cells would specifically aggregate with themselves in response to predation and not other species such as Gonium. To test this, we subjected Chlamydomonas, Gonium, and a mixture of the cells to predation and determined if aggregation occurs between similar cells only or if cells of different species will aggregate due to this environmental pressure. Our data shows that predator induced aggregates predominantly contain one species, and between species aggregate was rarely observed. Because the predator aggregative response is indeed species specific, our next step will be to determine its genetic basis and see how predator responsive genes have evolved in Chlamydomonas compared to Gonium.

Elizabeth S. Mays - The role of sterols in cellulose synthesis: genetic and evolutionary approaches (Mentors: Janet M. Paper, and Kathrin Schrick).

Sterols are essential to the health and growth of plants.  Derived forms of sterols, steryl glucosides (SGs), are hypothesized to be critical for the production of cellulose microfibrils. Mutations in genes that play a role in sterol and steryl glucoside production suggest the importance of sterols and their derivatives to plant growth. UDP-glucose:sterol glucosyltransferases (UGTs) are thought to produce SGs. Double mutants of ugt80A2,B1 show slowed growth rate and reduced steryl glucosides. The presence of residual SGs in ugt80A2,B1 mutants suggests that another enzyme compensates by creating SGs. GCS is known to form SGs in yeast, and a mutation in this enzyme causes seedling lethality. This study uses a genetic approach in Arabidopsis to investigate the function of of glucosylceramide synthase (GCS) in plants. The combined effects of mutations in UGT80A2,B1,C1 and GCS were analyzed. Out of 60 F2 progeny, 19 gcs heterozygotes were identified. Within this group, ugt80B1 mutants, ugt80B1,C1 heterozygotes, and a ugt80B1,C1 mutant were identified. Phenotypic analysis of mutants determined that the gcs homozygote phenotype was observed at a frequency less than expected. The survival of gcs homozygotes appears reduced in seedlings with multiple mutations. Quantification of the seedling phenotypes may indicate whether GCS and UGT80 mutants have a lower probability of embryonic or gametophytic survival.

A study of the evolution of the biosynthetic pathway of plant sterols may give insight into the mechanism of cellulose production. A marked advancement in the cell wall is seen beginning with Charophycean green algae, the closest clade of algae to land plants. There is currently a debate as to which branch within this clade is the closest sister lineage to land plants. To shed light on this question, we performed an evolutionary analysis of the 17 enzymes in the sterol biosynthetic pathway as well as GCS and UGT80 A2, B1 and C1. Homologs of Arabidopsis enzymes were found in the Charales, Chlorokybales, Coleochaetales, Klebsormidiales, Zygnematales and Mesostigmatales groups of Charophycean algae. Phylogenetic analysis supports the hypothesis that Zygnematales and Coleochaetales are two of the closest branches. The phylogeny of clades based on the sterol biosynthetic pathway matches the evolutionary phylogenies of algae, mosses and land plants established with a broader scope of predicted protein sequences. 

Bonnie Bernard - Cytotypic and cryptic morphological variation in and among Phlox hoodii populations of the Black Hills (Mentors: Carolyn Ferguson, and Mark Mayfield).

Phlox hoodii, which ranges from the Rocky Mountains and High Plains to the Intermountain West to the Arctic Steppe, exhibits extensive ecotypic variation that makes taxonomic description quite challenging. Our investigation specifically addresses P. hoodii populations of the Black Hills, where the high plains and open pine woodlands often serve as an eastern outpost for Rocky Mountain flora. In addition to its unique ecology, the Black Hills region is of particular interest because P. hoodii populations here exhibit notable morphological variation that warrants intensive, population-level study. Furthermore, preliminary evidence from flow cytometry data indicates that genome size varies among these populations, suggesting that some populations are diploid while others are polyploid. This morphological and cytotypic variation leads us to two questions: (1) Can we recognize distinct, consistent morphologies within Phlox hoodii in the Black Hills? And (2) if so, do these different morphological entities correspond to particular cytotypes? We collected material from 15 localities within Buffalo Gap National Grassland and Black Hills National Forest. From this material, we assessed 19 floral and vegetative characters from preserved flowers and herbarium specimens, respectively, and used Principal Coordinates Analysis to investigate associated suites of character traits. DNA ploidy level was inferred via flow cytometry, and, to calibrate these genome size estimates to chromosome number, we obtained chromosome counts via the pollen mother cell squash technique. Morphological variation among the sampled populations was largely partitioned into two groups: a smaller, more glabrous entity corresponding to diploid ploidy level; and a larger, more pubescent entity inferred to be hexaploid. Only one population was inferred to be tetraploid, and its morphology corresponded to that of the diploid populations. Interestingly, the morphology of these inferred hexaploid plants is consistent with material from the Black Hills that monographer E. T. Wherry recognized as a distinct taxon, P. diffusa ssp. scleranthifolia; however, few subsequent workers have recognized this subspecies of P. diffusa, and phylogenetic study in our lab shows that these populations are not closely related to P. diffusa but instead represent variation within P. hoodii. Further, our hexaploid entity may be affiliated with P. hoodii ssp. canescens, which exhibits similar morphology but has traditionally been recognized only in more western portions of the species’ range. Ultimately, a broader sampling of diploid and polyploid morphologies across the distribution of P. hoodii will inform a revised taxonomy of the P. hoodii polyploid complex.

Casie Lee - The Thrill of Victory, the Agony of Defeat: Developing and Testing a Standard Protocol for Field Estimates of Short Term Growth in Fish Predators (Mentor: Martha Mather).

In aquatic ecosystems, motile predators have the capacity to increase their growth rate and size by moving to areas with favorable conditions and avoiding those with adverse conditions. Understanding field estimates of short-term growth (days to weeks) can offer insight into organismal condition in relation to movement, other behaviors, and habitat variables such as temperature, physical structure, and availability of food. However, field-based estimates of short-term growth are limited. Diets offer hour-scale estimates of organismal nutrition but are highly variable. Stable isotope analysis can provide long-term approximations (months to years) of nutritional status but is not sensitive to temporal patterns. Alternatively, the ratio of RNA-DNA (RNA:DNA) may reveal an intermediate window of growth. In our study, we asked (1) if a standard protocol for RNA:DNA analysis existed in the literature for field-caught fish, (2) if this protocol yielded consistent results, and (3) how differences in sampling and analysis procedures contribute to sources of variability. We first searched the literature and compiled research papers on RNA:DNA analysis methods for both field-sampled fish and fish raised in controlled laboratory settings. From the literature, we summarized a standard protocol that reflected methods currently in use to evaluate short term growth of field-caught fish. This protocol was then tested on a set of hatchery-reared channel catfish, Ictalurus punctatus (170-300 mm TL). Subsequently, several sources of variability in sample collection, preservation, tissue processing, and nucleic acid analysis were examined. Based on trials of 332 fish, we obtained an improved, tested, and validated protocol that can be used to examine predator growth in Kansas reservoirs. However, although potentially very useful, the sensitivity of the analysis to environmental and methodological circumstance suggests that this tool be used and interpreted with caution.

Liliana N. Calderon - Effects of Fire Treatments and Slope Position on Macroinvertebrates in North American Tallgrass Prairie (Mentors: Ellen Welti, and Bruce A. Snyder).

The reduction of active management, such as prescribed fire, may have detrimental effects on the native biodiversity of non-insect macroinvertebrates of North American tallgrass praires. The reduction of active burning of the prairie can be described as a disturbance to this habitat. Such disturbances can allow for the establishment of non-native species which are known to displace native species. Habitat loss and invasive species are the two leading causes for the global biodiversity loss. In this study conducted on the Konza Prairie Biological Station located in North Eastern Kansas, macroinvertebrate biodiversity was recorded in order to measure how it varied with slope position (low, mid, high) and fire frequency (1, 4, and 20 year fire return intervals). We also measured the impacts of fire frequency on the native macroinvertebrate community. If the majority of the macroinvertebrates being surveyed are native to the prairie and also prefer moist soil, then the most taxonomic richness will be found at 1 year burns at the low slope position. The assumption is that more natives will be found at the 1 year burn site because this site best represents historic prairie-like conditions that native species have adapted to. Earthworms, millipedes, centipedes, isopods, opilionids, solpugids, and scorpions were surveyed by conducting timed hand searches and by digging soil monoliths (30x30x10 cm) at the three fire frequencies and slope positions. Bee bowls were utilized for the collection of bees. Contrary to what was predicted, taxonomic richness was observed to increase as fire frequency decreased. Taxonomic richness increased as slope position decreased as was predicted. No interaction between fire and slope position in regards to biodiversity was observed. Non-native species were only recorded at the 20 year burn site. These results suggest that fire frequency and slope position, as independent factors, impact macroinvertebrate biodiversity; fire frequency most likely impacts the presence or absence of non-native organisms.

Armand Cann - Small mammal responses in relation to vegetation recovery in patch-burn grazing pastures (Mentors: Drew Ricketts and Brett Sandercock).

Fire and ungulate grazing are two important disturbances that maintain tallgrass prairie.  Historically the interaction of fire and grazing by bison promoted a heterogeneous environment – a mosaic of different vegetative communities across the landscape.  However, agricultural production have replaced bison with cattle and changed the frequency of prescribed fire in the grassland ecosystem.  Annually burned pastures have become a common rangeland management practice in the tallgrass prairie.  Intensive fire and grazing have a negative effect on the biodiversity in grasslands – turning a once diverse community into a homogeneous environment.  For the last four years at Konza Prairie Biological Station, patch-burn grazing management has been implemented across three experimental units.  This alternative management promotes a changing vegetation structure across the pasture by implementing incremental burns on patches, rather than the entire pasture.  The goal of the patch-burn grazing model is to create a mosaic community comparable to historic rangeland management.  A mosaic community is hypothesized to facilitate a favorable environment for multiple plant species instead of the predominant grasses favored by the traditional treatment.  Our study evaluated small mammal species abundance and richness within the patch-burn grazing watersheds and two controls as they may fluctuate due to the vegetation responses across treatments.  These data were collected from July 2011 to June 2013.  We collected monthly small mammal samples by live trapping in 2 grids per watershed within 3 treatments:  annually burned and grazed, patch-burn grazed, and 4-year burned and not grazed.   To observe percent vegetation cover data was collected at 5 points along a series of 8 transects in each watershed.  Additionally, pH was measured in all watersheds to determine if differences in soil acidity affected vegetation cover – no significant difference was found.  However, significant differences among habitat variables were detected between treatments over the two-year time period.  Furthermore, small mammal species richness was significantly higher within the patch-burn grazing treatment (9 species), in comparison to the annual-burn and grazed (4.5 species).  Furthermore, a regression of vegetation cover and species abundances provides evidence for our hypothesis that differences in small mammal species richness are due to vegetative responses to the patch-burn grazing model.  However, more extensive study is required in order to further understand the mechanism behind vegetation recovery in the patch-burn model.

Alessandro Bartolo - Effects of Patch-Burn Grazing on Dickcissel Territory Size (Mentors: Bram Verheijen, and Brett Sandercock).

Following larger trends of avian decline, populations of many grassland songbirds in the United States have decreased significantly over the past 50 years. Several factors have been implicated in these declines, one of which is habitat loss on breeding grounds due to agricultural intensification. Modern methods of land management are thought to reduce the quality and heterogeneity of breeding habitat through annual burning and persistent grazing. In contrast, patch-burn grazing seeks to increase heterogeneity by reducing grazing intensity and increasing the time between burns. Because of their complex phenology and ingrained responses to environmental stimuli, songbirds in grassland habitats are vulnerable to rapid environmental changes. In particular, changes at breeding grounds may affect the ability of male birds to secure high quality territories. High quality territories are especially important for Dickcissels (Spiza americana) as courtship, mating, foraging, and nesting all occur within an established territory. The purpose of our study was to investigate how Dickcissel territory size changed in relation to patch-burn grazing and modern land management techniques. Male Dickcissels were mist netted and banded with a unique combination of colored leg bands. Territories of banded males were then mapped using a “flush mapping” technique on five Konza watersheds representing modern and patch-burn grazing land management regimes. Behaviors such as calling, foraging, alarming, and aggression toward other males were used to define territory boundaries. Perch heights were measured and identified to investigate perch affinity. Preliminary results indicate no significant difference in territory size among the five treatments, though potential correlations with elevation, time of season, breeding status, and proximity to roads are being investigated. Moreover, our data may suggest improvements to traditional flush mapping techniques.

Jenny Lohmiller - Vegetation architecture effects on the diversity and abundance of web-building spiders (Mentors: Jesús Gómez, and Anthony Joern).

Spiders are ubiquitous arthropod predators in grassland ecosystems. This study examined the effect of vegetation architecture on the abundance and diversity of web-building spiders at Konza Prairie. We hypothesized that plant architecture diversity and abundance is an important factor driving the abundance and diversity of web-building spiders at the landscape scale. Konza Prairie is subject to a variety of different burn frequencies and grazing treatments resulting in a mosaic of habitat types that differ in the vegetation architecture for webs to be constructed upon. Transects were set up in watersheds with 1, 4, and 20-year burn frequencies in both the ungrazed and bison-grazed areas. Each of the selected watersheds was burned within the past two years, but the vegetation architecture present reflected the burn history of the watershed. We censused four 100-meter transects that were set up in each ungrazed watershed, and two 100-meter transects were set up in each bison-grazed watershed. We recorded the orientation, height, diameter, and type of each web, the distance along the transects, and the presence or absence of the spider. Results show that spider abundance was greatest in the twenty-year burn watershed where abundance of structure for web placement and vegetation architectural complexity was higher. Spider abundance was lowest in the one-year burn watershed where the vegetation architecture was low. The bison-grazed watersheds showed greater abundance and diversity of web-building spiders in relation to their corresponding ungrazed watersheds. In the ungrazed watersheds, we tested experimentally the hypothesis that additional web-building spiders would inhabit an area where the habitat structure availability for webs was increased. The habitat was manipulated by adding ten bunches of dead dogwood spaced evenly down a quarter of each transect. Our results show that in the one-year burn watershed, spider abundance increased by 600% in the manipulated sections of the transects. The twenty-year burn watershed did not show a significant difference in web-building spider abundance before or after the habitat manipulation. This may be due to the already high abundance of structures for web placement making our manipulation effects week.

Division of Biology