LINKING PLANT AND SOIL NUTRIENT DYNAMICS IN TEMPERATE AND

TROPICAL MONTANE FORESTS

by

Claire Anne Qubain

A thesis submitted in partial fulfillment

of the requirements for the degree

of

Master of Science

in

Biological Sciences

MONTANA STATE UNIVERSITY

Bozeman, Montana

July 2018
 ©COPYRIGHT

by

Claire Anne Qubain

2018

All Rights Reserved

 ii

ACKNOWLEDGEMENTS

This work would not have been possible without the dedication and mentorship of

my advisor, Jia Hu, and my committee members, Yuriko Yano and Dave Roberts. Their

guidance and patience were invaluable throughout this process. Lab mates and

technicians were also instrumental in my work. Thank you to Justin Martin, Tim Clute,

Erik Anderson, Taylor Simpson, Chase Dart, Sara Nerby, and Corinne Moss. Jane

Klassen and Stefania Mambelli were incredibly helpful when performing chemical

analyses. Thank you to the Department of Ecology staff, Meghan Heim, Jim Barutha, and

Sarah Deaton, who guided my success.

This also would not have been possible without the support of friends and family.

Thank you to my mom, Pam, for recognizing the value of education and pushing me

through all my stages of learning, and to my dad, Phil, for instilling in me a love of

plants. Perry, my husband, deserves endless kudos for supporting me through my grad

school ups and downs, and my sister, Allison, will always be my support pillar. Thank

you to friends for all the encouragement. Nicole Hupp, Meryl Storb, Will Glenny, Cody

Deane, Kelli Roemer, Lindsey Fell, and Elise Otto gave invaluable support, in my

research and otherwise.

This work was supported by the Montana Institute on Ecosystems, the Critical

Zone Observatories and Science Across Virtual Institutes, the Montana Water Center,

and the USDA NIFA program (award number 2015-67020-23454).

 iii

TABLE OF CONTENTS

1. INTRODUCTION ...........................................................................................................1

2. LINKING NITROGEN ALLOCATION IN DOUGLAS-FIR TO SOIL

NITROGEN AVAILABILITY IN A WESTERN MONTANE FOREST ......................4
Contribution of Authors and Co-Authors ........................................................................4
Manuscript Information Page ..........................................................................................5
Abstract ............................................................................................................................6
Introduction ......................................................................................................................7
Methods..........................................................................................................................11
Site Description......................................................................................................11
Growth Fraction Collection and Analysis..............................................................11
Mass Balance: Whole Tree Harvest and Calculations ...........................................13
Soil N Sampling .....................................................................................................15
Statistical Analysis .................................................................................................16
Results ............................................................................................................................17
Growth Fraction %N and C:N ...............................................................................17
Mass Balance: Whole Tree Harvest and Calculations ...........................................19
Isotopic Approach to Estimate Uptake Versus Reallocation .................................21
Soil N Mineralization.............................................................................................22
Discussion ......................................................................................................................23
Leaf Level Measurements ......................................................................................23
Whole Tree Nitrogen Dynamics ............................................................................24
Spruce Budworm Outbreak....................................................................................27
Foliar δ15N to Predict Storage Versus Uptake .......................................................28
Soil N and Climate Conditions ..............................................................................29
Acknowledgements ........................................................................................................30
Tables .............................................................................................................................31
Figures............................................................................................................................32

3. CLIMATE AND INVASION DRIVE SOIL NUTRIENT DYNAMICS IN

TROPICAL MONTANE FORESTS OF THE GALÁPAGOS ARCHIPELAGO........42
Contribution of Authors and Co-Authors ......................................................................42
Manuscript Information Page ........................................................................................43
Abstract ..........................................................................................................................44
Introduction ....................................................................................................................45
Methods..........................................................................................................................48
Site Description......................................................................................................48
Meteorological Data...............................................................................................49
Soil Sampling .........................................................................................................50
Plant Sampling .......................................................................................................51
 iv

TABLE OF CONTENTS CONTINUED

Statistical Analysis .................................................................................................51

Results ................................................................................................................................50
Soil Sampling .........................................................................................................51
Foliar N in Natives Versus Non-Natives ...............................................................54
Discussion ..........................................................................................................................54
Nitrogen Pools .......................................................................................................55
Phosphorus Pools ...................................................................................................57
Why are non-natives successful in the Galápagos? ...............................................58
Implications............................................................................................................58
Acknowledgements ............................................................................................................60
Tables .................................................................................................................................61
Figures................................................................................................................................63

4. CONCLUSIONS AND FUTURE WORK ....................................................................66

REFERENCES CITED ......................................................................................................68

 v

LIST OF TABLES

Table Page

1.1 Biomass Regressions .......................................................................................31

2.1 Galapagos Site Descriptions ............................................................................61

2.2 Model Selection ...............................................................................................62

 vi

LIST OF FIGURES

Figure Page

1.1 Storage Versus Uptake .....................................................................................32

1.2 Collection Methods ..........................................................................................33

1.3 Meteorological Data.........................................................................................34

1.4 %N ..................................................................................................................35

1.5 C:N ..................................................................................................................36

1.6 Estimated Growth Fraction Biomass ...............................................................37

1.7 Total N .............................................................................................................38

1.8 δ15N ..................................................................................................................39

1.9 Resin Exchangeable N .....................................................................................40

1.10 Net N Transformation ....................................................................................41

2.1 Site Map ...........................................................................................................63

2.2 Meteorological Data.........................................................................................64

2.3 Soil Nutrients ...................................................................................................65

 1

CHAPTER ONE

INTRODUCTION

Nutrient cycling within watersheds is controlled by feedbacks between plant

nutrient use and soil nutrient availability (Chapin III et al. 2011). Nutrients enter

ecosystems through weathering of parent material (Walker and Syers 1976; Houlton et al.

2018), biological fixation of atmospheric nitrogen (N) (Houlton et al. 2008), and

deposition from rainfall and dust (Chadwick et al. 1999). Once nutrients enter a system, a

large portion cycle internally. Some nutrients, like phosphorus (P), can be tightly bound

to soil particles, and others, such as organic and inorganic N, can be immobilized and

metabolized by microbes in the soil or taken up by plants. Nutrients cycle back into the

soil through leaf abscission, and the litter is broken into its original components through

microbial decomposition, where it is again available for microbial or plant uptake. As

internal ecosystem cycling occurs, nutrients also exit the system through leaching and

erosion by wind and water, or through gaseous emissions. Nutrient cycling is not a

globally uniform process, however. Climate strongly controls the rate at which internal

nutrient cycling occurs, and nutrient cycling rates are much faster in wet and warm than

in cool and dry ecosystems. Environmental constraints, including variability in nutrient

cycling, influence plants’ capacity to grow.

Nutrients limit plant productivity to some degree in all ecosystems (Vitousek and

Howarth 1991). However, plants acclimate to this limitation by gaining mycorrhizal

symbionts, growing longer roots, altering the physiological mechanism of nutrient

 2

uptake, or storing nutrients (Chapin et al. 2011). Most of our knowledge of long-lived

tree nutrition is based upon seedling trees grown under artificial conditions, upon

variation in soil nutrient pools that are then assumed to correlate with plant nutrient

uptake, or upon changes in foliar nutrient concentrations that are assumed to reflect

nutrient uptake rates on an annual basis. However, plant nutrition in long-lived

evergreens may differ from our general understanding of plan nutrition that was

developed by testing seedlings grown in greenhouses. For example, some evidence

suggests that evergreens growing in low nutrient ecosystems are not nitrogen (N) limited

because they have low photosynthetic rates per unit of leaf N (PNUE) (Field and Mooney

1986; Reich et al. 1998; Warren and Adams 2004). Evergreens potentially alter their

physiology and store N in excess (Warren and Adams 2004), and storage could permit

evergreen growth to be independent of N uptake and availability (Proe and Millard 1994).

However, little research has directly examined when evergreens take in the bulk of their

N in natural systems, so patterns of evergreen N uptake in conjunction with seasonal soil

nutrient availability are virtually unknown (though see Chapin and Kedrowski 1983;

Socci and Templer 2011). While storage of nutrients within evergreenss has been

examined, we have yet to link spatial and temporal patterns of N availability in soils to N

uptake in mature trees in a field setting.

While soil nutrient dynamics influence plant nutrient uptake, storage, and use,

plant functions also influence soil processes. This is especially noticeable in invaded

ecosystems, where non-native species trigger positive feedbacks in nutrient cycling (Liao

et al. 2008). For example, often times during plant invasion, total plant biomass increases,
 3

positively influencing litter biomass, litter decomposition rates, and C and N stocks in

soil organic matter. Then, net N transformation rates increase, leading to higher

concentrations of ammonium and nitrate in the soil. The increase in soil N availability

enhances net primary productivity (NPP), initiating further increases in biomass and

driving the positive feedback between plant invasion and changes in soil nutrient

dynamics (Ehrenfeld 2010). While general patterns of ecosystem change under plant

invasion are established, some specific ecologically and culturally significant systems

experiencing invasion have not yet been examined (Percy et al. 2016). Invaders under

varying climate regimes may inflict unexpected changes in ecosystem dynamics, and

establishing how specific ecosystems respond to land use change is important to inform

management practices in those systems.

By combining techniques from biogeochemistry and plant physiology, I have

explored basic physical and biological controls on nutrient dynamics. In chapter two, I

explore how soil N availability varies in conjunction with N storage and allocation in

Pseudotsuga menziesii growing in a montane forest of western Montana. In chapter three,

I address how climate variability and plant invasion influence soil N and P dynamics in

tropical montane forests of the Galapagos Archipelago. Because anthropogenic climate

change, land use change, and invasion are three of the major global change processes that

threaten temperate and tropical montane forests, understanding these basic process will

become increasingly important as plant invasions intensify and as climate continues to

change.
 4

CHAPTER TWO

LINKING NITROGEN ALLOCATION IN DOUGLAS-FIR TO SOIL NITROGEN

AVAILABILITY IN A WESTERN MONTANE CONIFER FOREST

Contribution of Authors and Co-Authors

Manuscript in Chapter 2

Author: Claire Qubain

Contributions: CAQ collected samples, performed chemical and statistical analyses, and
wrote the first draft of the manuscript.

Co-Author: Yuriko Yano

Contributions: YY secured funding, developed the sampling protocol, collected samples,

and provided feedback on the manuscript.

Co-Author: Jia Hu

Contributions: JH secured funding, developed the sampling protocol, helped with sample
collection, and provided feedback on the manuscript.
 5

Manuscript Information

Claire Qubain, Yuriko Yano, Jia Hu

Oecologia

Status of Manuscript: [Put an x in one of the options below, delete this]

X Prepared for submission to a peer-reviewed journal
____ Officially submitted to a peer-reviewed journal
____ Accepted by a peer-reviewed journal
____ Published in a peer-reviewed journal

Springer Berlin Heidelberg

 6

LINKING NITROGEN ALLOCATION IN DOUGLAS-FIR TO SOIL NITROGEN

AVAILABILITY IN A WESTERN MONTANE CONIFER FOREST

Claire Qubain1, Yuriko Yano1, Jia Hu2

1
Ecology Department, Montana State University, 310 Lewis Hall, Bozeman, MT 59717,
USA
2
School of Natural Resources and the Environment, University of Arizona, 1064 East
Lowell Street, Tucson, AZ 85712, USA

Email addresses:
[email protected] (C. Qubain)
[email protected] (Y. Yano)
[email protected] (J. Hu)

Key words: evergreenss, Pseudotsuga menziesii, storage, uptake, availability

Abstract

It is widely accepted that temperate ecosystems are nitrogen (N) limited; thus, the

magnitude of N available in the soil influences plant N uptake and assimilation. The main

objective of this study was to explore whole tree N uptake dynamics across a season and

to link N uptake with soil available N. We used a whole tree N mass balance approach to

infer seasonal changes of total N between multiple needle and stem cohorts and bole

tissue (referred to as growth fractions). Foliar nitrogen isotopes (δ15N) and net N

transformation rates in the soil served as independent measurements to predict if trees

were drawing from storage versus uptake to support their new needle growth. We

detected that Douglas-fir (Pseudotsuga menziesii var. glauca (Mayr) Franco) trees
 7

accumulated 51.09 ± 1.06 g N/ tree in 2016 and 89.40 ± 1.07 g N/ tree in 2017 in their

new needle growth. However, no significant drawdown of N from previous years’ growth

fractions coincided with the accumulation of N, suggesting that the N in new needles was

not reallocated from storage within the growth fractions tested. The depletion of foliar

δ15N before bud break suggested that mycorrhizae mediated uptake of N from the soil.

However, Douglas-fir did not synchronize N uptake with periods of high soil N

availability. Overall, our results demonstrated that mature Douglas-fir are drawing the

bulk of their N from the soil, which indicates efficient use of available N in the

ecosystem.

Introduction

It is widely accepted that temperate ecosystems are nitrogen (N) limited; thus, the

magnitude of N available in the soil influences plant N uptake and assimilation (Chapin

et al. 1988). N availability in soils, in turn, is controlled by numerous abiotic factors. In

higher elevation western U.S. forests, snow is a dominant abiotic factor that drives N

cycling. Throughout the winter, snow insulates the soil and allows soil temperatures to

remain above freezing so that N ammonification and mineralization can occur (Maurer

and Bowling 2014). As a result, N accumulates in the soils over winter because plants are

not taking it up, and is subsequently released in a large pulse during snowmelt (Brooks et

al. 1998). Given that N is an important nutrient for photosynthesis and growth, plants

should take up this large pool of N as soon as it is readily available. However, conifers

may not be using this readily available pool to support new growth (Nasholm and

Ericsson 1990; Proe and Millard 1994). In one study, Picea sitchensis relied on N taken
 8

up the previous autumn to support new growth before bud break, suggesting that an

asynchrony could develop between when N is available versus when trees actually take

up N to support new growth (Proe and Millard 1994). However, given that many of these

studies were conducted in controlled greenhouses or plantations, it remains unclear how

prevalent this asynchrony is under field settings where snow is the dominant form of

precipitation.

While available N in the soil is relatively easy to measure across time, as

demonstrated across a range of ecosystems, including tropical and temperate forests,

grasslands, and arctic and alpine tundra systems (Vitousek and Matson 1988; Davidson et

al. 1992; Turner et al. 1997; Jaeger et al. 1999; Schimel et al. 2004; Campbell et al.

2014), N uptake by plants remains more difficult to measure. The in-situ depletion

method, where intact roots are immersed in a solution of known N concentration, can

offer a direct measure of the magnitude of N uptake across a season, but the method can

be time intensive and difficult to implement at a larger spatial scale. Alternatively, a mass

balance approach helps to track changes in foliar nutrient concentrations across an entire

year to assess if N accumulating in new tissues is translocated from parts of the plant or

directly taken up by the roots. By accounting for changes in biomass in the branches of

Picea mariana across a season, Chapin and Kedrowski (1983) found that by bud break,

the current season’s growth had already accumulated 70% of its maximum N

concentration. Applying the mass balance approach at the whole tree level might allow us

to determine if trees use newly acquired N or N from storage to support new growth.
 9

While N uptake is difficult to measure directly, plant N uptake is mainly

controlled by water availability and temperature. (Chapin and Kedrowski 1983; Gessler

et al. 2002; Socci and Templer 2011). Nutrients move through the soil to the root

interface mainly via diffusion, and when soils dry down, nutrient transfer to roots slows

(Lambers et al. 1998; Matson et al. 2002). Similarly, low water availability constrains

transpiration, indirectly inhibiting N uptake (Gessler et al. 2002). N uptake can also be

inhibited by low soil temperatures and root damage in the spring (Millard and Grelet

2010; Sanders-DeMott et al. 2018). Thus, because N uptake is limited by temperature

early in the growing season and by low soil moisture later in the season, stored N may be

an important source supporting new growth. Furthermore, given that many plant storage

and uptake studies have focused on tundra ecosystems and eastern U.S. deciduous

forests, where midsummer moisture limitation rarely occurs, a different pattern of N

storage and uptake may exist in ecosystems where mid-season moisture is limiting.

Plants may store N throughout different plant parts, including stems, branches, old

leaves, new leaves, buds or roots. In evergreen plants, the main N storage pool is likely in

old needles (Chapin 1980), in the form of protein, where it can represent up to 70% of

total N (Millard 1988; Näsholm and Ericsson 1990). Rubisco, which makes up 50% of

the total protein in C3 plants, is the main component of protein N storage (Chapin et al.

2011; Tegeder and Masclaux-Daubresse 2018). Along with protein storage, amino acids,

such as arginine, asparagine, and glutamine, as well as ribosomal proteins, play a role in

transient N storage across the season (Nasholm et al. 1994; Masclaux-Daubresse et al.

2017). However, it is still uncertain if these pools are always reallocated to support new
 10

growth, particularly in evergreenss growing in snow-dominated ecosystems with dry

midsummer conditions. The mass balance approach has been applied to track net changes

in foliar N in order to detect storage mobilization within plants (Chapin 1980). If trees

primarily use stored N to support new needle growth, then total N content in the past

season needles, the branches, or the bole are expected to decline as new growth

accumulates N throughout the season (Fig. 1.1a). However, if trees are primarily using

newly acquired N from the soil, then N in the past season needles, the branches, or the

bole are not expected to decline as new growth accumulates N throughout the season

(Fig. 1.1b).

Given that N uptake, storage, and remobilization are important processes that

regulate tree growth, refining our knowledge of these patterns is essential for our basic

understanding of how mature trees use N. Thus, the main objective of this study was to

explore whole tree N uptake dynamics across a season and to link N uptake with soil

available N. More specifically, we asked the following questions: 1) How much N is

remobilized from storage in evergreen trees versus how much N is newly acquired from

the soil in order to support new growth? 2) Do these patterns of N remobilization versus

uptake differ across an elevation gradient? 3) If trees do rely heavily on newly acquired

N, do seasonal patterns of N uptake synchronize with seasonal patterns of soil available

N? In order to address these questions, we applied an N mass balance approach in young

(40 – 80 yrs.) Douglas-fir trees growing in western Montana to estimate changes in N of

different growth fractions within an entire tree (Hansen et al. 1991). While this approach

has been successfully demonstrated in shrubs (Chapin III et al. 1980; Gray and
 11

Schlesinger 1983) and in trees grown on plantations (Nambiar and Fife 1991), this is one

of the first studies to use this approach on whole trees in the field.

Methods
Site Description

The study was conducted during the 2016 and 2017 growing seasons in a mixed

conifer forest of the North Fork of Elk Creek (NFEC) watershed at Lubrecht

Experimental Forest in western Montana, USA. We sampled both trees and soils on

southeast facing hillslopes at both a high (c. 1700 m) and a low (c. 1400 m) elevation site.

The mean temperatures from 1981 to 2010 were 4.2 °C and 3.0 °C at the low and high

elevation sites, respectively. Since 1970, snowfall represents 24% and 41% of annual

precipitation at low and high elevations, respectively (NRCS SNOTEL, stations 604 and

657). The soil types ranged from extremely gravelly sandy loam and gravelly sandy loam

to gravelly ashy loam, depending on sampling site and soil depth (NRCS, 2017). To

record air temperature, relative humidity, and soil volumetric water content (VWC) at

both sites, we used VP3 and 5TE sensors connected to EM50 data loggers recording at

30-minute intervals (METER group, Pullman, WA). To record precipitation, we used a

tipping bucket rain gauge (METER, Pullman, WA). The data logger at the high elevation

site malfunctioned between mid August and the end of September of 2017, so only low

elevation data is reported for that period.

Growth fraction collection and analysis

In order to examine seasonal changes in percent N (%N), carbon and N ratio

 12

(C:N), and δ15N among different plant parts, we collected different growth fractions,

including needles, buds, stems, litter, and bole tissue from 10 Douglas-fir throughout the

2016 and 2017 growing seasons. Five trees were located at the high elevation site and

five were located at the low elevation site. In 2016, we sampled 13 times, starting in mid-

April and ending in the beginning of November. In 2017, we sampled 12 times, starting

at the end of February and ending in mid-September. During the February sampling

period in 2017, only trees at the low elevation site were sampled because snow had not

melted at the high elevation site, and trees there were considered dormant. Litter samples

were collected using litter traps beginning in September 2016. During each sampling

period, we collected one approximately 3.4 cm long bole sample using a tree borer. We

also collected sun needles from three small branches in the middle canopy using a pole

pruner (Fig. 1.2-1). Directly after clipping, we divided needles and stems by cohort (Fig.

1.2-2). In 2016, the samples were divided between buds or current year (CY) needles,

(depending on the timing of bud break relative to sampling period), previous year (CY-1)

needles, CY-1 stems, and CY stems if present. In 2017, we divided samples into buds or

CY needles, CY-1 needles, two-year-old (CY-2) needles, CY-1 stems, CY-2 stems, and

CY stems if present. New stem growth did not suberize until August in both years, so

new stem growth was considered needle material until the August sampling date for both

years. We determined the beginning and end of each needle and stem cohort by the

annual bud-scale scars on the stems. If both buds and needles were present during a

collection period, both growth fractions were collected for that period. Bud break

occurred during the week of May 31 in 2016 and June 15 in 2017 at the low elevation
 13

site, and during the week of June 15th in 2016 and June 25 in 2017 at the high elevation

site.

After collection, all samples were transported back to the lab and dried at 60°C

for 48 hours. All growth fractions except for the bole tissue were homogenized and hand

ground using liquid nitrogen. Bole samples were ground in a cyclone sample mill (UDY

corporation, Colorado, USA) and then pulverized using a tissue lyser (Quiagen

TissueLyser II, Hilden, Germany) for 12 minutes. After all samples were ground and

weighed, the needles and buds were analyzed for total N, total C, δ15N, and δ13C content

using continuous flow dual isotope analysis with a CHNOS Elemental Analyzer

interfaced to an IsoPrime100 mass spectrometer at the University of California Berkeley

(Fig.1.2-3). Because woody tissues were typically too low in N for accurate isotope

analysis and because we did not think these fractions would be as dynamic across the

growing seasons, stem, bole, and needle litter samples were only analyzed for total N and

total C using a CHNOS Elemental Analyzer at Montana State University.

Mass balance: whole tree harvest and calculations

While most studies focus on using parameters such as percent N or C:N to infer

seasonal patterns of N allocation, we used a whole tree N mass balance approach by

multiplying percent N by the total estimated mass of the different growth fractions. In

order to estimate total biomass of each growth fraction, at the end of July 2016, we

harvested six trees, ranging from 7.5-15.8 cm diameter at breast height (DBH) (Fig 1.2-

4). We divided subsets of six branches from each tree (three each from the upper and

lower canopy) into separate growth fractions (bole, branches, CY needles, CY-1 needles,
 14

CY stems, CY-1 stems, buds, all other needles, and all other stems) and reserved a subset

of the bole. We weighed the entire cut tree in the field and then brought samples back to

the lab to dry at 60 °C. The needles and stems were dried for one week and the boles

were dried for two months.

Because we could not sample the entire tree, we estimated the mass of each

growth fraction in a whole tree from a subset of branches, three upper canopy branches

and three lower canopy branches) (Fig 1.2-4). From these six branches, we estimated the

total dry mass of each growth fraction for an entire tree. We then developed an allometric

relationship between DBH and each growth fraction’s dry mass (Fig 1.2-5). We applied

this allometric relationship to the 10 trees that we continuously sampled from (hereafter

referred to as N-collection trees) to estimate the dry mass of CY needles, CY-1 needles,

CY-1 stems, and bole tissue (Fig. 1.2-6). Because we only collected bole samples from

the outer 3.4 cm, we divided the total predicted bole dry mass by the tissue sampled

relative to the entire DBH of the tree. In order to calculate whole tree N content, we

multiplied the estimated biomass of each growth fraction by the N concentration of each

growth fraction from each sampling period. Dry mass estimates of CY-1 stems were

applied to CY-1 stems in 2016 as well as CY-1 and CY-2 stems in 2017. Dry mass

estimates of CY needles were applied to CY needles in 2016 as well as CY and CY-1

needles in 2017. Dry mass estimates of CY-1 needles were applied to CY-1 needles in

2016 and CY-2 needles in 2017.

We harvested trees in late July to ensure that new needles were fully elongated

(maximum biomass); however, we needed to estimate the change in biomass as new

 15

needles flushed. We used published data to estimate the percent of maximum elongation

in new growth as a function of the number of days after bud break (Emmingham 1977),

making the assumption that the trees in our environment followed similar patterns of bud

break phenology as those in a central Cascades environment. To estimate bud dry mass,

in March 2017, we collected one branch from the middle canopy of three separate trees at

the same site where trees were harvested in July 2016. We divided the branches between

CY buds, CY-1 needles, CY-1 stems, CY-2 needles, and CY-2 stems to record the wet

mass of each fraction. After drying, we estimated the mean ratio of bud dry mass to CY-

21 needle dry mass as 0.0757. We then calculated bud biomass estimates for the N-

collection trees by multiplying the CY-1 biomass predicted from the allometric

relationship times the bud dry mass ratio calculated.

Lastly, to scale litter N to the whole tree level, we estimated how many litter

traps, which were approximately 0.13 m2, could fit under each tree canopy and multiplied

that area times the total dry biomass of each litter sample after each collection. The crown

area of each tree was estimated using relationships developed between crown diameter

and DBH following Curtis and Reukema (1970).

Soil N Sampling

To measure N availability, we used an ion exchange resin (IER) probe method, as

well as the buried bag approach (Eno 1960). At both high and low elevation sites, every

month, from April until August 2016, eight soil IER probes (4 cation and 4 anion probes)

1
CY-2 needles sampled in 2017 and CY-1 needles sampled in 2016 are the same needle cohort
that flushed in 2015.
 16

were inserted at six locations on the same hillslopes where sampled trees were growing.

Additionally, monthly from April through October 2017, we inserted four IER probes (2

cation and 2 anion probes) and one buried bag of soil under the canopy of each N-

collection tree. After removal, the probes were kept cool and cleaned with distilled water

within 48 hours, refrigerated until the end of the season, and then analyzed for NH4-N

and NO3-N using flow injection analysis (Western Ag Inc.).

To carry out the buried bag approach, we collected pairs of soil samples to a 15

cm depth from under each N-collection tree from May until October 2017. One of the

samples was buried in a plastic bag under the canopy for a one-month incubation period,

and the other was cooled and transported back to the lab for processing. After the month

long incubation period was over, we removed the buried samples. To process both sets of

samples, we extracted the samples using 1M KCl within 48 hours of sampling. Extracts

were gravity filtered through p8 coarse paper filters then syringe filtered through 0.7 um

glass filter tips. After filtration, the samples were analyzed for ammonium (NH4+) and

nitrate (NO3-) using flow injection analysis (Lachat Quik-Chem Series 2400, Colorado).

Statistical Analysis

For the biomass scaling, we fit linear models to each separate growth fraction in R

(R Core Team 2017). We tested how dry mass in CY needles, CY-1 needles, CY-1 stems,

and boles varied as a function of DBH. In these models, and all following models,

normal-QQ plots and residual plots were used to visually assess how each model met the

assumptions of normality and constant variance, and both CY needle dry mass and CY-1

needle dry mass were log transformed to meet those assumptions. One outlier was
 17

removed from the CY-1 stem model because it had a Cook’s distance value greater than

one.

For the %N, total N, C:N, and δ15N response variables, we fit linear mixed effects

models using the lme function in R (Pinheiro et al. 2014; R Core Team 2017). To select

each model, we started with full models of three way interactions between fixed effects

of a second order polynomial of days after flush (where negative values were before bud

break and positive values were after bud break each growing season), growth fraction,

and elevation, and a random effect of individual tree. A parameter or interaction was not

included in the model if it had a p-value > 0.05. After using this stepwise model selection

process, we presented models testing how each response variable (%N, C:N, total N, and

δ15N changed as a function of an interaction between a 2nd order polynomial of days

after flush and growth fraction, after controlling for the random effect of individual trees.

All response variables except δ15N were log transformed to meet the assumption of

constant variance. In 19 bole samples, %N was too low to measure using the elemental

analyzer, so those samples were treated as not available (NAs) in the model. To report

contrasts in the response for each growth fractions across time, we used the predict.lme

function (Pinheiro et al. 2014).

We used linear mixed effects models to separately examine NH4+ and NO3- net

transformation rates collected using both the buried bag and IER probe methods. In each

model, we tested how the log transformation of both NH4+ and NO3- varied as a function

of time and elevation, after accounting for the random effect of sampling location. The N

transformation rates using the IER probe method were modeled separately for 2016 and
 18

2017 because the probes were collected from two different locations, and the net N

transformation rates using the buried bag approach were only collected and analyzed for

the 2017 season.

Results
Growth fraction %N and C:N

The timing of snowmelt between the high and the low elevation sites can differ by

almost one month (Yano et al., Under Review), leading to differences in the timing of

bud break. Therefore, in order to compare the %N and C:N ratios of the different growth

fractions, we normalized time on the x-axis to reflect before and after bud-break. We first

examined differences in %N between the two different elevations using the following

linear mixed effects model:

log(%N of growth fractions sampled) = ß0 + ß1time + ß2time2 + ß3Ig=CYn + ß4Ig=CYbd +

ß5Ig=CY-1n+ ß6Ig=CY-1s + ß7Ig=CY-2n + ß8Igrowth=CY-2s + ß9Ig=l + ß10Ig=b + ß11time2 * I g=CYn +
ß12time2 * Ig=CYbd + ß13time2 * Ig=CY-1n + ß14time2 * Ig=CY-1s + ß15time2 * Ig=CY-2n + ß16time2 *
Igrowth = CY-2s + ß17time2 * Ig=l + ß18time2 * Ig =b + ß19Ielev = low + ß20Ielev = high + treei + εij,
Treei ~ N(0, σ2), εij ~N(0, , σ2ε)

where g = growth fraction, CY = current year, n = needle, bd =bud, s = stem, l = litter, b

= bole, and elev = elevation, εij = residual variability of the jth occasion for the ith subject,
and N (0, σ2) denotes that errors are normally distributed with a mean of 0 and a variance
of σ 2.

Each year was modeled separately to better meet the assumptions of normality and

constant variance. We found that median %N in all the growth fractions was 1.13%

higher at the high elevation than at the low elevation site in both 2016 (χ2 (1, 8 D.F.) =

35.48, p-value < 0.001) and 2017 (χ2 (1, 8 D.F.) = 23.17, p < 0.0001). Furthermore,

median %N in each growth fraction was dependent on the number of days after bud break

(χ2 (12, 518 D.F.) = 27.38, p-value < 0.0001 in 2016 and χ2 (14, 737 D.F.) = 19.37, p-
 19

value < 0.0001 in 2017). Among the different growth fractions, %N was highest in buds

and CY needles during the bud break period, followed by CY-1 needles and CY-2

needles, and CY-1 stems (Fig. 1.4). Lowest %N was found in bole tissue, litter, and CY-2

stems (Fig. 1.4).

As trees increase biomass in the spring while also acquiring N to support new

growth, stoichiometric variability (e.g. C:N) among different growth fractions may

provide insight into the controls of biomass on N concentrations. We used the following

linear mixed effects model:

log(C:N) = ß0 + ß1time + ß2time2 + ß3Ig=CYn + ß4Ig=CYbd + ß5Ig=CY-1n+ ß6Ig=CY-1s + ß7Ig=CY-

2 2 2
2n + ß8Igrowth=CY-2s + ß9Ig=l + ß10Ig=b + ß11time * I g=CYn + ß12time * Ig=CYbd + ß13time *
2 2 2 2
Ig=CY-1n + ß14time * Ig=CY-1s + ß15time * Ig=CY-2n + ß16time * Igrowth = CY-2s + ß17time *
Ig=l + ß18time2 * Ig =b + ß19Ielev = low + ß20Ielev = high + treei + εij, Treei ~ N(0, σ2), εij ~N(0, ,
σ 2 ε)

Each year was modeled separately to better meet the assumptions of normality and

constant variance. We first examined differences in C:N between the high and the low

elevation and found that the absolute C:N ratio was 0.89 greater at the high elevation than

at the low elevation in 2016 (95% C.I. of 0.85 to 0.93) and 0.91 greater (95% CI of 0.87

to 0.95) in 2017 after accounting for growth fraction and time. We also examined C:N

among the different growth fractions and found that similar to %N, the C:N ratio in each

growth fraction was dependent on the number of days after bud break (χ2 (12, 534 D.F.) =

22.80, p-value < 0.0001 in 2016 and χ2 (14, 746 D.F.) = 22.02, p-value < 0.0001 in 2017).

In both years, the bole had the highest C:N ratio, followed by litter, CY-2 stems, CY-1

stems, CY-2 needles, and CY-1 needles (Fig. 1.5). Across the year, bud C:N declined

prior to bud break, reaching the lowest point just prior to bud break, but then increased
 20

following bud break; this suggests that the addition of new biomass following bud break

diluted the N signal. The C:N ratio in bole tissue also declined before and after bud break,

and C:N in CY-1, and to a lesser degree in CY-2 stems, increased (Table 1.1).

Mass balance: whole tree harvest and calculations

From our whole tree harvests, we developed allometric relationships between

DBH and mass of the different growth fractions (Fig. 1.5). Strong evidence suggested

that the dry mass of CY needles, CY-1 stems, and bole tissue varied as a function of

DBH, but weak evidence suggested that the dry mass of CY-1 needles varied according

to DBH (Table 1.1). For every 1 cm increase in DBH, dry biomass was predicted to

increase by 1.40 Kg in CY needles (95% CIs of 1.09 – 1.79), 1.35 Kg in CY-1 needles

(95% CIs of 90.09 to 1.98), 0.12 Kg in CY-1 stems (95% CIs of 0.02 to 0.22), and 4.0

Kg in bole tissue (95% CIs of 1.2 to 6.48 Kg).

Using a mass balance approach, we followed pools of N among different growth

fractions within an entire tree throughout two growing seasons. We used the following

linear mixed effects model:

log(Total N) = ß0 + ß1time + ß2time2 + ß3Ig=CYn + ß4Ig=CYbd + ß5Ig=CY-1n+ ß6Ig=CY-1s +

ß7Ig=CY-2n + ß8Igrowth=CY-2s + ß9Ig=l + ß10Ig=b + ß11time2 * I g=CYn + ß12time2 * Ig=CYbd +
ß13time2 * Ig=CY-1n + ß14time2 * Ig=CY-1s + ß15time2 * Ig=CY-2n + ß16time2 * Igrowth = CY-2s +
ß17time2 * Ig=l + ß18time2 * Ig =b + ß19Ielev = low + ß20Ielev = high + treei + εij, Treei ~ N(0, σ2),
εij ~N(0, σ2ε)

Each year was modeled separately to better meet the assumptions of normality and

constant variance. Overall, after accounting for an interaction between days after bud

break and growth fraction, and a random effect of individual tree, we did not find a

difference in total N between elevations (p-value > 0.05) as we did for %N and C:N.
 21

However, similar to the %N results, we found that differences in total N in each growth

fraction were dependent on the day after bud break (χ2 (12, 516 D.F.) = 187.97, p-value <

0.0001 in 2016 and χ2 (14, 737 D.F.) = 73.89, p-value < 0.0001 in 2017). Before bud

break in 2016, CY-1 needles maintained the largest N pool, but after bud break, the CY

needles accumulated the most N out of all the growth fractions (Fig. 1.6). In 2017, most

N was in CY-1 needles prior to bud break. However, unlike in 2016, total N in CY

needles post bud break never surpassed that in CY-1 needles, even after CY needles in

2017 had accumulated 100% of their N. For the other growth fractions, total N did not

significantly change before and after bud break, although there were differences in total

N between the different growth fractions. We found highest total N in CY needles,

followed by CY-1 and CY-2 needles, bole tissue, CY-1 stems, CY-2, and buds. We

calculated that post bud break, CY needles accumulated a median of 51.09 ± 1.06 g N/

tree in 2016 and 89.40 ± 1.07x g N/ tree in 2017.

Isotopic approach to estimate uptake versus reallocation

We also used an isotope approach as an independent method to support our

estimations of uptake versus reallocation of N by analyzing the δ15N of buds and needles.

We used the following linear mixed effects model:

δ15N = ß0 + ß1time + ß2time2 + ß3Ig=CYn + ß4Ig=CYbd + ß5Ig=CY-1n+ ß6Ig=CY-2n + ß7time2 * I

2 2 2
g=CYn + ß8time * Ig=CYbd + ß9time * Ig=CY-1n + ß10time * Ig=CY-2n + ß11Ielev = low + ß12Ielev =
high + treei + εij, Treei ~ N(0, σ ), εij ~N(0, σ ε)
2 2

We did not detect a difference in mean δ15N between elevations (χ2 (1, 8 D.F.) = 4.36, p-

value = 0.07). However, significant differences in δ15N in each growth fraction were
 22

dependent on the day after bud break (χ2 (6, 601 D.F.) = 14.16, p-value < 0.0001). For

both CY and CY-1 needles, δ15N began to decline prior to bud break, but then increased

sharply around the period of bud break and new needle flush. We did not find differences

in mean δ15N between needle cohorts, except at the low elevation in 2017, where buds

were more depleted than CY-1 and CY-2 needles (Fig. 1.7).

Soil N Mineralization & Nitrification

Using a linear mixed model, we tested how transformations of NO3- and NH4+

responded as a function of burial period and site, after accounting for a random effect of

the sampling location. From the IER probes, we found a difference in NO3-N across the

seasons in 2016 (χ2 (1, 11 D.F.) = 11.2, p-value = 0.006) and in 2017 (χ2 (1, 49 D.F.) =

6.70, p = 0.01); we found a difference between elevations in 2017 (χ2 (1,8 D.F.) = 8.30,

p-value = 0.02 in 2017), but not in 2016 (p-value = 0.8 in 2016). After every additional

burial period, the rate of NO3-N nitrification increased by 1.02 mg/m2/month in 2016 and

1.0 mg/m2/month in 2017 (95% C.I. of 1.01 to 1.04 mg/m2/month in 2016 and 1.01 to

1.15 mg/m2/month in 2017). In 2017, NO3-N was 2.01 mg/m2/month higher at the high

elevation site compared to the low elevation site (95% CI of 1.15 to 3.66 mg/m2/month).

From the IER probes, we detected a difference in NH4-N across time in 2017 (χ2

(1,49) = 10.42, p-value = 0.002 in 2017), but not in 2016 (χ2 (2, 10) = 0.8, p-value = 0.4

in 2016), and no differences between elevations in 2016 or 2017 (χ2 (1,4) = 0.81, p-value

= 0.4 in 2016, χ2 (1,8) = 4.09, p-value = 0.07 in 2017). For every additional burial period,
 23

the median NH4-N increased by 2.05 mg/m2/month in 2017 (95% CI 1.15 to 3.65

mg/m2/month), and it was 2.34 mg/m2/month greater at the high elevation compared to

the low elevation site in 2017 only (95% CI of 0.88 to 6.16 mg/m2/month).

From the buried bag method implemented in 2017, net NO3-N and NH4-N

transformations were not different across time or between elevations (p-value = 0.2 and

0.6, respectively, for NO3-N, and p-values = 0.95 and 0.73, respectively, for NH4-N).

Discussion

N is a limiting nutrient for many temperate plants, and evergreens can potentially

store large quantities, allowing them to be buffered from inter-annual variation in N

availability (Warren and Adams 2004; Du et al. 2018). However, in our study, we found

that Douglas-fir used N acquired from belowground during the present growing seasons,

not from storage in older growth fractions, to support new needles. Tissue N

concentrations and C:N ratios in separate growth fractions were lower in trees growing at

a lower elevation, but scaling nutrient content to the whole tree level removed any

elevation difference found in foliar N concentrations, which was just a function biomass

in whole trees. Patterns of seasonal N uptake seemed to synchronize with periods of high

N availability in the soil, as determined by observing foliar δ15N and soil N availability

across the growing seasons.

Leaf Level Measurements

Biomass accumulation, resulting from needle expansion during the growing season,
 24

was the most important factor controlling changes in foliar %N and C:N ratios. Biomass

accumulation is important because new needles become a carbon source for the rest of

the tree for the remainder of the growing season. In buds, %N increased until bud break,

when buds became CY needles and %N decreased, likely due to dilution by the addition

of biomass across the seasons (Chapin and Kedrowski 1983; Fife and Nambiar 1984)

(Fig. 1.4). C:N decreased in buds until bud break and then increased in CY needles until

the end of the season, further demonstrating that the addition of C diluted the foliar N

signal as needles flushed (Fig. 1.5).

Our findings are in line with reported N concentrations in studies of other evergreens.

Percent N in needles from Pinus radiata seedlings decreased from 2.6% at bud break to

0.8% when they were three years old (Fife and Nambiar 1982). Though minimum %N in

our study was around 1%, we still observed maximum values near those in the Pinus

radiata study directly after bud break. Our findings of %N in all the remaining needle

cohorts were about 0.5% greater than Pseudotsuga menziesii saplings raised in growth

chambers (Hobbie et al. 2001). Compared with Ledum palustre, an evergreen shrub

observed in taiga field settings, %N in our observations was 1.5% lower in CY needles

directly after bud break, but that cohort maintained similar dilution by C across the

growing season (Chapin et al. 1980). In the same study, %N in CY-1 needles and CY-1

stems were 0.5% and 1.0% higher, respectively, across the season than presented here. In

general, foliar nutrient concentrations fell within the range of 10 mg g-1 to 25 mg g-1

found for evergreens summarized from 92 studies (Aerts and Chapin 1999).

Whole Tree Nitrogen dynamics

 25

The storage of resources, including nutrients, is an important plant function; however,

some studies have found that evergreen species depend less on stored N and instead rely

on N reabsorbed from litter or newly acquired N from the soil (Chapin 1980; Aerts and

Chapin1999). Because total N did not decline in older needle and stem cohorts or in bole

tissue, we determined that most of the N supporting new growth in Douglas-fir was not

drawn from N stored in those growth fractions. Instead, our results suggest that the trees

were using newly acquired N from the soil to support new growth.

Evergreens do not always draw down N storage from old leaves to support new

growth (Chapin et al. 1990; Aerts and Chapin1999). In a tundra ecosystem, while 27 –

41% of the total N in new leaves of deciduous shrubs was drawn from storage, none of

the total N in the new growth of an evergreen shrub, Ledum palustre, was drawn from

storage in past season needles (Chapin et al. 1980), but no speculation as to where N was

provided from was presented. In a similar study, even after old leaves were manually

defoliated from evergreen species in tundra and Mediterranean environments, N

accumulation in new leaves still continued (Jonasson 1989), further demonstrating that N

to support new growth in evergreens was not derived from N stored in old leaves.

On the other hand, some studies have shown that evergreenss allocate N from storage

to support the current season’s growth, indicating an uncoupling of foliar N from soil

nutrient variability across seasons. For example, Ceanothus megacarpus, an evergreen

shrub, used stored N to avoid nutrient limitation during soil dry down or when soil

nutrients were not readily available (Gray 1983; Gray and Schlesinger 1983). Ceanothus

even stored more N than deciduous species (Chapin III and Shaver 1989), which is
 26

uncommon, though this could be due to the species’ N-fixation strategy (Gray 1983).

From a greenhouse study using an isotope tracer method, Picea sitchensis seedlings used

N from the previous season to support current season needles (Proe and Millard 1994).

Lastly, using a mass balance approach, Chapin and Kedrowski (1983) observed that old

needle N in Picea mariana decreased throughout the summer and increased again by

winter, indicating that those trees were using and then replenishing stored N, 70% of

which had already accumulated before bud break (Chapin and Kedrowski 1983).

Differences in storage versus uptake patterns between this study and others are mostly

likely due to differences in climate, growth habit, and life stages.

The mass balance approach has limitations (Nambiar and Fife 1991; Proe and Millard

1994). The method only allows for accounting net nutrient transfer within the plant, not

partitioning of N between each needle or stem cohort, potentially confounding the

detection of a draw down in N storage (Proe and Millard 1994). Secondly, if stored

instead of newly acquired N is supporting growth, the time when the stored N was taken

up can not be detected. This study also has specific limitations. We assumed that roots

and needles older than three years provided minimal N to support new growth because

total N in Pinus radiata roots did not fluctuate seasonally (Nambiar 1987), and because

nutrient pools in older needles are relatively inert (Aerts and Chapin III 1999). Some N,

however, could have been reallocated from roots or needles and stems older than three

years that we did not detect. Secondly, there were uncertainties surrounding our whole

tree biomass estimates. Because we only estimated biomass at one point in time, we had

to assume that the same amount of new biomass was added in 2017 as it was in 2016,
 27

including bud biomass, and that new needle elongation occurred at the same rate as

published values from tree populations from the central Cascade Mountains. Because

Douglas-fir needles live for six to eight years (Balster and Marshall 2000), we also

assumed no needle abscission occurred in CY-1 and CY-2 needles, likely leading to some

overestimation of total N in those fractions. Despite the assumptions made, the mass

balance approach still allowed us to estimate how much N mature trees growing under

field conditions accumulate across two growing seasons.

Spruce Budworm Outbreak

The year before study began, a western spruce budworm (Choristoneura

occidentalis) outbreak occurred across forests in the state of Montana, and a large

percentage of the CY-1 needles in 2016 were defoliated (Qubain, C., personal

observation). Spruce budworm can defoliate between 8% and 17% of a tree’s gross

volume in a given outbreak (Alfaro et al. 1985), and outbreaks are most severe in

drought-stressed trees with relatively low foliar N concentrations (Cates et al. 1983;

Redak and Cates 1984). During the season prior to our first year of sampling (2015),

defoliation from spruce budworm was severe, and during our two sampling seasons

(2016 and 2017), defoliation was present but minimal. The outbreak could have affected

our conclusions regarding tree N origins across growing seasons. We predicted that if

trees were drawing stored N to support new growth from the previous season’s needles,
 28

then we would detect little draw down in total N in CY-1 needles after severe defoliation

(2016) but significant draw down in the same needle cohort after mild defoliation (2017).

This pattern would suggest that the trees were not accessing N storage because that

cohort was nearly gone after the severe outbreak, not because the trees do not use stored

N in an undisturbed state, as we discovered. After not detecting a draw down of total N in

CY-1 needles following the severe or the mild defoliation events, and knowing that

artificial defoliation did not affect the use of stored N in Pinus radiata (Jonasson 1989),

we concluded that budworm defoliation did not influence whether the trees studied here

were using stored N or not. However, because total N in CY-1 needles during 2016 was

so much lower than in 2017 (Fig. 1.7), we did determine that the outbreak affected the

total size of the N pool in that needle cohort and could have affected the trees’ ability to

fix C.

Foliar δ15N to predict storage versus uptake

Using an alternative and independent isotope method to assess N acquisition by

Douglas-fir trees, we found further support that trees were using newly acquired N from

the soil to support new growth. We observed that foliar δ15N became more negative

between the beginning of the season and bud break, and then became more positive

between bud break and the end of the growing season (Fig. 1.8). Though naturally

abundant 15N is notoriously difficult to interpret (Hogberg 1997), the consistent temporal

pattern in foliar δ15N fractionation between needle cohorts, elevations, and seasons we

detected indicates a clear pattern of N uptake. The temporal patterns in foliar δ15N
 29

fractionation likely reflect mycorrhizal mediation of N from the soil, where mycorrhizae

were readily transferring N to the trees before bud break and then slowed the transfer

between bud break and the end of the season After uptake from the soil, mycorrhizae

incorporate the lighter isotope into proteins and amino acids because it is kinetically

easier to use a lighter molecule. The N transferred to the plant from the mycorrhizae is

relatively depelted in 15N, which is expressed in the foliar N (Hobbie and Högberg 2012).

This finding is supported by a study examining mycorrhizal presence in a similar forest

community in western Montana, where ectomycorrhizal root tips were most abundant in

May and June (around the timing of the depletion event in this study) and least abundant

in July and August (which correlates with the enrichment event after bud break here)

(Harvey et al. 1978). Fractionation does not occur during plant uptake from bulk soil

when N concentrations are low, as they were here, or when mycorrhizae are not present

or active, so we would not have detected uptake without mycorrhizal mediation (Handley

and Raven 1992; E. Dawson et al. 2002; Hobbie and Högberg 2012). While reallocation

of stored N from old to new leaves in deciduous species did cause fractionation (E.

Dawson et al. 2002; Kolb and Evans 2002), we did not detect a movement of stored N in

this study, so we assumed that no fractionation occurred within the tree once the N was

acquired from the soil.

Without examining mycorrhizal activity directly, other factors, such as variation in

rooting depth or uptake of different forms of N could have contributed to variation in

foliar δ15N in this study (E. Dawson et al. 2002). However, these processes are unlikely to

have occurred. Douglas-fir trees at this site use water from the upper meter of the soil
 30

profile (Martin, J.T., Personal Communication), and because nutrient uptake is so closely

linked to water use, the trees studied are likely drawing nutrients from the upper meter or

higher of the soil. Secondly, because plants take up the form of N that is most readily

available in the soil (Lambers et al. 1998), and NH4+ is an order of magnitude more

concentrated than NO3 – at this site (Yano et. al. Under Review), NH4+ is likely the main

N form used across the season. So, variation in rooting depth and the form of N used

from the soil likely had little influence on the temporal variation in the δ15N patterns we

observed across the seasons.

Soil N and climate conditions

Concentrations of N belowground must be high enough to supply the trees with

their estimated annual N budget. The buried bag approach indicated that microbial

immobilization of N was more dominant than mineralization in this ecosystem (Fig. 1.9).

Further, the rate of NH4+ and NO3 – adsorption to ion exchange resins increased across the

growing season, indicating that mineralization rates were highest towards the end of the

growing season and lowest during the beginning of the season (Binkley and Matson

1983; Krause and Ramalal 1987). Thus, soil N availability and Douglas-fir N

accumulation did not synchronize. Nevertheless, they were still able to accumulate most

of their N from belowground, even when N availability was low.

In conclusion, our findings suggest that evergreen trees growing in snow-

dominated ecosystems rely upon N taken up from the soil during the present growing

season to support new needle growth. While soil N was adequate to support the trees’

accumulation of N in new growth, they did not synchronize soil N availability with N
 31

uptake. By accessing N from the soils, trees preclude the loss of nutrients from the

system they grow in, maintaining relatively closed cycling of N within the watershed.

Acknowledgements

Thank you to technicians E. Anderson, T. Simpson, C. Dart and C. Moss for their

hard work collecting and processing samples. Thank you to J. Klassen for her guidance

with chemical analysis.

Tables

Growth Equation F-stat D.F. P-Value R2

Fraction
CY Needles Log(Dry Mass) = ß0 + ß1DBH 14.76 1, 4 0.01 0.78
CY-1 Needles Log(Dry Mass) = ß0 + ß1DBH 5.06 1, 4 0.08 0.56
CY-1 Stems Dry Mass = ß0 + ß1DBH 15.5 1, 3 0.02 0.83
Bole Dry Mass = ß0 + ß1DBH 20.09 1, 4 0.01 0.83

Table 1.1 – Output from linear models used to predict the biomass of each growth
fraction in the N-collection trees. CY stands for current year and DBH stands for
diameter at breast height.
Figures

N reallocated from past to

current season growth (a)
Total N in Growth Fraction

N from uptake to current

season growth (b)

32
Past season
needle, bole, or
stem
Current season
needle
50 300
Day of Year

Figure 1.1 – Conceptual figure of potential expected outcomes, where a) represents a drawdown of stored N from the past season
growth and b) represents that the tree bypasses storage and instead uses newly acquired N from the soil.
 N-Collection trees Harvest Trees
n = 10 n=6
1. 4.

x3 x3
2.

3. Analyze for
%C and %N

33
Dry Mass (Kg)
5.
6.
%N x Estimated growth fraction dry mass
= Total N in whole tree growth fraction

DBH (cm)

Figure 1.2 – Biomass harvesting methods used to calculate whole tree total N.
 34
Figure 1.3 – 5-day moving averages of temperature (˚C) and relative humidity (%) are shown in the top two panels, and daily
cumulative precipitation (mm) and the daily volumetric water content (VWC) averaged by 10 cm, 30 cm, and 50 cm soil depths are
shown in the bottom two panel. The broken x-axis between years represents the overwinter period.
 35
Figure 1.4 - Tissue %N is represented across time relative to bud break (n = 5) with standard errors. The top panel presents the
changes of %N in separate growth fractions at the low elevation site, and the bottom panel presents the changes of %N at the high
elevation site. Day 0 represents the day of bud break.
 36
Figure 1.5 – C:N at the low elevation site (top panel) and at the high elevation site (bottom panel) show that dilution of N from an
increase in biomass, C, occurs as new needles grow. Points are mean of 5 ratios, and error bars represent the standard errors. Note the
split y-axis and different y-axis scales between the bole tissue and all other growth fractions.
 37
Figure 1.6 – Predicted biomass of each growth fraction in the trees that N was sampled. Values were extracted from the allometric
relationships developed from growth fraction biomass as a function of diameter at breast height. Note: total biomass for the bole only
represents the outer 3.4 cm).
 38
Figure 1.7 - New needles at both the low elevation site (top panel) at and at the high elevation site (bottom panel) accumulated N
without drawing down N from older growth fractions. Points are means of 5 samples, and error bars represent the standard errors.
 39
Figure 1.8 - δ15N in all growth fractions sampled became more depleted around bud break and then more enriched following the
depletion event. Points are means of 5 samples, and error bars represent the standard errors.
 40
Figure 1.9 – Exchangeable N from the IER probes at the low elevation (top panel) and at the high elevation (bottom panel) was
generally low during the early growing season and then increased towards the end of the season. Points represent the means of five
samples buried for a month long period, and error bars represent the standard errors.
 41
Figure 1.10 – Net transformation rates from the buried bags at the low elevation (left panel) and the high elevation (right panel)
showed no statistical differences across the 2017 season. Each point represents the mean of five samples buried for a month long
period, and error bars represent the standard errors.
 42

CHAPTER THREE

CLIMATE AND INVASION DRIVE SOIL NUTRIENT DYNAMICS IN

TROPICAL MONTANE FORESTS OF THE GALAPAGOS ARCHIPELAGO

Contribution of Authors and Co-Authors

Manuscript in Chapter 3

Author: Claire Qubain

Contributions: CAQ secured funding, developed the sampling protocol, collected

samples, performed the chemical and statistical analysis, and wrote the first draft of the
manuscript.

Co-Author: Diego Riveros-Iregui

Contributions: DR-I developed the sampling protocol, collected samples, secured

permits, and provided feedback on the manuscript.

Co-Author: Jia Hu

Contributions: JH secured permits, developed the sampling protocol, collected samples,

and provided feedback on the manuscript.
 43

Manuscript Information

Claire Qubain, Diego Riveros-Iregui, Jia Hu

Ecology

Status of Manuscript:
X Prepared for submission to a peer-reviewed journal
____ Officially submitted to a peer-reviewed journal
____ Accepted by a peer-reviewed journal
____ Published in a peer-reviewed journal

Ecological Society of America

 44

CLIMATE AND INVASION DRIVE SOIL NUTRIENT DYNAMICS IN

TROPICAL MONTANE FORESTS OF THE GALAPAGOS ARCHIPELAGO

Authors: Claire Qubain1, Diego Riveros-Iregui2, Jia Hu3

Institutions: 1Montana State University, 2University of North Carolina-Chapel Hill

3
University of Arizona

Keywords: climate, invasion, phosphorus, nitrogen

Abstract

We examined how climate and plant invasion influence soil nitrogen (N) and

phosphorus (P) in tropical montane forests on San Cristóbal Island, Galápagos. We

collected soils at the end of the warm and wet season and at the end of the cool and dry

season along an elevational gradient and under native and non-native plant canopies. The

elevation gradient represents a climosequence of temperature, precipitation, relative

humidity, and volumetric water content on San Cristóbal. We also analyzed plant tissues

for foliar N concentrations to compare native and non-native plant nutrient use.

Ammonium (NH4+) and phosphate (PO43-) increased along the elevational gradient while

nitrate (NO3-) was most concentrated at the high elevation. Compared to under non-native

plant canopies, NH4+ was 32% more concentrated under native canopies while NO3- was

43% more concentrated under native canopies. We did not detect any differences in PO43-

pools under native versus non-native plant canopies. Leaching and denitrification likely

caused export of NO3- during the wet season and at wetter elevations, while rapid

turnover rates and plant uptake induced seasonal and elevational differences in PO43-. We

hypothesized that NH4+ was not as variable seasonally because it is not as strongly
 45

controlled by leaching as are NO3- and PO43-. Overall, understanding nutrient dynamics

in the archipelago will allow land managers to decide how to control non-native plants as

well as to maintain an understanding of nutrient dynamics in a heavily invaded ecosystem

in the future.

Introduction

The processes of soil formation and biogeochemical cycling are driven by five

state factors: topography, parent material, time, climate, and potential biota (Jenny 1941).

Topography influences nutrient dynamics through leaching and erosion (Weintraub et al.

2014), while different parent materials alter the nutrient composition of overlying soils

(Yavitt 2000). Phosphorus is most abundant in young soils, while nitrogen (N)

transformation by microbes increases in older soils (Lambers et al. 2008b). Climate

influences nutrient dynamics both directly and indirectly through weathering, leaching

and erosion as well as through influencing microbial activity and nitrogen transformation

(Richardson et al. 2004; Chapin et al. 2011). In addition to the influence of climate, plant

community composition can also change biogeochemical cycling under vegetation

canopies (Zinke 1962; Sturm et al. 2005). Island ecosystems have served as natural

laboratories to conduct studies of pedogenesis because each formation process can be

more easily isolated. Islands present strong elevation gradients that drive climate

variability across a small area, develop along chronosequences within archipelagos, and

host different microbial and vegetation communities across space. While the Hawaiian

Islands have served as a central site for many studies of soil formation, other

archipelagos, such as the Galápagos, have similar characteristics but have received less
 46

attention. On San Cristóbal Island in the Galápagos Archipelago, climate and biota are

two key state factors that influence nutrient dynamics.

Climate patterns in the Galápagos are quite variable. The archipelago experiences

two seasons, the cool season (June –December), and the warm season (January – May),

due to an inter-annual migration of the Inter-Tropical Convergence Zone. Temperature

and precipitation vary along the elevation gradient and between the leeward and

windward sides of the islands (Snell and Rea 1999). The climo-sequence is divided

between the very arid to arid zone at low elevations, the transition zone, and the humid to

very humid zone at high elevations (Percy et al. 2016). Higher elevations are cooler and

receive more precipitation in the form of fog (Violette et al. 2014), while lower elevations

are dryer and typically only receive rainfall. However, the climate regime in the

Galápagos is predicted to change due to intensification of the El Niño Southern

Oscillation (ENSO) (Trueman and d’Ozouville 2010). The Galápagos will experience

more intense drought in La Niña years followed by prolonged rainfall events during El

Niño years. Longer droughts and prolific rainstorms will likely alter rates of parent

material weathering and soil development (White and Blum 1995), but we lack thorough

knowledge of soil processes on the islands to detect those changes. Therefore,

establishing basic knowledge of soil N and P chemistry on the islands will become more

important as the climate regime intensifies.

In addition to climate effects, plant invasions influence nutrient cycling in tropical

island ecosystems. In general, plant invasion can increase N availability, mineralization

rates, and litter decomposition rates (Ehrenfeld 2003). Invasive plants not only directly
 47

affect nutrient cycling, but they also trigger indirect positive feedbacks in soil nutrient

dynamics. Added nutrients and accelerated biogeochemical cycling due to introduced

species increase the abundance of additional non-natives (Ostertag and Verville 2002).

While most research suggests that invasive plants increase soil nutrient availability and

decomposition rates, not all areas respond following these patterns; some studies find

declines or no change in soil nutrient availability after plant invasion (Ehrenfeld and

Scott 2001). Therefore, examining invasion on an individual species or location basis will

allow for the detection of potentially unpredictable effects of plant invasion on ecosystem

processes. Furthermore, studying these basic nutrient dynamics could influence how land

managers implement native plant restoration.

In the Galápagos, plant invasions may alter biogeochemical processes; on one of

the older islands, San Cristóbal, 70% of the island is impacted by land use change and

plant invasion, and since 1987, 77% of the native vegetation across the islands has been

converted to non-native (Villa and Segarra 2010). Many farmers have abandoned their

crops to work in the more lucrative tourism industry, and as a result, agricultural species,

like guava (Psidium guajava) and blackberry (Rubus niveus), grow uncontrolled. The

non-native vegetation may be influencing nutrient dynamics on San Cristóbal, but their

biogeochemical impact has not been thoroughly examined. To date, only a handful of

studies have focused on characterizing soil N or P concentrations in the Galápagos.

(Kitayama and Itow 1999; Chacón 2010; de la Torre 2013; Jäger et al. 2013). To our

knowledge, no studies have examined soil N and P availability across the strong elevation

gradient on San Cristóbal. These observations leave many avenues for discovery of
 48

physical and biological controls on soil nutrient dynamics.

The main objective was to characterize seasonal soil N and P concentrations on

San Cristóbal, Galápagos. We evaluated how climate controls seasonal nutrient

concentrations through the influence of temperature, precipitation and soil moisture, and

we characterized how non-native plant species mediated changes in soil N and P

concentrations across the island’s elevation gradient. Few studies have examined these

processes thoroughly in the Galápagos, and further investigation is necessary to inform

the management of ecosystem change in the Galápagos.

Methods

Site Description

The Galápagos Archipelago is located 1000 km off the coast of Ecuador and is comprised

of 13 main islands. They formed when the Nazca Plate passed over a hot spot in the

Pacific Ocean, causing volcanic eruptions, and eventually islands (Geist et al. 2014). Of

the islands in the archipelago, this study was performed on San Cristóbal Island, one of

the oldest, dated to 2.35 ma (Geist et al. 2014). On the leeward, drier side of the island,

we sampled soils along an elevation gradient at low (300 m above sea level (a.s.l.)), mid

(500 m a.s.l.) and high (650 m a.s.l.) elevation sites under both native and invasive plant

canopies. Bursera graveolens (torchwood), Zanthoxylum fagara (cat’s claw), and

Miconia robinsoniana (miconia) are dominant native plant species at the low, mid, and

high elevations, respectively. However, non-native species, such as Psidium guajava

(guava) and Rubus niveus (blackberry), are dominant at all elevations sampled.

Study sites are managed to varying degrees. The low and high elevation sites are
 49

managed to maintain native species, while the mid elevation site is relatively unmanaged.

The low elevation site is a managed farm where in the last 3-4 years, in one plot, non-

native species were removed and native species were replanted, and in another plot, non-

native species were not removed. In the restored plot at the low elevation, non-native

crop plants were planted and manure was applied as fertilizer on the crop plants. At both

the mid elevation and high elevation, native and non-native plants are interspersed, but at

the high elevation, non-native eradication efforts are taking place and Miconia ribsoniana

was replanted in the last 5-10 years.

Typical of many tropical islands, the Galápagos experience a warm and wet

season (January–May) and a cool and dry season (June–December). From 1977 to 1983

on San Cristóbal at c. 6 m.a.s.l, the mean annual temperature was 24.8°C, and the mean

temperature during the cool season was 23.5°C and was 26.5°C during the warm season

(Violette et al. 2014). The estimated mean annual rainfall on San Cristóbal was 2961

mm/year above 650 m.a.s.l., and the measured mean annual rainfall at c. 6m a.s.l was 368

mm/year (Pryet 2011; Violette et al. 2014). Based on evidence from Santa Cruz island, an

additional 26% of the median annual rainfall in the highlands occurs in the form of occult

precipitation (fog), but fog is rarely present at the low elevations (Pryet et al. 2012).

Meteorological Data

EM50 data loggers recorded air temperature, relative humidity, precipitation, and

volumetric water content (VWC) at 15-minute intervals at each of the three sites

(METER Group, Pullman, WA). To measure VWC, EC-5 sensors were buried at 10, 20,

and 50 cm at the mid and high elevation sites and at 10, 20, and 30 cm at the low
 50

elevation site. Due to sensor malfunctions at the high elevation site, air temperature and

relative humidity were not recorded in 2017. However, temperature inside the data logger

was recorded. In order to estimate the air temperature at the high elevation site, I modeled

outside air temperature as a function the temperature inside the data loggers from both the

low and mid elevation sites. I then used that model to predict air temperature at the high

elevation site. Further, at the low elevation site, two of the three VWC probes

malfunctioned, so only values from the 30 cm depth were reported. The average VWC

from all three depths was presented for the mid and high elevation sites.

Soil Sampling

At each of the three sites, we collected 15 samples at a depth of 15cm from under

native plant canopies and 15 samples from under non-native plant canopies, for a total of

30 samples per site. Samples were collected in May 2017 at the end of the wet

season/start of dry season and in December 2017 at the end of the dry season/start of wet

season. Within 24 hours of sample collection, ammonium (NH4+) and nitrate (NO3-) were

extracted from the soil samples in a solution of 2M KCl for one hour. All live plant

material and rocks were manually removed from the soil cores. Samples were inverted by

hand every 15 minutes across the one-hour extraction period, and then they were gravity

filtered with p8 coarse paper filters and syringe filtered through 0.7 µm glass tips. After

extraction, samples were stored frozen until analysis. Remaining soil was reserved,

transported, and then air-dried for one month. We extracted phosphate (PO43-) in a

solution of ammonium fluoride and hydrochloric acid following a Type I Bray’s

extraction procedure. Soil solutions were shaken by hand for five minutes and then
 51

filtered in the same manner as the N extracts. All extracts were frozen for storage and

then analyzed using flow injection analysis (Lachat Quik-Chem Series 2400, Colorado).

Plant Sampling

During each sampling period, we collected leaf samples from the vegetation we

sampled soils under. All samples were transported back to the lab and dried at 60°C for

48 hours. Leaves were hand ground using liquid nitrogen and then weighed into tin

capsules. Then the samples were analyzed for %N and %C using a CHNOS Elemental

Analyzer. Due to small sample size, statistical analyses were not conducted. However,

more samples will be analyzed in the future.

Statistical Analysis

In order to examine spatial and temporal patterns of NH4+, and NO3, and PO43-

concentrations, we fit linear mixed effect models using the lme function in R (Pinheiro et

al. 2014; R Core Team 2017). To select each model, we used a stepwise AIC process

(Table 2). We started with a full model testing a three-way interaction between elevation,

plant canopy type, and season, after accounting for the random effect of sampling

location. We removed variables or interactions one at a time and selected the model with

the lowest AIC score. After visually assessing if each model distribution met the

assumptions of normality and constant variance using residual and normal-QQ plots, the

concentrations of each nutrient were log transformed.

 52

Results

Soil Sampling

We examined differences in NH4+, NO3-, and PO43- across three elevations (300m,

500m, and 650 m), between the wet and the dry season, and under native and non-native

plant canopies. For each analysis, we first fit a full linear mixed effects model including a

three-way interaction between elevation, season, and plant type (native or non-native),

after accounting for a random effect of sampling location. We then used a stepwise AIC

process to select each model (Table 2). For NH4+, we selected the following model:

Log(NH4+) = ß0 + ß1I300m + ß2I500m + ß3I650m + ß4Iw + ß5Id + ß6In + ß7Iin + ß8I300m * Iw +

ß9I500m * Iw + ß10I650m * Iw + ß11I300m * Id + ß12I500m * Id + ß13I650m * Id + ß14Iw * In +
ß15Iw * Iin + ß16Id * In + ß17Id * Iin + locationi + εij, locationi ~ N(0, σ2), εij ~N(0, σ2ε),

where 300m, 500m, and 650m refer to the site elevation expressed in meters; w and d
refer to wet or dry season; n and in refer to native or invasive vegetation, εij = residual
variability of the jth occasion for the ith subject, and N(0, σ2) denotes that errors are
normally distributed with a mean of 0 and a variance of σ2.

At the mid and high elevation sites, median NH4+ was higher during the dry

season, but at the low elevation site, median NH4+ was higher during the wet season. In

the dry season, NH4+ increased along the elevation gradient from 5.47 mg Kg-1 (95%

confidence interval (CI) of 4.48 to 6.68 mg Kg-1) at the low elevation to 11.59 mg Kg-1 at

the high elevation (95% CI of 9.02 to 14.15 mg Kg-1, respectively). However, during the

wet season, NH4+ did not vary across the elevations. During the dry season, there was no

difference in NH4+ concentrations under native or non-native plant canopies. However,

during the wet season, NH4+ concentrations were 2.69 mg Kg-1 greater under native

versus non-native plant canopies (95% CI of 2.54 to 3.28). We also found an interaction
 53

between elevation and season (χ2 (2,81 df) =3.70, p-value = 0.03), suggesting that

elevational differences in median NH4+ concentrations depended on the season sampled.

Furthermore, we detected evidence for an interaction between NH4+ concentrations and

vegetation type (χ2 (1,81 df) = 4.86, p-value = 0.03), suggesting differences in soil

median NH4+ concentrations between plant types also depended on the season sampled.

To examine differences in NO3- concentrations between elevations, seasons, and

plant type, we selected the following model:

Log(NO3-) = ß0 + ß1I300m + ß2I500m + ß3I650m + ß4Iw + ß5Id + ß6In + ß7Iin + ß8I300m * Iw +

ß9I500m * Iw + ß10I650m * Iw + ß11I300m * Id + ß12I500m * Id + ß13I650m * Id + locationi + εij,
locationi ~ N(0, σ2), εij ~N(0, σ2ε)

During the dry season, NO3- did not vary across elevations and ranged from 4.9

mg Kg-1 at the low elevation (95% CI of 4.05 to 6.04 mg Kg-1) to 6.0 mg Kg-1 at the high

elevation (95% CI 4.95 to 8.17 mg Kg-1). However, during the wet season, the median

NO3- concentration at the low elevation was 6.04 mg Kg-1 (95% CI of 4.95 to 7.39 mg

Kg-1), which was 17 times greater than at the high elevation and 35 times greater than

that at the mid-elevation. The median NO3- concentration under native plant canopies was

0.59 mg Kg-1 greater than under non-native plant canopies (χ2 (1,86 df) =32.10, p-value <

0.0001). We detected an interaction between NO3- and elevation, suggesting that

differences in median NO3- concentrations between elevations depended on the season

sampled (χ2 (2, 82 df) = 103.06, p-value < 0.0001).

We selected the following model to examine differences in PO43- concentrations

between elevations and seasons:

 54

Log(PO43-) = ß0 + ß1I300m + ß2I500m + ß3I650m + ß4Iw + ß5Id + ß6I300m * Iw + ß7I500m * Iw +

ß8I650m * Iw + ß9I300m * Id + ß10I500m * Id + ß11I 650m * Id + locationi + εij, locationi ~ N(0,
σ2), εij ~N(0, σ2ε)

For PO43-, we found that plant type did not influence PO43- concentrations (p-

value > 0.05), so it was not included in the final model. We still detected that the

elevational differences in median PO43- concentrations depended on the season sampled

(χ2 (2, 87 df) = 4.57, p-value= 0.01). During both seasons, PO43- was most concentrated at

the high elevation site but did not differ between the low and mid-elevations. The median

PO43- concentration at the high elevation during the wet season was 51.93 mg Kg-1 (95%

CI of 40.44 to 66.69 mg Kg-1) and was 134.30 mg Kg-1 during the dry season (95% CI of

99.48 to 148.41 mg Kg-1). The seasonal differences at the low and mid-elevations each

differed by less than three mg Kg-1.

Foliar N in natives versus non-natives

Preliminary examinations showed that foliar %N was higher in native compared

to non-native species at two of the three elevations sampled (Table 1). At the low

elevation, the mean foliar %N of three native species, Bursera graveolens, Chiocacca

alba, and Leocarpus darwinii, was 0.35% greater than that of the non-native species

Psidium guayaba. At the mid elevation, the foliar %N in the native species Zanthoxylum

fagara was 1.0% greater than in the mean %N of two non-native individuals, Psidium

guayaba and Pennisetum purpureum. At the high elevation, when five samples from the

dominant native species, Miconia ribsoniana, and the dominant non-native species,

Psidium guayaba, were compared, the mean %N was 0.55% greater in the non-native.
 55

Discussion

Seasonal and elevational changes in climate and the presence of non-native plants

influenced soil N and P pools in the tropical montane forests on San Cristóbal Island,

Galápagos. Soil N and P varied between elevations but typically depended on the season

sampled, with precipitation, temperature, and soil moisture influencing soil nutrient

dynamics (Jenny 1941; Vitousek and Chadwick 2013). On the other hand, the influence

of non-native plants on soil N and P pools in this study did not agree with the general

consensus that nutrient pools increase under non-native canopies (Liao et al. 2008).

Instead, we discovered that soil N pools decreased under non-native canopies while

available P did not change between the two plant types.

Nitrogen Pools

Climate controlled nutrient dynamics on San Cristóbal Island, but the effect sizes

depended on the season sampled. This result is expected, since rainfall and temperature

gradients also determine thresholds of soil development in other ecosystems (Ewing et

al.; Chadwick et al. 2003; Vitousek and Chadwick 2013). Here, NH4+ increased with

elevation in the dry season but did not vary with elevation during the wet season. NO3-

concentrations decreased with elevation during the dry season. During the wet season,

NO3- was most concentrated at the low elevation and least concentrated at the mid

elevation. Leaching and denitrification likely caused export of NO3-, a highly mobile ion,

out of the system during the wet season and at wetter elevations (Chapin III et al. 2011;

Weintraub et al. 2014), but NH4+ was less variable across the elevation gradient and

between seasons, presumably because it is not as prone to leaching and denitrification as

 56

NO3-. Interestingly, Chacón (2010) observed that both NH4+ and NO3-, not just NH4+,

increased along the elevation gradient on neighboring Santa Cruz Island. However, N

concentrations in our study still fell within the N concentration ranges observed in other

soils in the Galápagos (Kitayama and Itow 1999; Chacón 2010; Jäger et al. 2013). The

differences in N pools across elevation found between this study and others could have

been due to the age of the islands where observations were made, to the sampling season,

or to varying levels of disturbance or land use where sampling took place. Some of the

studies focused their sampling on one island zone or on one season. Further, sampling

could have occurred in sites with greater or lesser disturbance or land use change, but

there is little way to compare between studies without knowing specific sites.

We detected that NH4+ and NO3- were more concentrated under native than under

non-native canopies. However, most studies examining how invasion influences nutrient

dynamics find that soil nutrient pools are more concentrated under non-native canopies

(Vitousek et al. 1987; Scott et al. 2001; Ehrenfeld 2003, 2010; Liao et al. 2008; Vilà et al.

2011). Because the magnitude and direction of changes in nutrient pools in invaded

systems varies greatly depending on plant functional traits, ecosystem types, and N-

fixation status of invaders, Liao et al. performed a meta-analysis to generalize how

invasion influences ecosystem properties. Based on 94 studies, they determined that

NH4+ and NO3- pools under non-native plant canopies were 30% and 17% greater,

respectively, compared to under native plant canopies (Liao et al. 2008). In this study,

however, NH4+ and NO3- concentrations were 33% and 42% lower, respectively, under

non-native canopies than native canopies.

 57

In other studies conducted in the Galápagos, non-native plants typically increased

soil N pools. In the arid zone on San Cristóbal Island, percent N (%N) in bulk soil was

highest in a pasture restored with native species and lowest in actively managed

agricultural lands containing non-natives (de la Torre 2013). In the humid zone on Santa

Cruz, NH4+ was more concentrated under non-native canopies, but they detected no

differences in NO3- under different canopy types (Jäger et al. 2013). Whereas we

examined numerous non-native and native species growing in all climate zones on San

Cristóbal, other studies carried out on the Galápagos either followed the influence of a

single non-native species or of plant communities in single climatic zones, which may

have influenced the magnitude of non-natives’ influence on soil chemistry. Increases in

soil N pools after invasion are far from universal, however, and decreases or no

differences, as we saw in this study, were also observed (Ehrenfeld and Scott 2001;

Svejcar and Sheley 2001; Martin et al. 2009; Scharfy et al. 2009; Ehrenfeld 2010).

Phosphorus Pools

Climate was the strongest control of PO43- dynamics in the Galápagos. PO43-

concentrations were significantly higher at the high elevation site, suggesting that greater

soil moisture at the high elevation could have increased the organic P turnover rates, in

turn elevating the P pool (Turner et al. 2011). However, this is opposite the result that

Chacón (2010) observed on Santa Cruz, where PO43- concentrations decreased with

increasing elevation. PO43- varied seasonally as well, suggesting that plant uptake

controlled P pools along shorter time intervals. After the cool, dry season, PO43- was
 58

almost double the concentration of PO43- after the warm, wet season. Under drier

conditions, diffusion of PO43- from the soil matrix to root depletion shells slow (Gahoonia

et al. 1994), and this process could have caused the build up of PO43- during the cooler,

drier season. Further, cooler temperatures may have caused plant activity, including

nutrient uptake, to decrease, leaving more P available in the soil pool (Lambers et al.

2008a).

We did not observe differences in PO43- concentrations in soils under native and

non-native plant canopies. Kueffer et al. found a similar result in the Seychelles Islands,

where three non-native trees did not induce changes in total soil P under their canopies

(Kueffer et al. 2008). However, even though soil nutrient pools may not change, non-

natives may still impact litter concentrations or decomposition rates. For example, total P

increased in litter mass from Hieracium pilosella, a non-native forb growing in New

Zealand (Scott 2001), and litter P and litter decomposition rates increased in invaded

plots in Hawaii (Allison and Vitousek 2004). Though we did not detect changes in soil P

pools, changes in plant litter or alteration of decomposition rates may still be apparent if

we were to observe those processes.

Why are non-natives successful in the Galápagos?

Even though non-native species do not benefit from increasing soil nutrient pools

on the island, they are still more successful than native plants. Both soil and plant

processes observed here suggest that escaping physiological constraints is not what

allows non-natives to succeed in this ecosystem. Instead, non-native plants have escaped

dispersal limitations (Belyea and Lancaster 1999). In the 1980s, the feral goat population
 59

on the islands grew to around 100,000 individuals, and as a result, rapid plant community

changes took place (Coblentz 1978). Goats acted as seed vectors after eating guava and

blackberry fruits, and the plants were widely dispersed. Non-native do not escape nutrient

limitation in this system, yet they are ubiquitous and abundant because they have escaped

dispersal limitations.

Implications

While it is important for land management efforts in the Galápagos to understand

soil nutrient dynamics, observations of soil dynamics or vegetation communities are

executed under a shifted baseline because the islands are so heavily influenced by

invasion (Villa and Segarra 2010; Bush et al. 2014). The Galápagos National Park and

some private landowners have made strong efforts to restore native plant species in the

last 10 years. While the separate entities are managing land for different practices, such

as agriculture or tourism, they have the same goal of mitigating non-natives’ impact. This

may have influenced the results we found here through legacy effects. At two of our three

field sites, land managers and owners had been trying to eradicate non-native species, and

restoration efforts are succeeding to varying degrees. Non-native plants could have

legacy effects in the soil even after they are eradicated (Jäger et al. 2013; Abraha et al.

2018), or the presence of non-native understory species that had not been controlled may

have influenced our findings. Instead of testing soils under native and non-native plants

that are sometimes interspersed, native and non-native treatment plots could be more

heavily maintained for longer periods to test the influence of non-native plants on soil

processes in a more coordinated manner.

 60

Nonetheless, these findings, along with the work of Chacón (2010), present the

most complete synopsis of nutrient dynamics in the Galápagos to date. More work is

needed to illucidate patterns of soil development along the archipelago’s chronosequence,

as was performed in the Hawaiian archipelago (Vitousek et al. 1983, 1995; Crews et al.

1995). Further, the Galápagos presents a unique dichotomy between human and natural

systems which should be explored in order for conservation efforts to be effective in the

future. Overall, understanding nutrient dynamics in the archipelago will allow land

managers to decide how to control non-native plants as well as maintain an understanding

of nutrient dynamics under a shifted baseline for the future.

Acknowledgements

Thank you to technicians S. Nerby and C. Moss for their hard work collecting and

processing samples. Thank you to J. Klassen and C. Gabrogge for their guidance with

chemical analysis and to A. Gomez, S. Schmidt, and M. Percy for sharing their site

information and meteorological data.

Tables

Site Name Site Elevation Soil Type* Soil Mean Native foliar Non-native
Texture* Soil pH* %N foliar %N
Mirador 300 m Ultisol Sandy loam, Sandy clay loam, and Clay 6.06 2.22% 1.87%
Cerro Alto 500 m Ultisol Loamy sand and Clay 6.02 3.28% 2.16%

El Junco 650 m Oxisol Sandy loam 4.05 1.59% 2.14%

Table 2.1 – Site Descriptors - metrics with a * are described by Percy et al., In Prep.

61
Analyte Fixed effects AIC
NH4+ Elevation * Season * Plant Type 355.5612
Elevation*Season + Plant Type *Season 345.837*
Elevation * Season + Plant Type 346.826
Elevation + Season + Plant Type 346.808
Elevation * Season 346.758
Elevation + Season 346.755
NO3- Elevation * Season * Plant Type 375.051
Elevation * Season + Plant Type *Season 370.6681
Elevation * Season + Plant Type 367.063*
Elevation + Season + Plant Type 483.803
Elevation * Season 390.261
Elevation + Season 497.262
PO43- Elevation * Season * Plant Type 390.238
Elevation * Season + Plant Type *Season 387.382

62
Elevation * Season + Plant Type 387.164
Elevation * Season 382.595*
Elevation + Season 384.67

Table 2.2 – Results from the stepwise AIC model selection processes. We selected the models with AIC scores listed with an *
Figures

San Cristóbal Island

63
300 m
500 m
650 m
Galapagos Science Center

Figure 2.1 – Map of study sites along the elevation gradient with photos demonstrating the difference in vegetation communities along
the climosequence. Zanthoxylum fagara (left) and Scalesia gordilloi (right) pictured at the 300m site, and El Junco lake (l) and
Psidium guyaba (r) at the high elevation site. Arrow on map inset shows location of the Galápagos Archipelago.
 25.0 Elevation
600
300m
Elevation

Cumulative Daily Rainfall (mm)

500m
300m
22.5 650m
500m
Temperature(˚C)

400 650m

20.0

200
17.5

0
15.0
Jan 2017 Apr 2017 Jul 2017 Oct 2017 Jan 2018 Jan 2017 Apr 2017 Jul 2017 Oct 2017 Jan 2018

120 Elevation 100 Elevation

300m 300m
Daily Max. Relative Humidity (%)

500m 500m

64
650m 75 650m

100

VWC (%)
50

80
25

60 0

Jan 2017 Apr 2017 Jul 2017 Oct 2017 Jan 2018 Jan 2017 Apr 2017 Jul 2017 Oct 2017 Jan 2018

Time

Figure 2.2 - Meteorological measurements of (clockwise) mean daily air temperature, cumulative daily rainfall, VWC, and mean
daily maximum RH. Note that the air temperature at the 650 m elevation site is modeled from the temperature inside the data logger,
and the VWC at the 300 m elevation site is from the 30 cm depth while the VWC from the 500 m and 650 m elevation sites are from
the 20 cm depth.
 65
Figure 2.3 - NH4+, NO3-, and PO43- concentrations across elevations and between native or non-native plant canopies sampled under
with measurements from the warm season in the left column and values from the cool season in the right column. Lower cases letters
denote differences in nutrient pools between elevation and between season and upper case letter denote differences between pools
under native and non-native canopies.
 66

CHAPTER FOUR

CONCLUSIONS AND FUTURE WORK

I built on our fundamental understanding of ecosystem function by examining

how climate variability influences feedbacks between plant processes and soil nutrient

dynamics. At Lubrecht Experimental Forest, I examined how variability in snow depth,

precipitation, and soil moisture influenced seasonal nitrogen allocation in Douglas-fir. I

then examined if N cycling within Douglas-fir synchronized with patterns of N

availability in the soil. In this case, N availability in the soil influenced plant nutrient

dynamics. On the other hand, on San Cristóbal Island in the Galápagos Archipelago,

plants fed back and influenced soil nutrient dynamics. Changes in precipitation, soil

moisture, and temperature strongly controlled nutrient concentrations in the soil, and to a

lesser degree, plant community type determined nutrient concentrations, especially N

concentrations, in the soil.

While these projects elucidated patterns of basic ecosystem function, further work

could augment the findings’ impact. For example, while we know that Douglas-fir trees

must be accessing most of their N from the soil, it is still uncertain if they are actually

using that N to photosynthesize. Answering this question would allow us to conclude if

evergreens are N limited or not by testing the hypotheses drawn by Warren and Adams

(2004). To examine N-limitation in Douglas-fir, I would measure metabolically active

and metabolically inactive Rubisco content and then relate those measurements to trees’
 67

photosynthetic rates across a season. This would allow me to determine if trees are

actually using the N they are taking up from the soil across the season.

In the Galápagos, further work is needed to inform restoration of the landscape.

However, the restoration must be carried out in a socially minded manner. Residents

value some invasive species, like orange trees and guava, for their products, and native

species restoration should be carried out in concert with agricultural production. Not all

land, though, can be used for production, and the Galapagos National Park must direct

and implement most of the restoration efforts. Scientists must guide land management in

order for restoration efforts to become effective, and results presented here can inform

land managers of impacts that their restoration will have going forward.
 68

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