Abstract:

Riparian corridors maintain regional biodiversity where they occur. Within the last century the floristic composition of riparian communities in the Southwest has drastically changed following introduction of the exotic tree Tamarix spp. In an attempt to control Tamarix spp. populations the Tamarisk leaf beetle (Diorhabda spp.) has been utilized as a biological control agent. Multiple years of data collection at our study sites along Fountain Creek (Fountain, CO) allowed us to characterize the response of Tamarix spp. to invasion by the biological control agent during the summer of 2013. In analyzing data collected before, during, and after the beetle invasion we saw a significant effect of foliar herbivory on Tamarix spp. physiology and survival strategy. During the beetle invasion Tamarix spp. experienced increased stomatal conductance (low abscisic acid sensitivity), and diminished foliar chlorophyll content and flower production. Phenotypic selection analysis on functional traits changed before, during, and after the beetle invasion. Before the invasion successful individuals exhibited high stomatal conductance and wasted water. During and after the invasion, selection was detected for increased chlorophyll content. These results suggest that Tamarix spp. experienced decreased photosynthetic potential during the invasion, and required high stomatal conductance to facilitate gas exchange for photosynthesis. It appears that Tamarix spp. responds to defoliation by increasing water use in an attempt to sustain photosynthate allocation to reproductive structures and below ground biomass. These results have implications for management strategies. As a facultative phreatophyte Tamarix spp. has garnered attention due to its prolific water use in areas of low water accessibility. The leaf beetle may increase the tendency of Tamarix to waste water during the multiple growing seasons needed for defoliation to be an effective management technique, which could have drastic effects on both shallow and deep groundwater reserves and water availability for native species.

Introduction:

Species of the nonnative genus Tamarix L. (Tamaricales: Tamaricaceae; hereafter

Tamarix) are involved in one of the most serious invasions by noxious weeds within the

United States (Robinson 1965; Stein and Flack 1996; DeLoach et al. 2000). Tamarix is a small tree, or shrub from Eurasia that aggressively invades riparian ecosystems. Tamarix can completely exclude native species from riparian habitats by forming monocultures of dense stands with high leaf area (Sala et al. 1996). The implications of Tamarix competition with native species for water and light are widespread. Consequently, riparian community structure and fluvial ecosystem processes are transformed following

Tamarix introduction and establishment.

Tamarix invasion is aided by both biotic and abiotic factors. The invasive nature of Tamarix has particularly benefited from anthropogenic disturbance to riparian ecosystems (i.e. dam construction and flow regulation). Varying management techniques have been utilized in an attempt to control and manage Tamarix, with the hope of restoring riparian ecosystems and salvaging water lost through evapotranspiration. This paper will focus on the impact of the biological control agent, the Tamarisk leaf beetle,

Diorhabda spp. (Coleoptera: Chrysomelidae), on Tamarix functional traits and reproductive fitness.

Tamarix introduction and naturalization:

Horticulturists performed the first introductions of Tamarix during the early 19th century from sources in Europe, Asia, and North Africa (Horton 1964; Gaskin and Shaal, 2002;

Gaskin and Kazmer 2006). Since the early 1820’s, up to 12 species from the genus

Tamarix have been introduced into North America (Baum 1967; Crins 1989). Qualities such as ease of propagation and the ability of Tamarix to survive in saline and xeric soils were deemed beneficial, and dissemination of this information led ranchers, farmers, and range managers to plant Tamarix for erosion control, as a windbreak, and shade tree.

During the mid-1800’s Tamarix was planted by the Army Corps of Engineers along streams as a bank stabilizer (Bean et al. 2013). By the early 1900’s populations became established in the Colorado Plateau region (Graf 1978). Certain species (Tamarix ramosissima Ledeb., T. chinensis Lour. and their hybrids) have exhibited the ability to successfully invade western riparian areas (Gaskin and Schaal 2002; Gaskin and Kazmer

2006).

Tamarix escaped cultivation in the 1930’s and has since invaded vast swaths of land throughout the western United States (Robinson 1965). Tamarix has invaded riverine ecosystems of the lower Colorado River, resulting in a displacement of up to 90% of the riparian communities historically dominated by species in the genera Salix and Populus

(Malpighiales: Salicaceae) (Crines et al. 1989). Tamarix has become naturalized along many waterways throughout the western U.S., and established populations can be found from the border with Mexico north to Minnesota, and from Kentucky west to California,

Idaho, and Oregon (tamariskmap.org). During the era of dam building on western U.S. waterways (ca. 1940-1960), novel habitats were generated along riverbanks and reservoir margins, which facilitated a period of rapid Tamarix expansion (Robinson 1965).

Tamarix invasibility:

Abiotic factors that promote the invasion of Tamarix, such as the cumulative impact of river diversions, irrigation projects, dam construction, and flow regulations on riparian ecosystems, are mostly of anthropogenic origin (Sala et al. 1996). Dams disrupt natural flood regimes and alter fluvial processes (i.e. stream bank erosion and sediment deposition), which are integral to the establishment of many native riparian species

(Fenner et al. 1985; Stromberg et al. 1991, Auble et al. 1994). Salts leach into waterways from surrounding agricultural fields, and solutes become concentrated in reservoirs with low flows and high evaporation rates (Lee and Bell 1999; Havel et al. 2005).

Consequently, waterways downstream of dams experience higher salinity levels, which aid in the establishment of salt tolerant species (e.g. Tamarix) and inhibit the establishment of salt sensitive species (Hagan and Roberts 1973; Ohmart et al. 1988;

Siegle and Brock 1990; Busch and Smith 1995, Shafroth et al. 1995, Lovell et al. 2009).

Moreover, riparian species that are sensitive to water availability are negatively impacted by reduced water table height from river diversions and flow regulations (Smith et al.

1991; Stromberg et al. 1991).

Tamarix struggles to invade riparian ecosystems that experience natural or naturalized flood regimes (Tyson 2010). Overbank flooding produces crucial fluvial disturbances that flush salts from floodplains and inundate sandbars (Nagler et al. 2011). The spring snowmelt regime is more pronounced on waterways without flow regulations. Native species (e.g. Populus and Salix) have evolved to coordinate flowering phenology and seed dispersal with these flooding events (Karrenberg et al. 2002). Tamarix flowering phenology lags slightly behind Populus and Salix (Shafroth et al. 1998). Therefore,

Populus and Salix seedlings are able to establish earlier in the growing season relative to

Tamarix. Rapid stem elongation results in shading of Tamarix seedlings and consequentially higher Tamarix mortality rates at viable germination sites (Tomanack and Zeigler 1962; Brock 1984; Shafroth et al. 1998). Tamarix establishment is greater along waterways that do not have naturalized flood regimes.

Certain key biological characteristics have contributed to Tamarix invasability.

Tamarix disperses small, light, comose seeds in vast quantities (Merkel and Hopkins

1957; Warren and Tuner 1975; Sisneros 1991). Flower production and seed dispersal is abundant and continuous throughout the growing season (Brock 1984; Tomanack and

Zeigler 1962). Earlier snowmelt could shift the important spring flood events that provide

Salix and Populus a competitive advantage over Tamarix with respect to flowering phenology (Hersch and Reiberg 2012; Shafroth et al. 1998). Continuous seed dispersal results in complete colonization of viable germination sites by Tamarix seedlings, and shading during the subsequent growing season inhibits local establishment by native species. Seeds produced by Tamarix are able to germinate in soils with high salinity levels (Brotherson and Winkle 1986; Shafroth et al. 1995). Seedlings are able to tolerate xeric and inundated soils for finite periods of time (Sala et al. 1996). Early growth strategy results in resource allocation to belowground biomass accumulation, and over time the development of shallow and deep roots structures augments the capacity of

Tamarix to operate as a facultative phreatophyte (Busch et al. 1992).

As a facultative phreatophyte, Tamarix has the ability to access the water table as well as shallow ground water reserves (Tomaso 1998). Tamarix successfully invades riparian ecosystems in the U.S. and competes with Salix and Populus species for available resources, most importantly light and water. Early growth strategies differ between these three species. Tamarix exhibits relatively slow aboveground biomass accumulation due to resource allocation to belowground biomass, while Salix and Populus experience rapid stem elongation as resources are allocated to above ground biomass accumulation (Karrenberg et al. 2002; Sher et al. 2002). Mature Tamarix individuals have deep and wide roots, which are especially advantageous for acquiring water when stream bank height is high and when water resources are limited (Li-xue and

Yan 2008).

Tamarix water use:

As water has become an increasingly contentious issue in arid regions in light of westward expansion and population growth, concerns have been raised regarding

Tamarix water use. Popular press articles often report that Tamarix can transpire up to

200 gallons of water per day (Holdenback 1987). This statistic has been perpetuated by policy makers and management agencies and has been the basis for much discussion and legislative actions (Owens and Moore 2007). This number is a serious overestimate;

Tamarix water use is approximately 2 gallons of water per day per plant (Sala et al.

1996). Some studies have shown that Tamarix water use is among the highest of any phreatophyte assessed in the southwestern U.S. (Brotherson and Winkle 1986), including native riparian trees (Busch and Smith 1995; Neill 1985). Other studies have challenged these earlier assumptions. Claims have been made that Tamarix is not invariably a high- water use plant (Owens and Moore 2007; Lovell et al. 2009; Nagler et al. 2013). Cleverly et al. (2002) reported water use values between 0 and 20 mm d-1 from 18 studies for

Tamarix, which is within the scope of water use reported for native species (Scott et al.

2004). Studying Tamarix water use provides the opportunity to examine which factors are driving variable water use patterns. Understanding how key factors affect Tamarix water use will allow management agencies to more appropriately manage Tamarix to salvage water and restore riparian ecosystems. Local environmental conditions, population density, and water table depth can influence Tamarix water use. The genetic structure of a population predisposes each individual to respond to environmental conditions in a particular manner. Therefore, genetic structure dictates survival strategy. Each individual must balance the tradeoff between gas exchange and photosynthetic rate. When stomata are open gas exchange occurs and carbon dioxide is made available for photosynthesis. Consequently, stomatal conductance increases while stomata are open which results in water loss. The phytohormone abscisic acid plays an important role in drought response by influencing stomatal aperture and transpiration rates (Larcher 2003). Chlorophyll harnesses energy from the sun to fix carbon, making it an integral part of photosynthesis (Krause and

White 1991). Investigating foliar chlorophyll and abscisic acid content in conjunction with stomatal conductance provides insight into the tradeoff between gas exchange and photosynthetic rate and drivers of variable water use patterns. Studying these functional traits across many individuals can shed light on survival strategies at the population level.

The habitat and density of Tamarix stands appears to greatly influence water use tendencies. High water use by Tamarix is largely due to the high leaf area index of

Tamarix communities compared to other riparian populations (Sala et al. 1996). In areas of high density or where monocultures form, Tamarix has the ability to dry up springs, drain small ponds, and even desiccate perennial streams (Johnson 1987). Previous data suggests that relative to other riparian species, Tamarix may be able to maintain relatively higher carbon assimilation rates when water is plentiful, and conserve more water under drought conditions (Cleverly et al. 2006; Lovell et al. 2009). These results indicate that

Tamarix has greater water use plasticity than Salix or Populus. Controlling Tamarix stands with high leaf area may salvage water, although the vegetation that replaces

Tamarix will determine the magnitude of water salvage (Shafroth et al. 2005).

Ultimately, riparian restoration will benefit from the control of Tamarix stands that aggressively monopolize resources, as competitive dominance shifts to other species.

Tamarix control:

Varying management techniques have been employed to control Tamarix, with the hope of reducing the negative impacts of Tamarix on riparian ecosystem structure and processes. Traditional control strategies, such as mechanical removal, prescribed burning, and aerial herbicide application have been utilized to control Tamarix populations. These techniques are costly, can have unintended ecological impacts on the establishment and productivity of native plant and soil communities, and are not always successful in the long term (Hultine et al. 2010). Due to Tamarix representing the sole genus from the family Tamaricaceae in North America, biological control with the Tamarisk leaf beetle was tested as a viable management technique (Gaskin et al. 2004).

The success of a biological control program is usually defined as the ability of the biological control agent to inflict damage and increase mortality rates of the target species, which ultimately causes a reduction in abundance of the target organism

(Denslow and D’Antonio 2005; Thomas and Reid 2007). Following secure cage trials, the Tamarisk leaf beetles were released experimentally at seven field sites during the spring of 2001 (DeLoach et al. 2003, 2004). Since the original introductions occurred, the beetles have successfully spread from the original release sites and defoliated thousands of acres of Tamarix stands (Carruthers et al. 2008). The success of the Tamarisk leaf beetle at dispersing to and invading Tamarix dominated riparian ecosystems has the potential to make the beetles one the most widespread biological control agents in recent history (Snyder et al. 2010). Although the Tamarisk leaf beetle has succeeded at defoliating extensive Tamarix stands, the physiological impacts of the biological control agent on Tamarix are still widely unknown, and could produce ecosystem wide changes

(Denslow and D’Antonio 2005).

Objectives:

The objective of this study is to determine the response of Tamarix to invasion by the biological control agent, the Tamarisk leaf beetle (Diorhabda spp.). Several studies have attempted to determine the effect of the biological control agent on Tamarix physiology

(Hudgeons et al. 2007a,b; Cunningham et al. 2009; Shafer et al. 2010; Snyder et al. 2010;

Pattison et al. 2011a, b). Fewer studies (none to our knowledge) have examined the impact of the biological control agent on physiological traits beyond water use and resource reserves. We set out to examine the response of Tamarix to invasion by the

Tamarisk leaf beetle with respect to a suite of functional traits and reproductive fitness.

We measured stomatal conductance, foliar chlorophyll and abscisic acid content, proportion of alive stems, and total flower production. We carried out phenotypic selection analysis on functional traits to deduce how the biological control agent impacted Tamarix life history strategy. We asked the following research questions:

1) How is Tamarix impacted by the biological control agent? – How are functional traits impacted? – How is reproductive fitness impacted? 2) How is Tamarix survival strategy impacted by the biological control agent? – Does phenotypic selection change between invasion time periods? – Does life history strategy change between invasion time periods?

Materials and Methods:

Study system:

Riparian forests in Colorado have historically been comprised of a diverse assemblage of forbs and graminoids interspersed among two dominant woody species in the Salicaceae family, the plains cottonwood (Populus deltoides) and the sandbar or coyote willow

(Salix exigua) (Reichenbacher 1984; Scurlock 1998). Populus is a large, tall, deciduous tree with broad leaves that forms the canopy of riparian forests. The understory is primarily made up of Salix, which forms tall stands of individual shoots with narrow leaves (Gregory et al. 1991). Both species have evolved to rely on flood events during the spring snowmelt flood regime for seed dispersal and seedling establishment (Mahoney and Rood 1998; Sher et al. 2001; Karrenberg et al. 2002; McBride and Strahan 2006).

Moreover, Salix and Populus coordinate flowering and seed production with these flood events. During this time an increase in streamflow and height occurs, which erodes stream banks and deposits sediment on sandbars (Scott et al. 1996; Merritt and Cooper

2000; Caskey et al. 2015). Following a flood event, sediment deposition coupled with a slow decline in water table height and soil moisture content provides a fertile substrate with sufficient water availability for germination and subsequent seedling establishment

(Bradley and Smith 1986; Mahoney and Rood 1998; Taylor et al. 1999; Johnson 2000;

Karrenberg et al. 2002).

Tamarix ramosissima (hereafter Tamarix) is a non-native, deciduous shrub or small tree native to the Old World (Baum 1967). Tamarix is characterized by reddish stems, pale green foliage, and distinctive minute, pink flowers. The native range of

Tamarix is extensive and ranges from southeastern Europe to Asia (Baum 1967). Tamarix can be found in saline and xeric soils along riparian corridors in arid regions

(Decker 1961; Brotherson and Winkle 1986; Ladenburger et al. 2006). Tamarix is the only genus from the family Tamaricaceae found in North America, although species within the same order (Caryophyllales) do exist (e.g. Frankenia salina) (DeLoach et al.

2004). Up to twelve species from the genus are found in the U.S. (Baum 1967). Certain species readily interbreed and form hybrids (Gaskin and Kazmer 2009). Introgression and hybridization supports Tamarix invasion and adaptation to environmental stressors in novel environments (Gaskin and Schaal 2002; Gaskin and Kazmer 2009).

Competition for light dictates establishment dynamics of Tamarix, Salix, and

Populus seedlings. On waterways experiencing natural flood regimes Tamarix struggles to establish among native species as they undergo rapid stem growth and eventually shade Tamarix seedlings (Sher et al. 2002; Beauchamp and Stromberg 2007; Mortenson and Weisberg 2010). Tamarix establishment is greater along waterways that do not have early growing season flood events (Lovell et al. 2009). Continuous seed dispersal results in complete colonization of viable germination sites by Tamarix seedlings. Shading by established Tamarix seedlings inhibits local establishment by native species during the subsequent growing season.

Management agencies have struggled to control the spread of Tamarix and reintroduce native species to areas dominated by Tamarix monocultures. Aerial herbicide application, mechanical removal, prescribed burning, and biological control are some of the more effective management techniques (Hughes 2003; McDaniel and Taylor 2003;

Hart et al. 2005). The Tamarisk leaf beetle (Diorhabda spp.) has been introduced as a biological control agent in an attempt to control Tamarix via foliar herbivory. These attempts have been frustrated by the evolution of novel Tamarix genotypes, which the introduced beetles did not evolve with in their native range (Gaskin and Kazmer 2009).

Introduction of beetles from distinct taxonomic groups and biogeographic origins has aided in reducing these frustrations throughout the widely varying biogeographic regions of the western US.

The biological control program has utilized four species from the genus

Diorhabda, although only three have established populations in North America. The most likely species found at our study site along Fountain Creek is Diorhabda carinulata.

Diorhabda carinulata (the northern Tamarisk leaf beetle) is from central Asia and is adapted to higher latitudes. Two populations have become widespread throughout

Colorado, Montana, Wyoming, Utah, and Nevada, and have established in Idaho and

Oregon as well. Following secure cage trials in 2001 (Dudley et al. 2001) experimental releases were carried out at seven sites, including a site near Pueblo, CO. Defoliation at the Pueblo, CO site was less dramatic than the other sites (e.g. Lovelock, NV). Predation by ants and other predators on the beetles and their larvae, and challenging weather patterns are attributed to producing these results (DeLoach et al. 2004; Bean et al. 2007).

Beetles dispersing from the Pueblo introduction site most likely aggregated at our

Fountain Creek study site during the 2013 field season. Beetles aggregate to trees following an initial period of male beetle colonization (Cosse et al. 2006). If the host tree is deemed viable, males will release pheromones to attract additional males and females.

These pheromones combine with green leaf volatiles produced by Tamarix, and together may act synergistically to signal the presence of feeding adult beetles (Cosse et al. 2006).

Extremely high densities of beetles aggregate to the host tree in response to the production of these substances. Mating and oviposition occurs, and both adults and larvae predate upon the host tree. Once resources are locally depleted, beetles will fly up into wind currents and alight at distances several kilometers from the original host plant.

Observations suggest that populations can travel up to 40 km in a season. Bean et al.

(2013) applied aggregation pheromones to a sticky trap on a mesa 1,500 ft. above a stand of Tamarix, and over 20 beetles flew up to the trap. It is possible that anomalous winds could disperse beetles over greater distances. Pheromones, kairomones, and green leaf volatiles attract nearby beetles, and re-aggregation to the selected host tree is integral to generating a successful breeding population following dispersal (Cosse et al. 2005).

Successfully managing Tamarix populations via leaf beetle herbivory hinges on several factors. Leaf beetles feed by scraping the cuticle away from leaves and stems, at which point the mesophyll and vascular system is exposed. Beetles feed on these tissues and cause water loss and leaf desiccation, which induces leaf drop and therefore a loss of photosynthetic area (Dudley 2005). Repeated defoliation events over several growing seasons are needed to increase tree mortality, as carbon starvation occurs and reduces growth and leaf production (Hudgeons et al. 2007b; Dudley and Bean 2011). Beetle herbivory challenges fundamental tradeoffs in Tamarix by stressing the balance between resource allocation (e.g. photosynthate and nutrients) to growth, reproduction, internal storage, or defense. Ultimately, the genetic structure of Tamarix individuals determines susceptibility to herbivory and consequentially the extent of defoliation.

Study sites:

Three sandbars along Fountain Creek were chosen as study areas. Fountain Creek originates in Woodland Park and passes through Teller, El Paso, and Pueblo Counties before joining the Arkansas River. The Fountain Creek watershed drains approximately

930 square miles of parts of the aforementioned counties in southeastern Colorado. Land use includes forests, rangelands, military reservations, urban areas, and agriculture within the watershed. Impervious area within the watershed has increased with urbanization. As impervious area increases, infiltration decreases, runoff increases, and stream bank erosion intensifies as the receiving stream experiences a quicker hydrologic response

(Stogner 2014). These factors contribute to the dynamic nature of the Fountain Creek fluvial ecosystem.

The Fountain Creek watershed experiences seasonally varying flow regimes.

These flow regimes include: base flow, snowmelt, and summer flow. Base-flow begins in late September or early October and continues into the spring until April. Stream flow is generally uniform during the base flow period, and fluctuates around 100 cubic feet per second (Stogner 2000). Depending on precipitation and temperature during the winter, the snowmelt period begins in early to mid-April and extends into June. Streamflow peaks in mid-May following a considerable increase from base-flow conditions. Summer streamflow is highly variable during the summer monsoon season. Streamflow fluctuations are driven by storm runoff during the afternoons and evenings. During the summer flooding events alter streamflow dynamics as channel widening and deepening occurs, as well as stream bank erosion and sediment deposition on sandbars (Stogner

2014).

Three sandbars along Fountain Creek were chosen as study areas within our study site near Fountain Creek Regional Park (38°42’07.51” N, 104°43’02.25” W). These sandbars are identified as northern, middle, and southern; the northern, middle, and southern sandbars vary in size, with approximate areas of 0.797 miles (11.5 acres), 0.572 miles (5.68 acres), and 0.584 miles (10.7 acres), respectively (Google Maps Engine).

From the northern to southern sandbar the elevation changes from approximately 1,702 meters to approximately 1,697 meters (Google Earth). These sandbars are relatively uniform in nature, although some distinct differences exist.

The northern and middle sandbars experience higher light levels compared to the southern sandbar. A mixed canopy of Populus and Tamarix provides shade and decreases light penetration to the understory and soil surface. All three sandbars have well drained soils comprised of sand and coarse sediment. The northern sandbar has scattered groups of Tamarix individuals with one dense community located approximately 25 meters from the waterway. Most Tamarix individuals on the middle sandbar are found in a mixed community of Salix and Tamarix that stretches approximately 270 meters along the waterway at a maximum distance of approximately 35 meters from the bank. The middle sandbar also has scattered groups of Tamarix individuals up to approximately 125 meters from the water way. The southern sandbar has a dense forest of Tamarix individuals towards the north, with groups of Tamarix individuals forming a roughly contiguous community that stretches approximately 250 meters to the southern limit of the sandbar.

The southern sandbar is located near a severe bend in the waterway relative to the middle and northern sandbars. During high water periods, water from Fountain Creek is able to encroach into the sandbar, producing flood channels and occasional periods of inundation. The middle and northern sandbars are most likely less affected by periods of high water. Therefore, the southern sandbar is more disturbed by heavy streamflow periods than the middle or northern sandbars. It is worth noting that an irrigation canal enters into Fountain Creek near the middle of the northern sandbar. When the canal is actively discharging water this section of the northern sandbar and the northern section of the middle sandbar are likely impacted.

Experimental Design:

Tree selection and site environment measurements

During the 2013 field season a total of 356 Tamarix individuals from the three sandbars were tagged with bright tape and labeled with an identification number. A 50-meter open reel fiberglass measuring tape was used to measure the distance of each tagged tree from the waterway. Distances were measured from the base of the trunk to the perpendicular intersection with the stream bank. If the distance exceeded 50 meters, the terminal end of the tape would be marked on the soil and a subsequent measurement was performed from the marked point. This process was repeated as necessary. Distances were measured to the nearest decimeter when possible.

Stem diameter measurements were collected for each tagged individual during the

2013 field season. Vernier calipers were used to measure stem diameter 15 cm above the soil surface (Scienceware, Bel-Art Products, Wayne, NJ). Debris was brushed away to reveal the soil surface when a dense mat of leaf litter was encountered. Measurements were recorded to the nearest millimeter, and estimated to a tenth of a millimeter by best fit on the tick mark scale. Care was taken to ensure that the caliper opening was not altered as it was removed from the trunk.

Light levels at the stem and at one meter from the stem were collected in addition to soil moisture content for each sandbar. Of the 356 trees tagged during the 2013 field season, light levels were recorded for 109 individuals. A LightScout quantum meter (Spectrum Technologies, Inc) was used to measure light levels at the stem and at one meter from the stem. The device was held at breast height on the stem and at one meter facing south to collect each measurement. Light measurements were recorded five seconds after the device was placed in the recording position. Care was taken to ensure that body positioning while taking measurements did not inhibit light penetration or cause the canopy to be displaced from its natural position. Volumetric water content (VWC) was measured using a TDR Moisture Meter at each of the sandbars (Campbell Scientific,

Logan, UT). The device was inserted to a uniform depth at each measurement point in an attempt to minimize error. During the 2013 and 2014 field season 40 and 14 soil moisture measurements were recorded, respectively.

Functional trait measurements

Stomatal conductance measurements were collected to determine transpiration rates for

Tamarix individuals. A Steady State Diffusion Leaf Porometer was used to collect all stomatal conductance measurements (Decagon Devices, Pullman, WA). Measurements were collected between 10:00 and 14:30 when light levels were greater than 1200 µmol photons m-2 sec-1. Leaf selection for stomatal conductance measurements was carried out according to the following protocol. From the trunk, the first main branch was selected.

The first red stem (indicative of new growth at our study site) was selected off of the first main branch. Terminal leaves (our estimate of leaves which have fully expanded recently) on the first red stem were selected for clamping. Two to three leaves were grouped and clamped in an attempt to cover the porometer sensor head aperture. For saplings with non-dominant branching, we sampled two to three non-flowering leaves that will not become stems at the top of the individual. During the 2013 field season stomatal conductance was measured for 308 individuals between 6/24-6/28 and on 7/2 and 7/5. During the 2014 field season stomatal conductance was measured for 188 individuals on 7/7 and 7/9. Stomatal conductance data were adjusted for day and time of day with regression.

Leaves were collected from select Tamarix ramosissima individuals to quantify foliar chlorophyll content. For each individual the first main branch was followed until the first red stem was found. The first red stem was followed to the terminal region, where five to six leaves were removed and placed directly on ice in a cooler. Samples were transferred to a dark, -20°C freezer immediately upon returning to the laboratory.

Leaf samples were stored in labeled Ziploc bags in the freezer until chlorophyll extraction and quantification was carried out. During the 2013 and 2014 field seasons 129 and 46 individuals were sampled for chlorophyll analysis, respectively.

Chlorophyll was extracted and quantified according to the following protocol.

Leaves were stripped from the petiole/midrib and weighed in a weigh boat. Total fresh weights ranged from 300-350 mg. Samples were transferred to a centrifuge tube

(Nalgene) containing 10 mL of cold, pure, spectrophotometric grade acetone. Samples were ground with a Polytron grinder at setting “4” for 25 seconds. Samples were allowed to rest on ice for at least 5 minutes. A Genesys thermospec 20 was blanked at 647 nm with 2 mL of cold, pure, spectrophotometric grade acetone (Thermo Fisher Scientific,

Waltham, MA). Two, one milliliter aliquots of the extraction solution were transferred to a cuvette. Special care was given to removing the extraction solution from the top of the supernatant column, to avoid disturbing the ground plant material on the bottom of the centrifuge tube. To quantify foliar chlorophyll content absorbance values were measured at 647 nm and 664 nm. The following equation was used to quantify the amount of foliar chlorophyll in the leaf samples:

[(18.4∗퐴푏푠@647 푛푚)+(6.4∗퐴푏푠@664 푛푚)∗퐸푥푡푟푎푐푡 푣표푙 (푚퐿)] 퐶ℎ푙표푟표푝ℎ푦푙푙 (푚𝑔) = 푠푎푚푝푙푒 푓푟푒푠ℎ 푤푒𝑖푔ℎ푡 (푚푔)

Foliar abscisic acid (ABA) was extracted and quantified from leaf samples collected from frozen leaf samples collected during the 2013 and 2010 field seasons.

Samples collected in 2010 were weighed and stored in 1.5 mL microcentrifuge tubes.

From the stored 2010 leaf samples, those chosen for extraction (n=32) had the largest weights. Leaf samples from the 2013 field season were collected according to the same protocol utilized for chlorophyll analysis. From the leaf samples collected during the summer of 2013, 51 were randomly chosen for ABA extraction. Leaves were stripped from the woodier petiole, weighed (~300mg), cut to reduce size, and immediately placed in 1.5 mL centrifuge tubes before being stored in a defrosting freezer. Leaf samples were stored in the dark until lyophilization could be carried out.

All samples were lyophilized in open centrifuge tubes inside a vacuum chamber.

The vacuum chamber was placed inside a -20°C freezer for the duration of the lyophilization. Leaf tissues were lyophilized in the dark for 21-24 hours. Samples were stored in sealed centrifuge tubes in a dark, defrosting freezer until extractions were performed.

Abscisic acid (ABA) was extracted from lyophilized leaf samples using a

Polytron grinding instrument with minimal lighting. Leaf samples were transferred from

1.5 mL centrifuge tubes to large (Nalgene, Thermo Fisher Scientific, Waltham, MA) centrifuge tubes containing 3 mL of ABA extraction buffer. Samples were ground with a

Polytron grinder on setting “4” for 1 minute, and then leaf material that had adhered to the side of the tube was resuspended in the extraction buffer. The extraction solution was then ground for another minute. Two 700 µL aliquots were immediately removed with a micropipette and placed in a new, labeled 1.5 mL microcentrifuge tube. The sample was then stored in a dark box in a -20°C freezer until centrifugation was carried out. Between samples the Polytron was cleaned.

Before centrifugation, samples were placed in an operating fume hood with the microcentrifuge tube open. After an hour of evaporation in the dark, samples were centrifuged at 2,200 g for 3 minutes. Following centrifugation the samples were placed on ice and 100 µL of supernatant was removed and placed in a new, labeled 1.5 micro centrifuge tube on ice. 900 µL of TBS (with MgCl2 in the TBS) was added to each sample, and then vortexed on setting “5” for 5 seconds. Samples were then centrifuged at

2,200 g for 10 minutes. Following centrifugation, 700 µL of the supernatant was removed and placed in a new, labeled micro centrifuge tube before being stored in a freezer in the dark until ELISA could be carried out.

ABA was quantified in leaf samples using ELISA (Agdia, Inc., Elkhart, IN). ABA standards (mixed isomers) were prepared to gather data for the construction of a standard curve. Serial dilutions were carried out to create standards for ELISA. Creating the standard (A1) from the filter paper resulted in an ABA concentration of 1 x 10-6 M.

Serial dilutions were initiated by adding 200 µL of A1 to 1.80 mL of 1X TBS in a new labeled (B1) 1.5 ml microcentrifuge tube. For each subsequent standard (C1-G1), 500 µL of the preceding dilution was added to 2.00 mL of 1X TBS. The resulting standard concentrations ([ABA]) were: A1=1 x 10-6 M, B1=1 x 10-7 M, C1=2 x 10-8 M, D1=4 x

10-9 M, E1=8 x 10-10 M, F1= 16 X 10-11 M, G1=32 x 10-12 M. For each plate wells were loaded in the dark on ice. Solutions were vortexed before being loaded into wells. Following the incubation period, the wells were dumped.

Wells were washed three times by adding 200 µL of the 1X PBST wash solution to each well before dumping. The substrate solution was vortexed before adding 200 µL to each well. The plate was then sealed and placed inside a dark, humid box. The test wells were incubated at 37°C for 1.5 hours. The plate was removed from the incubation box before placing into the plate reader. We used an Optima plate reader to gather data for the wells at 405 nm. Optical densities were recorded for the standards and samples.

To estimate tree health during the 2013 and 2014 field seasons the number of alive and dead stems was recorded on 308 and 168 individuals, respectively. From this data we calculated the proportion of alive to dead stems. Stems were meticulously counted and recorded as either “dead” or “alive” according to the following protocol. We moved up the trunk counting stems on major branches. Stems were defined as lateral growth from a branch with woody character near the node. “Alive” stems had at least

50% of the stem covered in healthy, green foliage. “Dead” stems were those which were not classified as “alive”. We moved along the branch and counted out along lateral growth to census all stems present.

Fitness estimates

Inflorescences were counted and used to estimate total flower number for each individual. We note that flower number does not equate with reproductive fitness; however, because of high selfing and outcrossing rates, most flowers in our populations do become fruits (Drummond and Heschel, unpublished data). We define an inflorescence as a cluster of 6-8 racemes. Moving up the trunk, stems containing inflorescences were counted on each major branch. To increase accuracy branches were bent or moved and counts were performed while moving along stems methodically. From this we were able to determine an estimate of the total number of inflorescences present on each individual.

We measured 10 racemes per individual in order to calculate mean raceme length.

From the trunk the first major branch was chosen. From the first major branch, the first red stem was followed to the terminal region. Five terminal racemes from the terminal inflorescence were chosen and measured to the nearest millimeter. This measurement was repeated on the second red stem on the first major branch.

To estimate total flower production raceme lengths were averaged for each individual. To estimate the number of flowers on each raceme, average raceme length was multiplied by 7.0477387, and then added to 7.4210996. These values were generated by measuring the length and total flower number of five racemes at the terminus of the lowest branch of 40 plants, and then regressing raceme length against total flower number to generate a linear model (R2=0.72).

Statistical Analysis:

All statistical analyses were performed with JMP version 7.0.2 (SAS Institute, Cary, NC).

ANOVAs were used to test for functional trait and fitness estimate differences between invasion periods; invasion period and sandbar location were considered fixed factors.

Phenotypic selection analyses were used to determine the effects of functional traits on flower production for each invasion period. Data was relativized and standardized for each invasion period before performing phenotypic selection analysis. Fitness was estimated with flower number and relative fitness was determined by dividing total flower number by the grand mean for each invasion period. Traits were standardized to a mean of zero with a standard deviation of one. Linear regression was used to perform phenotypic selection analysis. Differential analysis was a simple linear regression of the standardized trait on relative fitness. Gradient analysis was a multiple regression of all standardized traits on relative fitness. For all statistical models, the distributions of residuals were examined and the data were log-transformed where necessary.

Results:

Environmental Profile:

United States Geological Survey (USGS) data for Fountain Creek, CO collected by station number 07106000 shows differences in mean gage height between invasion periods. Gage heights and maximum gage heights were averaged from June to August for each invasion time period. The post invasion time period had the highest gage height (x̄ =

1.46 m), while during the beetle invasion gage height was the lowest (x̄ = 0.973 m)

(Figure 1). The post beetle invasion time period was characterized by an increase in flood events leading to high channel height and stream flow. Maximum gage height was the highest during the post-beetle invasion time period (x̄ =1.62 m) and lowest during the beetle invasion (x̄ =1.15 m) (Figure 2).

Weather data for Ft. Carson, CO (<10 mi from study site) shows differences in temperature, precipitation, and growing degree days between the invasion periods.

Temperature and precipitation values for each invasion period were averaged for June and July, while growing degree days were averaged from June to August for each invasion period. Temperature was highest during the beetle invasion (x̄ =21.4°C) and lowest during the post-beetle invasion time period (x̄ =20.6°C) (Figure 3). Precipitation increased from the pre-beetle invasion time period to the post-beetle invasion time period

(Figure 4). Precipitation was lowest during the pre-beetle invasion time period (x̄ =6.98 cm) and highest during the post-beetle invasion time period (x̄ =9.16 cm). Mean growing degree values were highest during the beetle invasion (x̄ =630) and lowest during the post-beetle invasion time period (x̄ =569) (Figure 5). Mean growing degree days for the pre and post-beetle invasion time periods were similar and did not differ significantly.

Conditions appear to be the most optimal during the post-beetle invasion period, with high precipitation and low temperatures. This time period did have the fewest growing degree days, which may have impacted recovery following the invasion.

Precipitation amounts were similar between the pre-beetle and during-beetle invasion time periods, although during the invasion temperatures were higher. Conditions were challenging for Tanarix during the beetle invasion. Although these parameters are a good indication of environmental quality, they cannot be considered definitive measures.

Tamarix Water Use:

Stomatal Conductance

During the 2013 field season stomatal conductance was measured for 308 individuals during late June and early. For the northern sandbar, volumetric water content ranged between 2-4%, with light levels ranging from 91 to 2,000+ µmol photons m-2 sec-1 during the collection period (stomatal conductance measurements were not taken unless light levels exceeded 1200 µmol photons m-2 sec-1). Temperatures ranged from 25-38.4°C. For the middle sandbar, volumetric water content measurements ranged from 2-8%, with light levels ranging from 30-2000+ µmol photons m-2 sec-1. Temperatures ranged from

31.8-39.2°C. For the southern sandbar, volumetric water content ranged from 3-11%, with light levels ranging from 20-2,000+ µmol photons m-2 sec-1. Temperatures ranged from 29.0-38.1°C.

During the 2014 field season stomatal conductance was measured for 157 individuals during early July. For the northern and middle sandbars, temperature measurements ranged from 39-43°C. Humidity was measured at 24%. Light levels ranged from 1200-1,800 µmol photons m-2 sec-1. For the southern section light levels ranged from 400-1820 µmol photons m-2 sec-1. Temperature measurements ranged from

22-33°C, with an average humidity of 48%. Volumetric water content measurements ranged from 5-6%.

Mean stomatal conductance data for each invasion period differed significantly from the others (Figure 6). Invasion by the Tamarisk leaf beetle may have direct effects on Tamarix stomatal conductance. Invasion period had a significant effect on stomatal conductance (Table 1). Conductance was lowest during the pre-beetle invasion time period (x̄ =1.98417 mmol H2O•m-2 s-1, SE=0.01148761) and highest during the beetle invasion (x̄ =2.3026 mmol H2O•m-2 s-1, SE=0.01188679). Stomatal conductance decreased between the invasion and post-invasion time periods (x̄ =2.20781 mmol

H2O•m-2 s-1, SE= 0.0167245).

Foliar Abscisic Acid Content

Foliar abscisic acid content differed between the pre-beetle invasion time period and during the invasion, but these differences were not significant (Figure 7). Invasion period did not have a significant effect on foliar abscisic acid content (Table 1). Foliar abscisic acid content was lower during the pre-beetle invasion time period (x̄ =1.873E-10 M ABA/ mg fresh leaf weight, SE= 3.5518E-10) compared to during the beetle invasion (x̄ =8.971E-10 M ABA/ mg fresh leaf weight, SE= 8.6582E-10). The Tamarisk leaf beetle most likely does not have an effect on foliar abscisic acid content, but it appears as if sensitivity to ABA may change between invasion periods.

Foliar Chlorophyll Content

Invasion period had a significant effect on foliar chlorophyll content (Table 1). Foliar chlorophyll content differed significantly between invasion time periods (Figure 8).

Chlorophyll content declined across the invasion periods; foliar chlorophyll content was highest before the beetle invasion (x̄ =2.04988, SE=0.054423), decreased during the beetle invasion (x̅ =1.64303, SE=0.028069), and decreased again following the beetle invasion (x̄ =1.45303, SE=0.043164). The Tamarisk leaf beetle may have a direct effect on Tamarix foliar chlorophyll content and consequentially photosynthetic potential.

Proportion Alive

Proportion alive measurements differed significantly between the beetle invasion and post-beetle invasion time periods (Figure 9). There was a significant effect of invasion period on the proportion of alive stems (Table 1). During the beetle invasion there was a higher proportion of stems alive (x̄ =0.630566, SE=0.01233) than during the post-beetle invasion time period (x̄ =0.519896, SE=0.017316). The pre-beetle invasion period was characterized by trees which leafed out early in the season and had a high proportion of alive stems (observational and photographic data, pers. comm. SH).

Tamarix Fitness Estimates:

Flower Production

Invasion period had a significant effect on total flower number (Table 1). Total flower number was highest during the pre-beetle invasion period and differed significantly from the other invasion periods (x̄ =4618.88, SE=274.6356). Total flower number was lowest during the beetle invasion (x̄ = 2183.81, SE= 603.44738) and did not differ significantly with total flower number during the post-beetle invasion time period (x̄ =2629.1,

SE=461.8752) (Figure 10). The Tamarisk leaf beetle may have direct effects on Tamarix flower production.

Phenotypic Selection Analysis:

It was assumed that total flower number is a proxy for Tamarix reproductive fitness.

During the pre-beetle invasion time period a positive selection differential was detected for stomatal conductance (S=0.143887), foliar chlorophyll content (S=0.0610618), and a negative selection differential was detected for foliar abscisic acid content (S=-0.420167)

(Table 2). This indicates that individuals with higher stomatal conductance and foliar chlorophyll content had greater fitness, and that individuals with higher foliar abscisic acid content had lower fitness. Differential selection was significant for foliar abscisic acid content (p=0.0440), marginally significant for stomatal conductance (p=0.0602), and not significant for foliar chlorophyll content (p=0.4456). A positive selection gradient was detected for stomatal conductance (β=0.755974), while a negative selection gradient was detected for foliar chlorophyll (β=-0.598682) and foliar abscisic acid content (β=-

0.432772). Gradient selection was marginally significant for stomatal conductance

(p=0.0805) and foliar abscisic acid content (p=0.091). Gradient selection was not significant for foliar chlorophyll content (p=0.1217).

During the beetle invasion time period a positive selection differential was detected for foliar chlorophyll content (S=0.4423897) while a negative selection differential was detected for stomatal conductance (S=-0.177225) and foliar abscisic acid content (S=-1.129388) (Table 2). This indicates that individuals with higher foliar chlorophyll content, low stomatal conductance, and low foliar abscisic acid content had greater fitness. Differential selection for foliar chlorophyll content was moderately significant (p=0.0949); selection was not significant for stomatal conductance (p=0.132) and foliar abscisic acid (p=0.3396). A positive selection gradient was detected for foliar chlorophyll content (β=0.3336222) while negative gradient selection was present for stomatal conductance (β=-0.225071) and foliar abscisic acid content (β=-1.762159).

Gradient selection was not significant for stomatal conductance (p=0.5665), foliar chlorophyll content (p=0.3031), or foliar abscisic acid content (p=0.318).

During the post-beetle invasion time period a positive selection differential was detected for stomatal conductance (S=0.0181809) and foliar chlorophyll content

(S=0.2854682) (Table 2). This indicates that individuals with high stomatal conductance and foliar chlorophyll content had greater fitness. Differential selection was significant for foliar chlorophyll content (p=0.0403) and not significant for stomatal conductance

(p=0.844). A positive selection gradient was detected for stomatal conductance

(β=0.0116577) and foliar chlorophyll content (β=0.3005745). Gradient selection was marginally significant for foliar chlorophyll content (p=0.0968) and not significant for stomatal conductance (p=0.9418).

Discussion:

Invasion by the Tamarisk leaf beetle during the 2013 field season impacted Tamarix functional traits and reproductive fitness. During invasion by the biological control agent stomatal conductance significantly increased, foliar chlorophyll content significantly decreased, abscisic acid content decreased, and total flower number significantly decreased, with respect to the 2013 field season. Following the invasion stomatal conductance increased, foliar chlorophyll content significantly decreased, proportion of alive stems significantly decreased, and total flower number increased. Our results suggest that herbivory by the leaf beetle may not only impact Tamarix during a defoliation event, but also during the subsequent growing season. Furthermore, phenotypic selection and life history strategy are most likely impacted by the biological control agent.

Phenotypic selection and life history strategy changed between invasion periods.

Before the invasion, significant selection was detected for low abscisic acid content and high stomatal conductance. Successful individuals wasted water and maximized photosynthetic rate. During the invasion, selection was detected for high chlorophyll content and low stomatal conductance. Successful individuals maintained carbon fixation rates with high photosynthetic potential while conserving water to mitigate additional water loss. After the invasion selection was detected for high chlorophyll content and high stomatal conductance. Successful individuals had high photosynthetic potential and high stomatal conductance and maximized photosynthetic rate.

How does the biological control agent impact Tamarix water use?: The beetle invasion had a significant impact on stomatal conductance. During the invasion intense herbivory resulted in Tamarix defoliation. The beetles feed by scraping away the cuticle to access mesophyll and vascular tissues within the leaves. The cuticle is a morphological adaptation to drought and maintains foliar water content by reducing cuticular transpiration (Schreiber and Riederer 1996). Loss of the cuticle comprises the structural integrity of leaves and allows water to escape. Consequently, cuticular transpiration and evaporation rates increase. To mitigate additional water loss from damaged leaf tissues, Tamarix abscises masticated leaves (Snyder et al. 2010).

Consequently, Tamarix must increase stomatal conductance to compensate for water lost from tissues damaged via foliar herbivory.

Our results provide an interesting comparison to previously reported findings on

Tamarix water use in response to the biological control agent. Snyder et al. (2015) demonstrated in a controlled greenhouse environment that stomatal conductance increased in plants with beetles present. Moreover, their data indicated that beetle herbivory decreased photosynthesis and produced leaves that were unable to regulate water loss. They further conclude that beetle herbivory may make Tamarix less drought tolerant (Snyder et al. 2010). Our field results corroborate their findings in a controlled environment.

Our findings of decreased proportion of alive stems, foliar chlorophyll content, and total flower production during the post-beetle invasion time period are supported by studies addressing similar physiological questions. The results found by Pattison et al.

(2011) suggest that repeated defoliation reduces the growth and water relations of

Tamarix. They claim that this occurs through a decrease in resource allocation to belowground biomass (e.g. roots) (Pattison et al. 2011). Additional support for this hypothesis comes from Hudgeons et al. (2007b). They found that nonstructural reserves in the root crowns of trees in the 2002DA and 2003DA, measured in 2005 were significantly lower than those in the 2004DA or in non-defoliated trees (DA can be equated with beetle release date). Additional studies need to be performed to understand the long-term effects of the biological control agent on Tamarix water use and growth. Herbivory by the Tamarisk leaf beetle results in defoliation and a loss in photosynthetic area. To compensate for a loss of photosynthetic area, Tamarix may increase chlorophyll production to enhance photosynthetic potential in remaining photosynthetic tissues, or produce more leaves to increase photosynthetic area. While experiencing intense herbivory, both response strategies require a costly energetic input.

Our results show that disturbance by the Tamarisk leaf beetle decreases chlorophyll content during the invasion, and also during the post-invasion time period. Therefore, photosynthetic potential is reduced.

A tradeoff exists between drought response and photosynthetic rate. During the invasion, reduced photosynthetic potential required Tamarix to open stomata for extended periods of time. When stomata are open, sufficient gas exchange can occur to maintain carbon fixation rates. Consequently, transpiration and stomatal conductance increases as stomata remain open. To meet physiological demands, individuals experiencing diminished photosynthetic potential must waste water to maintain carbon fixation rates and photosynthate production. During the invasion, successful individuals were able to maintain high photosynthetic rates with high foliar chlorophyll content, while mitigating additional water loss with low stomatal conductance. Herbivory by the leaf beetle stresses the tradeoff between drought response and photosynthetic rate.

We found that ABA increased slightly during the invasion. This increase may have been in response to water stress or to facilitate abscission of leaves damaged via leaf beetle herbivory. Stomatal conductance also increased during the invasion. Individuals experiencing increased ABA content and increased stomatal conductance exhibit low

ABA sensitivity (cf. Heschel and Hausmann 2001). These individuals compromise drought response by wasting water to maintain carbon fixation rates. Under these conditions Tamarix may “steal” water from neighboring individuals, which can have impacts on water accessibility for native species.

How does the biological control agent impact Tamarix fitness?:

Decreased photosynthetic potential impairs photosynthate production and challenges fundamental tradeoffs in Tamarix. The balance between resource allocation to growth, reproduction, internal storage, and defense is stressed. In order to produce flowering structures and reproduce, additional physiological demands may suffer from reduced photosynthate availability. Our results show that the leaf beetle significantly impacts reproductive fitness by reducing total flower number. Ultimately, seed production is affected, which may impact Tamarix recruitment during the succeeding growing season.

Although our proportion alive measurement is a coarse estimate of Tamarix health, it appears that intense defoliation during one growing season impacts Tamarix during the subsequent growing season. This is likely due to decreased resource allocation to belowground biomass, possibly in favor of generating reproductive structures. Intense foliar herbivory by the Tamarisk leaf beetle during a defoliation event diminishes the ability of Tamarix to accumulate nonstructural carbohydrate reserves in belowground biomass. During the beginning of the growing season, foliar growth (e.g. bud break and leaf out) relies on stored nonstructural carbohydrate reserves. Without a sufficient supply of stored resources from the previous growing season, Tamarix growth is inhibited.

Moreover, Tamarix flowering phenology may be delayed, which will allow native species that established earlier in the growing season to shade Tamarix and decrease survivorship. In order to be an effective management technique, the biological control agent must intensely defoliate Tamarix over multiple growing seasons to inhibit storage of nonstructural carbohydrates and the extent of root structures, which ultimately impacts resource reserves and water accessibility.

How does the biological control agent impact Tamarix phenotypic selection and life history strategy?:

Phenotypic selection and life history strategy changed between invasion periods. Before the invasion, significant selection was detected for low ABA content and high stomatal conductance. Successful individuals wasted water and maximized photosynthetic rate.

During the invasion selection was detected for high chlorophyll content, and low stomatal conductance. Herbivory by the leaf beetle contributes to water loss from masticated tissues, and defoliation causes a decrease in photosynthetic area. Successful individuals maintained carbon fixation rates with high photosynthetic potential, while conserving water to mitigate additional water loss. After the invasion selection was detected for high chlorophyll content and high stomatal conductance. The population was no longer experiencing intense herbivory so water could be wasted. Successful individuals combined high photosynthetic potential with high stomatal conductance and maximized photosynthetic rate.

It appears that foliar chlorophyll content may drive the response of Tamarix functional traits to invasion by the biological control agent. During the invasion, we detected selection for low stomatal conductance, high chlorophyll content, and low ABA content. Individuals lacking high chlorophyll content required increased stomatal conductance to maintain carbon fixation rates. These individuals displayed a life history strategy contradictory to that seen in Tamarix under water stress (see Lovell et al. 2009); Tamarix conserves water under drought conditions and wastes water when abundant.

During the leaf beetle invasion, compromised photosynthetic potential via low chlorophyll content challenges established life history strategies in Tamarix. Most importantly, herbivory by the leaf beetle reduces belowground biomass accumulation and resource reserves which adversely affects the ability of Tamarix to recover from disturbance events.

What are the management implications of this study?:

Given its impact on fitness, the biological control agent will exert a selective pressure on

Tamarix. Herbivory by the leaf beetle may alter the evolution of life history in Tamarix populations. During a defoliation event, Tamarix individuals will utilize differing life history strategies depending on foliar chlorophyll content. Individuals with high foliar chlorophyll content will have high photosynthetic potential and will mitigate additional water loss from herbivory with low stomatal conductance. Individuals with low foliar chlorophyll content will waste water in an attempt to maintain carbon fixation rates. Low water availability will exacerbate water stress in individuals with low foliar chlorophyll content.

Given that on average, beetle herbivory results in decreased chlorophyll content and increased water use, beetle release may be selecting for drier habitats. Our results complicate the decision to use the biological control agent to manage Tamarix. We have shown that herbivory causes a significant increase in stomatal conductance. Although successful individuals conserved water during the invasion, water use at the population level was relatively high compared to the other field seasons. Repeated defoliation events over multiple growing seasons are needed to control Tamarix, which could severely impact water relations in riparian communities as Tamarix wastes water in an attempt to maintain carbon fixation rates. High transpiration rates by dense stands of Tamarix can severely impact the water table, shallow groundwater reserves, and consequentially water availability for native species (Johnson 1987).

Loss of Tamarix monocultures along waterways will have widespread effects on riparian corridors. Risk of erosion will increase following the loss of Tamarix root structures along waterways; stream bank architecture will be impacted and increased sedimentation may occur on downstream sandbars. Following Tamarix mortality the risk of fire encroachment into riparian buffer zones increases as the presence of desiccated above ground biomass increases. In communities where Tamarix stands contribute strongly to habitat structure and understory/overstory dynamics, trophic cascades may occur following the loss of Tamarix. The fundamental role of primary producers cannot be ignored in these ecosystems.

In areas where populations of Tamarix experience high mortality rates, riparian community structure can be impacted on a large scale. On waterways where steam-flow regulation prevents natural flood events establishment following loss of Tamarix is impeded. Riparian restoration following Tamarix mortality necessitates revegation with native species, so that shading can inhibit reestablishments by Tamarix and invasion by nonative ruderals such as Kochia spp. (Shafroth et al. 2005). Moreover, where beetle invasion does not result in high mortality rates, shifts in Tamarix life history strategy may further reduce water availability. We suspect that successful Tamarix individuals (i.e. water wasters) will disproportionately recruit to populations experiencing defoliation, which may cause Tamarix to have higher transpiration rates in arid regions. Figure Labels: (appendix A):

Fig. 1 Mean gage heights recorded by USGS station number 07106000 for Fountain Creek near Fountain, CO Mean values were calculated for pre, during, and post beetle invasion time periods. Fig. 2 Maximum gage heights recorded by USGS station number 07106000 for Fountain Creek near Fountain, CO. Means values were calculated for pre, during, and post beetle invasion time periods. Fig. 3 Mean temperature values in degrees Fahrenheit for each invasion period. Data gathered from historical records on weatherunderground.com. Fig. 4 Mean precipitation values for each invasion period. Data gathered from historical records for June on weatherunderground.com. Fig. 5 Mean growing degree days (base 50) for each invasion period. Data gathered from historical records on weatherunderground.com. Fig. 6 Mean stomatal conductance measurements plotted (+1SE) for Tamarix individuals during the pre, during, and post-beetle invasion time periods. Conductance values were adjusted for measurement time. Fig. 7 Mean foliar abscisic acid content (M ABA per mg fresh leaf weight) (+1SE) for the Fountain Creek Tamarix population for pre and during beetle invasion time periods. Fig. 8 Mean foliar chlorophyll content for the Fountain Creek Tamarix population for pre and during beetle invasion time periods. Fig. 9 Mean proportion alive data (+1SE) for the Fountain Creek Tamarix population for invasion and post-beetle invasion time periods. Fig. 10 Mean total flower numbers plotted (+1SE) for the Fountain Creek Tamarix population for the pre-, during-, and post-beetle invasion time periods.

1.6

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Figure 1. Mean gage heights recorded by USGS station number 07106000 for Fountain Creek near Fountain, CO Mean values were calculated for June and July for each invasion time period.

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Figure 2. Maximum gage heights recorded by USGS station number 07106000 for Fountain Creek near Fountain, CO. Means values were calculated June and July for each invasion time period. 21.6

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C) ° 21

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Figure 3. Mean temperature values in degrees Celsius calculated for June and July for each invasion period. Data gathered from historical records on weatherunderground.com.

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Figure 4. Mean precipitation values calculated for June and July for each invasion period. Data gathered from historical records on weatherunderground.com.

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550 Growing Degree Days 50) Degree Days (Base Growing 540 530 Pre During Post Invasion Period

Figure 5. Mean growing degree days (base 50) for each invasion period. Data gathered from historical records for June, July, and August on weatherunderground.com.

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Figure 6. Mean stomatal conductance measurements for Tamarix individuals plotted (+1SE) during the pre, during, and post-beetle invasion time periods. Conductance values were adjusted for measurement time. 1E-08

9E-09 8E-09 7E-09 6E-09 5E-09 4E-09 3E-09 2E-09 1E-09

0 ABA Content (mg ABA/mg FLW) ABA/mg (mg Content ABA Pre During Invasion Period

Figure 7. Mean foliar abscisic acid content (mg ABA per mg fresh leaf weight) (+1SE) for Tamarix individuals plotted for the pre and during beetle invasion time periods. 2.5

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Figure 8. Mean foliar chlorophyll content for Tamarix individuals plotted for the pre and during beetle invasion time periods.

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Figure 9. Mean proportion alive data (+1SE) for Tamarix individuals plotted for the invasion and post- beetle invasion time periods. 6000

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Figure 10. Mean total flower numbers plotted (+1SE) for Tamarix individuals plotted for the pre, during, and post beetle invasion time periods.

Table Labels: (appendix B): Table 1. ANOVA for treatment (pre-, during-, and post-beetle invasion) and site (south and north) on physiological traits. Conductance values (gst) were adjusted for measurement time, and log transformed. Invasion period and sandbar location were considered fixed factors. ABA=foliar abscisic acid concentration per milligram fresh leaf mass.

Log gst ABA Chlorophyll Total Flower Number Proportion Alive Treatment 189.179**** 0.2726 25.4445**** 10.6210**** 25.5742**** Site (south & north) 0.1462 0.8159 2.8953* 3.1140* 8.2657***

Table 1. Data are F values reported from ANOVA for treatment (pre-, during-, and post-beetle invasion) and site (North or South) on physiological traits. Conductance values (gst) were adjusted for measurement time, and log transformed. ABA=foliar abscisic acid concentration per milligram fresh leaf mass. F-values reported from effect tests. *P < 0.1 ** P < 0.05 *** P < 0.01 **** P < 0.0001

Table 2. Selection analysis for functional traits for pre-, during-, and post-beetle invasion time periods. Differential selection analyses (S) were linear regressions of the standardized trait on relativized fitness. Gradient selection analyses (β) were multiple regressions of all three traits and site on relative fitness. * P < 0.1 ** P < 0.05

Pre-Beetle During-Beetle Post-Beetle S β S β S β

gst 0.143887* 0.755974* -0.177225 -0.225071 0.0181809 0.0116577 Chlorophyll 0.0610618 -0.598682 0.4423897* 0.3336222 0.2854682** 0.3005745* ABA -0.420167** -0.432772* -1.129388 -1.762159 N/A N/A Table 2. Selection analysis for stomatal conductance (gst), foliar chlorophyll content, and foliar abscisic acid content per milligram fresh leaf mass (ABA) for pre-, during-, and post-beetle invasion time periods. Differential selection analyses (S) were linear regressions of the standardized trait on relativized fitness. Gradient selection analyses (β) were multiple regressions of all three traits and site on relativized fitness. * P < 0.1 ** P < 0.05

Table 3. Selection analysis for functional traits for pre-, during-, and post-beetle invasion time periods. Up arrows represent positive selection, or selection for a higher value for the trait. Down arrows represent negative selection, or selection for a lower value for the trait Pre-Beetle During-Beetle Post-Beetle S β S β S β

gst ↑ * ↑ * ↓ ↓ ↑ ↑ Chlorophyll ↑ ↓ ↑ * ↑ ↑ ** ↑* ABA ↓ ** ↓ * ↓ ↓ N/A N/A

Table 3. Selection analysis for stomatal conductance (gst), foliar chlorophyll content, and foliar abscisic acid content per milligram fresh leaf mass (ABA) for pre-, during-, and post-beetle invasion time periods. Differential selection analyses (S) were linear regressions of the standardized trait on relativized fitness. Gradient selection analyses (β) were multiple regressions of all three traits and site on relativized fitness. * P < 0.1 ** P < 0.05 Sources: Auble, G.T., J.M. Freidman, and M.L. Scott. 1994. Relating riparian vegetation to present and future streamflows. Ecological Applications 4:544-554

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