EMPLOYING STABLE ISOTOPES TO INVESTIGATE THE IMPACTS

OF INVASIVE SPECIES ON HAWAIIAN STREAM FOOD WEBS

______

A Thesis

Presented

to the Faculty of

California State University, Chico

______

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

in

Biological Sciences

______

by

Megan Layhee

Spring 2011 EMPLOYING STABLE ISOTOPES TO INVESTIGATE THE IMPACTS

OF INVASIVE SPECIES ON HAWAIIAN STREAM FOOD WEBS

A Thesis

by

Megan Layhee

Spring 2011

APPROVED BY THE DEAN OF GRADUATE STUDIES AND VICE PROVOST FOR RESEARCH:

______Katie Milo, Ed.D.

APPROVED BY THE GRADUATE ADVISORY COMMITTEE:

______Michael P. Marchetti, Ph.D., Chair

______Don Miller III, Ph.D.

______Daniel Pickard, M.S. DEDICATION

To my parents and my sister for always telling me that anything is possible.

And to Robert, for standing by my side.

iii ACKNOWLEDGEMENTS

I would like to thank Dr. Micael P. Marchetti, Tag Engstrom, and Dan Pickard

(CDFG, Aquatic Bioassessment Laboratory), for their encouragement and help

throughout the duration of the project. A big thanks to Dr. Sudeep Chandra (U of

Nevada) for lending me his expertise and assistance with data analysis. Thanks to

Sudeep’s grad students for help processing samples. A big thank you to Dr. Don Miller for agreeing to be a part of the completion of this thesis. Thanks to Anne Brasher (USGS) for all of her help in the first field season. Thanks to all the people and organizations that made stream sampling possible; D. Heacock, S. Smith, and Limahuli Botanical Gardens.

Thanks to UC Davis Stable Isotope Facility. Field work and collection of samples would

have been impossible without the help of Chris Arnett, Heather Bowen, Duryea

Delacroix, Sarah DelPaine, Becky Desfor, Sandy Fikes, Liz Leyvas, Megan Masonik,

Will McCall, Sierra Sisk, and Molly Thompson. Finally would like to thank Glenn

Woodruff for so much of his help in the field and in the lab throughout much of the

project.

iv TABLE OF CONTENTS

PAGE

Dedication...... iii

Acknowledgements...... iv

List of Tables ...... vii

List of Figures...... viii

Abstract...... xi

CHAPTER

I. Introduction ...... 1

Background...... 1

II. Methods...... 5

Study System ...... 5 Field Sites...... 7 Field Collection and Tissue Preparation...... 7 Analysis of Food Webs...... 9

III. Results ...... 14

Changes in Overall Food Web Structure ...... 14 Shifts in Native Species δ13C and Trophic Positioning...... 23 Variation in Food Web Trophic Diversity and Trophic Redundancy...... 24

IV. Discussion ...... 27

Disturbance in Hawaiian Streams...... 27 Ecosystem-Level Processes Affecting Overall Food Web Structure...... 27

v CHAPTER PAGE

Community-Level Process Affecting Overall Food Web Structure...... 29 Shifts in Native Species Trophic Space in Variably Disturbed Streams...... 33 Trophic Diversity and Redundancy in Hawai’i’s Disturbed Stream Food Webs...... 35 Implications for Hawai’i’s Native Freshwater Communities ...... 37

Cited References ...... 40

vi LIST OF TABLES

TABLE PAGE

1. Stream Consumers Sampled at Each Site with Varying Levels of Disturbance, in 2008 and 2009 ...... 16

2. Primary Producers Sampled at each Stream in 2009 and 2009...... 19

3. Results of Kruskal-Wallis Test Comparing Native Consumer Species Mean Δ13c and Mean Trophic Positions between Stream Sites with Varying Levels of Disturbance and between Sampling Years ...... 24

4. Calculated Mean Centroid Distance (Cd), Mean Nearest Neighbor Distance (Nnd), and Standard Deviation of Nearest Neighbor (Sdnnd) for All Streams Sites with Varying Levels of Disturbance in Both 2008 and 2009 Sampling Years ...... 26

vii LIST OF FIGURES

FIGURE PAGE

1. Map Detailing the Locations of the Four Streams on the Island of Kaua’I, HI, USA ...... 8

2. Mean δ13C and TP (±standard deviation) values of consumers in the lesser disturbed stream, Limahuli, including, a) upper site in 2008,b) upper site in 2009 and c) lower site in 2009 ...... 15

3. Mean δ13C and TP (±standard deviation) values of consumers in the moderately disturbed streams; a) upper Kapa’a site in 2009, b) lower Kapa’a site in 2009, c) lower Hulē’ia site in 2008 and d) lower Hulē’ia site in 2009...... 21

4. Differences in mean isotopic signatures of primary producers in 2008 and 2009 across sites with varying levels of disturbance including results of Kruskal-Wallis Test below. a) δ13C filamentous algae (2008, H=15.16, P=0.001), and b) δ15N filamentous algae (2008, H=15.16, P=0.001), c) δ13C periphyton (2008, H=15.16, P=0.001; 2009, H=15.83, P=0.007), d) δ15N periphyton (2008, H=10.89, P=0.004; 2009, H=15.83, P=0.007), e) δ13C leaf litter (2008, H = 7.41, P = 0.025; 2009, H=14.99, P=0.01), and f) δ15N leaf litter (2008, H=11.66, P =0.003; 2009, H=16.25, P=0.006)...... 22

5. Mean δ13C and TP (±standard deviation) values of consumers in the highly disturbed stream, ‘Opaeka’a, in the a) upper site in 2008 and b) upper site in 2009...... 23

6. Comparison of mean trophic positions and mean δ13 signatures (± standard deviation)of a-b) Awaous guamensis, c-d) Eleotris sandwicensis, and e-f) Atyoida bisulcata across sites with lesser (Limahuli sites) and moderate (Kapa’a and Hulē’ia sites) levels of disturbance in 2009...... 25

viii ABSTRACT

EMPLOYING STABLE ISOTOPES TO INVESTIGATE THE IMPACTS

OF INVASIVE SPECIES ON HAWAIIAN STREAM FOOD WEBS

by

Megan Layhee

Master of Science in Biological Sciences

California State University, Chico

Spring 2011

Anthropogenic disturbance around the world is restructuring ecosystems and changing interactions within ecological communities. In the Hawaiian Islands, one of the most significant forces of disturbance is the widespread presence of invasive species. In this study stable isotopes of carbon and nitrogen were used to examine trophic and energy flow changes occurring in Hawaiian stream communities due to biotic disturbances including invasive species. On the island of Kaua’i, we extensively sampled all members of the stream community, including primary producers and consumers, from four streams with varying levels of disturbance. The streams chosen in this study included Limahuli,

Kapa’a, Hulē’ia and ‘Opaeka’a. δ13C, δ15N signatures, and trophic positions of stream members were calculated to determine differences in overall food web structure, changes to native consumer isotopic signatures, and differences in food web diversity and trophic redundancy. Comparison of streams with varying levels of disturbance showed that

ix 1) overall food web structure varied dramatically among locations, 2) natives were nearly absent in heavily disturbed and highly invaded streams while at the same time a suite of non-native consumers are replacing the trophic roles left by the natives, 3) isotopic signatures and trophic position of native consumers were not significantly different across streams, and 4) heavily disturbed and highly invaded streams had more trophic diversity and generally lower trophic redundancy. Our study was able to quantitatively depict and compare differences in both the structure and trophic interactions of Hawaiian freshwater ecosystems webs due to varying levels of disturbance and species invasion.

x CHAPTER I

Introduction

Background

Modern human societies are drastically altering the physical, chemical, and biological processes that drive natural systems around the world (Vitousek et al. 1997). These alterations may alternatively be described as a form of global, anthropogenic disturbance, which is reorganizing the very ecological structure and functions of planetary ecosystems. The introduction of invasive species is one of the major forces of disturbance influencing these changes. Unfortunately the probability of successful invasion is assisted by both the current ease of global transport and the pervasive habitat modification described above (Hobbs et al. 2006; Lockwood et al. 2007). Once established in new systems, invasive species can in turn alter biotic interactions through a suite of forces including things like novel predation and competition pressures (Cox 1999; Hobbs et al.

2006; Mooney and Cleland 2001; Vander Zanden et al. 1999; Vander Zanden et al.

2003). There is no general consensus as to the characteristics that make an invader successful, and therefore the specific ramifications of invasive species to community function also remains unclear (Lockwood et al. 2007). Given that, it is not surprising that the degree of impact from invasive species may depend on inherent or site specific characteristics of the system (Lockwood et al. 2007; Sax et al. 2007).

1 2

On the Hawaiian Islands, invasive species pose a serious threat to the island’s biota and to the unique structure of the islands ecological communities (Brasher 2003). The

Hawaiian archipelago is the most isolated island chain in the world and the endemic biota have evolved in this remote landscape with few natural biotic introductions (McDowell

2003). As a result the island’s native ecological communities are characterized by low diversity and high endemism (Brasher 2003; McDowell 2003). Yet, over the last two centuries, the introduction of non-native species has occurred at an unprecedented rate, and it is estimated that over 3,000 non-native species have established on the islands

(Gagnè 1988). In particular, 50 species of macroinvertebrates, herptofauna, and fish species have successfully established in Hawai’i’s freshwater ecosystems where only seven freshwater fish species are native (Yamamoto et al. 2000). This suite of aquatic invaders has had large ecological impacts on the Hawaiian ecosystems including aggressive interspecific competition and increased predation on native species (Eldredge

1994; Eldredge 2000).

In addition to the flood of aquatic invasive species, Hawai’i has suffered a host of additional anthropogenic disturbances that in turn impact the native freshwater ecosystems. For example, Hawaiian freshwater species have evolved to exist within a unique set of geographical characteristics, including steep cascading stream channels, highly variable flow regimes, extensive waterfalls, and fluctuating water temperatures due to high levels of rainfall (Brasher 2003; McDowall 2003). Yet urban growth and extensive agricultural development, have altered the natural flow regimes on the islands, reduced water quality through waste water input, and degraded riparian ecosystems

(Brasher 2003). Resh et al. (1988) defines disturbance in stream ecosystems as a discrete 3 event outside a predictable range that disrupts the overall ecological structure and or function of a system. The geographical isolation of the Hawaiian Islands coupled with the island’s characteristically low biodiversity and high endemism make Hawai’i’s ecological communities uniquely susceptible to the effects of disturbance (Brasher 2003).

Stable isotope analysis has become a useful tool used for examining the consequences of invasive species on ecological communities (Mercado-Silva et al. 2008; Vander

Zanden et al. 1999; Vander Zanden and Rasmussen 1999; Vander Zanden et al. 2003).

Stable isotope analysis can be used as a time-integrated measure of food web interactions based on isotope transfer from food source to consumer (Fry 2006; Peterson and Fry

1987; Schmidt et al. 2007; Vander Zanden and Rasmussen 1999). Stable isotope analysis has illustrated numerous effects of invasive species including changes to food web structure, alterations of native species interactions, and changes to species preferred niche space (Vander Zanden et al. 1999; Vander Zanden et al. 2003). Stable isotope analysis is also useful in depicting overlap in trophic sourcing, changes in native species preferred food sources, and alterations in trophic positions and levels (Vander Zanden et al. 1999;

Vander Zanden et al. 2003).

The freshwater streams in Hawai’i are an excellent place to look for the effects of disturbance on aquatic trophic systems using stable isotopes because the intensity of disturbance varies dramatically among watersheds (HDAR and Bishop Museum 2008a,

2008b) and there is a low diversity of native aquatic biota (Brasher 2003). In Hawai’i, it has also been shown that non-native species are more prevalent in human manipulated streams due to their ability to tolerate altered aquatic conditions (Brasher et al. 2006).

Over a two year period we examined six sites in four streams on the island of Kaua’i 4 across a gradient of disturbance and used stable isotope analysis to address the following questions: (1) Does food web structure change with increased levels of disturbance? (2)

Do the isotopic signatures of native species change with increased levels of disturbance?

(3) Is there increased trophic diversity and trophic redundancy among consumers in food webs with increased levels of disturbance? CHAPTER II

Methods

Study System

Kaua’i is the oldest inhabited island in the Hawaiian archipelago. Hawaiian freshwater species have evolved within a unique set of geographical characteristics, including highly variable rainfall, high elevation gradients, and extensive waterfall barriers (Brasher 2003;

McDowall 2003) and therefore the freshwater fish assemblage is fairly limited and includes four Gobiidae species, Awaous guamensis, Lentipes concolor, Stenogobius hawaiiensis, Sicyopterus stimpsoni, one Eleotridae species, Eleotris sandwicensis, and two euryhaline fish species, Kuhlia sandvicensis and Mugil cephalus, which venture into

freshwater as juveniles (Yamamoto et al. 2000). The five freshwater Hawaiian gobies and

eleotrid are amphidromous; as adults they live and spawn in freshwater, as juveniles they

travel to sea, and migrate back into freshwater upon maturation (Kinzie 1990). Three of the goby species have fused-pelvic fins which assist them in navigating Hawai’i’s characteristically steep, fast-moving streams and waterfalls (Yamamoto et al. 2000). The two fish species lacking fused fins (Stenogobius hawaiiensis and Eleotris sandwicensis)

have relatively poor climbing abilities and are restricted to lower reaches of Hawai’i’s

streams (Kido 2007; Kinzie 1988; Maciolek and Timbol 1981). This has created spatially

explicit habitat zones for different species of Hawaiian freshwater fish and as a result,

5 6

Hawaiian freshwater communities differ along the continuum of a single watershed

(Kinzie 1988).

We examined four freshwater streams on Kaua’i that had varying degrees of biotic disturbance (Figure 1). Limahuli, the least disturbed stream, is a 2nd order perennial

stream, 4.8 miles (7.8km) long with a drainage area of 1.8 square miles (4.7km2) (HDAR

and Bishop Museum 2008a). Limahuli watershed is managed entirely for protection and conservation and has 9 documented non-native aquatic species present in the watershed

(HDAR and Bishop Museum 2008a; Kido 2007). Kapa’a, a 4th order perennial stream, is

59.2 miles (95.3km) long with a drainage area of 16.5 square miles (42.7km2) (HDAR

and Bishop Museum 2008b). Nearly half of Kapa’a watershed is managed for agriculture

and rural-urban development, leaving the other 50% of the watershed under conservation management, and has 30 documented non-native aquatic species (HDAR and Bishop

Museum 2008b). Hulē’ia, a 4th order perennial stream, is 89.7 miles (144.4km) long with

a drainage area of 28.2 square miles (73km2) (HDAR and Bishop Museum 2008c).

Nearly 70% of Hulē’ia watershed is managed for agricultural purposes, less than 1% for urban development, and the remaining land protected, and has 29 documented non-native aquatic species (HDAR and Bishop Museum 2008c). ‘Opaeka’a, the most disturbed of

the four streams (determined from visible assessment in the field), is a tributary of the

Wailua watershed which is a 5th order perennial stream with a drainage area of 52.6

square miles (136.2km2) (HDAR and Bishop Museum 2008d). A little over 30% of the

Wailua watershed is managed for agriculture and development, while the remainder of

the watershed is under conservation management, and has 27 documented aquatic non-

native species (HDAR and Bishop Museum 2008d). 7

Field Sites

In March of 2008 three sites were sampled which included a mid reach site on

Limahuli stream, a low reach site on Hulē’ia stream, and a mid reach site on ‘Opaeka’a stream (Figure 1). In March of 2009, the previous year’s sites were sampled again along with three additional locations including a site in the lower reaches of Limahuli, and two sites on Kapa’a stream, at low and mid reach locations (Figure 1). Upper and lower reach sites were sampled in order to capture the continuum of native communities that inhabit different reaches of Hawaiian streams and to compare different stream systems at similar reaches (Kido 2007; Kinzie 1988; Maciolek and Timbol 1981).

Field Collection and Tissue Preparation

The main goal of the field collection was to acquire tissue samples that covered all of the macro biota of the stream food webs at each field site. At each location various capture methods were used to collect community members from all trophic levels including primary producers, native consumers, and non-native consumers. Periphyton was scraped off the surface of instream rocks using a clean toothbrush. Filamentous algae was hand-picked from stream and rinsed in clean water. Instream leaf litter was hand- picked from backwater areas of each site and macro biota were rinsed off. Dip nets, minnow traps, turtle traps, and a backpack electro-shocker were used to collect various macroinvertebrates including crustaceans, mollusks, flatworms and aquatic insects.

Collection of herptofauna and fish included the use of hand seines, cast nets, minnow traps, turtle traps, fishing line, and the backpack electro-shocker. Field collection using a backpack electro-shocker was used in the second sampling year, 2009 but not in 2008. 8

Figure 1. Map detailing the locations of the four streams on the island of Kaua’i, HI, USA. In 2008 sampling year, a mid reach site location on Limahuli stream (Site 1 - 22° 12’ 51.1”N, 159° 34’ 40.5”W), a low reach site location on Hulē’ia stream (Site 2 - 21° 56’ 34.8”N, 159° 23’ 51.3”W), and a mid reach site location on ‘Opaeka’a stream (Site 3 - 22° 03’ 24.4”N, 159° 22’ 12.8”W). In 2009, the three stream sites (Site 1-3) sampled in the previous year were sampled again in addition to three more sites, including a lower reach site on Limahuli (Site 4 - 22° 13’ 14.8”N, 159° 34’ 34.7”W), an upper reach site on Kapa’a stream (Site 5 - 22° 06’ 11.0”N, 159° 19’ 49.7”W), and a lower reach site on Kapa’a stream (Site 6 - 22° 06’ 13”N, 159° 21’ 49.4”W).

All organisms collected (other than periphyton, filamentous algae, and leaf litter) were placed in 70% ethanol in the field and were identified to species in the lab on the day of collection. For each unique taxa, approximately 5-10 grams (or more) of tissue (wet weight) was collected. 9

Primary producer samples (filamentous algae, periphyton, and leaf litter) were dried in a low temperature oven at 80°C for several hours on the same day they were collected. In the laboratory, all other samples were dried in an oven at 60°C for 24 hours, and then ground into a fine powder using a mortar and pestle. For plant tissue, 2 to 3mg was packed into tin capsules. For animal tissue (invertebrates, fish, and herptofauna),

1±0.2mg was packed into tin capsules. In some instances it was necessary to combine several individuals of the same species into one sample to meet the dry weight requirements; otherwise each tin capsule was comprised of tissue samples from one individual organism.

Analysis of Food Webs

Stable isotope analysis (via mass spectrometer) was conducted at the UC Davis Stable

Isotope Facility in Davis, California. Natural abundance analysis was conducted to determine the carbon and nitrogen isotopic signature of individual samples. Every biotic entity has a specific ratio of heavy to light isotopes of carbon (13C/12C) and nitrogen

(15N/14N), termed isotopic composition (Fry 2006). An individual’s isotopic signature, or δ (delta) value (denoted δ13C for carbon and δ15N for nitrogen), is the ratio of heavy to light isotope based on a standard; PeeDee limestone for carbon and atmospheric nitrogen gas for nitrogen (Fry 2006; Peterson and Fry 1987). An individual’s carbon isotopic signature (δ13C) is an indication of its ultimate food source since there is little change in δ13C between food source and consumer (Fry 2006; Peterson and Fry1987).

Isotopic fractionation is a change in the isotopic ratio of heavy to light isotopes between food and consumer assimilation (Fry 2006; Peterson and Fry 1987). Isotopic fractionation 10 most often occurs in δ15N enrichment (i.e. heavy isotope retention) between food source and consumer, therefore δ15N isotopic signature is an indication of trophic position (Fry

2006; Peterson and Fry1987). Isotopic signatures (δX) were calculated as follows:

δX (‰) = (Rsample /Rstandard – 1) x 1000 where X is the amount of 13C or 15N heavy isotope in parts per thousand, Rsample is the ratio of heavy to light isotopes of tissue sample, and Rstandard is the ratio of heavy to light

isotope of the standard (Fry 2006).

Inherent differences exist in the carbon and nitrogen isotopic signatures of primary

producers across systems due to unique biogeochemical processes (Cabana and

Rasmussen 1996; Hobson et al. 2007). In order to accurately compare differences in

trophic positioning of consumers across communities with various levels of disturbance,

δ15N signatures of consumers had to be standardized, and were converted to trophic

position. Trophic positions (TP) for all consumers were calculated as:

TPconsumer = 2+ (3.4/ (δ15Nconsumer - δ15Nbaseline))

where 3.4 is the per trophic level enrichment in δ15N, 2 is the trophic level position of

primary consumers, δ15N consumer is the consumer’s raw nitrogen signature, δ15Nbaseline is the δ13C-δ15N relationship of primary consumers using linear regression analysis

(Vander Zanden and Rasmussen 1999; Vander Zanden et al. 2003). Linear regression analysis was used to find the δ13C-δ15N relationship of the primary invertebrate consumers in each food web; primary vertebrates were used as well if sampling size of primary invertebrates was not sufficient for analysis (Vander Zanden et al. 2003). For each consumer, δ15Nbaseline was calculated using the consumer’s δ13C and stream food

web specific primary consumer equation (Vander Zanden et al. 2003). If food webs 11 lacked a sufficient number of primary consumers at time of sampling, the δ13C-δ15N relationship of the primary consumers from a food web in the same stream but a different reach was used as a surrogate; including Lower Limahuli and the primary consumer baseline equation from Upper Limahuli in the same year (2009) was used as the surrogate.

For each stream food web, consumer bi-plots were constructed with mean δ13C signatures plotted against mean trophic positions (also termed niche space diagrams)

(Coat et al. 2009; Mercado-Silva et al. 2008; Vander Zanden et al. 1999; Vander Zanden et al. 2003). In each stream food web the range of δ13C signature and range of trophic positions were calculated for all consumers present, based on mean values for each species (Layman et al. 2007; Mercado-Silva et al. 2008). The range of consumer δ13C signatures provides an indication of the diversity of primary producers being utilized

(Layman et al. 2007). Typically the range of mean δ15N is calculated and considered an indication of trophic diversity, a higher δ15N range infers more trophic levels, but in this study the focus is on consumer trophic position which is a substitute for the estimation of trophic level diversity and trophic structuring of the food web (Layman et al. 2007;

Mercado-Silva et al. 2008). In addition to food web consumers, isotopic signatures of primary producers were also compared within and among sites to estimate variation in isotopic makeup of basal resources among stream sites with varying levels of disturbance

(Coat et al. 2009; Saito et al. 2008).

In order to measure shifts in native consumer isotopic signatures amongst streams with varying levels of disturbance, mean δ13C and mean trophic positions of single species 12 were compared across stream sites and across sampling years (Vander Zanden et al.

1999; Vander Zanden et al. 2003).

Within each food web trophic diversity and trophic redundancy were estimated by calculating centroid distance, nearest neighbor distance and standard deviation of nearest neighbor distance (Layman et al. 2007). Centroid distance (CD), or food web dispersion, represents the degree of trophic diversity (Layman et al. 2007; Mercado-Silva et al.

2008). The centroid is the center of the food web, or the average δ13C and trophic position value for all food web consumers and, the mean distance to the food web’s centroid is the average of all consumer distances to the centroid (Layman et al. 2007).

Mean nearest neighbor distance (NND) is a measure of species concentration and an indication of trophic niche redundancy, where smaller NND values suggest higher trophic redundancy (Layman et al. 2007). To calculate food web’s nearest neighbor distance, the closest neighbor for each species (in δ13C and trophic position space) is determined then the average of the distance all of species to their nearest neighbor is estimated (Layman et al. 2007). Standard deviation of nearest neighbor distance (SDNND) represents the evenness of species concentration in a food web, where lower SDNND implies a more evenly distributed trophic niches (Layman et al. 2007). This metric is calculated by finding the standard deviation of the mean nearest neighbor distance for the entire food web (Layman et al. 2007). Trophic diversity and trophic redundancy of each food web can be compared to other food webs across space or time to determine differences among community structure (Layman et al. 2007).

Published dietary information was used in conjunction with the estimates of isotopic signatures and trophic positions from this study to infer food sources of native and non- 13 native consumers (Yamamoto et al. 2000). Non-parametric Kruskal-Wallis test was used to compare mean δ13C and δ15N of primary producers across multiple sites and to compare different primary producer taxa within each site. The Kruskal-Wallis test was also used to compare mean δ13C and trophic positions of native consumers across stream sites and across years. All statistical analyses were done in Minitab 16. CHAPTER III

Results

Changes in Overall Food Web Structure

We found that the four streams sampled in this study had varying levels of disturbance

and the overall structure of food webs were different in the following ways; number of

non-native species present, proportion of non-natives making up food web, range of

consumer’s mean δ13C signatures and mean trophic positions, species inhabiting the top

trophic level, and isotopic structuring at the base of food web.

Limahuli, the least disturbed stream, contained two non-native species across both sites and years, and the number of native species was proportionally higher than non-natives;

33% of food webs were non-native species (Figure 2a-c). The range of consumer δ13C signatures in Limahuli was as low as 4.4 at the lower reach site (-23.06 to -20.54‰), the smallest range of all streams in this study (Table 1). The smallest range of trophic positions in this study was 0.98 at Upper Limahuli in 2009 (1.95 to 2.93) (Table 1). The top trophic level was inhabited by native omnivorous freshwater gobies including

Awaous guamensis and Lentipes concolor (Figure 2a-c).

At the base of the food web, filamentous algae was the most δ13C-enriched primary

producer in Upper Limahuli in 2008 (no comparison in 2009, due to low algal standing

crops during field season), periphyton was the most δ15N-enriched in both years, while

leaf litter was the most δ13C- depleted and δ15N-depleted in all sites of Limahuli (Table 2).

14 15

a)

b)

c)

Figure 2. Mean δ13C and TP (±standard deviation) values of consumers in the lesser disturbed stream, Limahuli, including, a) upper site in 2008,b) upper site in 2009 and c) lower site in 2009. Symbols represent type of organism; triangle=fish, square=herptofauna, diamond=crustacean, circle=mollusks, and line=aquatic insect/flatworms. Black represents s native consumer taxa and white represents non- native consumer taxa. 16

Table 1. Stream consumers sampled at each site with varying levels of disturbance, in 2008 and 2009 Stream site (reach)/ δ13C δ15N TP Disturbance level/ Year Type of Organism Web Member ID n Status Feeding Niche (mean±SD) (mean±SD) (mean±SD) Limahuli (upper) Insect/worms Cheumatopsyche pettiti (Hydropsychid caddisfly) HC 1 I herbivore -25.65 3.31 1.995 Low disturbance Turbellaria sp. (Flatworm) FW 2 N carnivore -22.81 ± 0.588 5.69 ± 0.041 2.39±0.08 2008 Mollusks Neritina granosa (Hīhīwai, Wī) HW 2 N herbivore -21.41 ± 0.184 4.9 ± 0.275 2±0.1 Crustaceans Macrobrachium lar (Tahitian prawn) TP 3 I omnivore -22.79 ± 1.95 6.57 ± 0.354 2.64±0.26 Fish Awaous guamensis (‘O‘opu nākea goby) ONG 3 N omnivore -22.62 ± 1.73 8.01 ± 0.259 3.05±0.11 Lentipes concolor (‘O‘opu ‘alamo‘o goby) ALG 3 N omnivore -17.16 ± 0.765 8.1 ± 0.326 2.48±0.18 Limahuli (upper) Insect/worms Cheumatopsyche pettiti (Hydropsychid caddisfly) HC 2 I herbivore -24.94 ± 1.87 4.2 ± 0.432 1.95±0.07 Low disturbance Turbellaria sp. (Flatworm) FW 1 N carnivore -23.79 6.31 2.45 2009 Crustaceans Atyoida bisulcata (Mountain ‘Ōpae shrimp) MOS 1 N omnivore -23.54 6.16 2.38 Macrobrachium lar (Tahitian prawn) TP 3 I omnivore -21.14 ± 0.529 7.51 ± 0.269 2.53±0.13 Fish Awaous guamensis (‘O‘opu nākea goby) ONG 3 N omnivore -21.99 ± 1.88 8.2 ± 0.719 2.82±0.36 Lentipes concolor (‘O‘opu ‘alamo‘o goby) ALG 1 N omnivore -17.86 10.02 2.93 Sicyopterus stimpsoni (‘O‘opu nōpili goby) ONOG 4 N herbivore -18.25 ± 1.56 6.81 ± 0.108 2.03±0.13 Limahuli (lower) Insect/worms Turbellaria sp. (Flatworm) FW 1 N carnivore -23.06 7.21 2.64 Low disturbance Crustaceans Atyoida bisulcata (Mountain ‘Ōpae shrimp) MOS 1 N omnivore -20.54 4.36 1.54 2009 Macrobrachium lar (Tahitian prawn) TP 3 I omnivore -22.87±0.165 7.32±0.45 2.65±0.14 Fish Awaous guamensis (‘O‘opu nākea goby) ONG 1 N omnivore -21.91 9.14 3.09 Eleotris sandwicensis (‘O’opu ‘akupa goby) OAG 3 N carnivore -22.95±0.412 7.57±0.49 2.74±0.19 Mugilogobius cavifrons (Mangrove goby) MGG 3 I carnivore -21.61±3.287 7.01±0.273 2.43±0.27 Kapa’a (upper) Insect/worms Cheumatopsyche pettiti(Hydropsychid caddisfly) HC 1 I herbivore -25.91 6.21 1.96 Moderate disturbance Turbellaria sp. (Flatworm) FW 1 N carnivore -24.38 7.87 2.29 2009 Mollusks Corbicula fluminea (Asiatic freshwater clam) AFC 3 I detritivore -23.01 ± 0.43 5.62 ± 0.31 1.48±0.12 Melanoides tuberculata (Thiarid snail) TS 3 I herbivore -24.15 ± 0.27 7.01 ± 0.81 2.01±0.25 Crustaceans Atyoida bisulcata (Mountain ‘Ōpae shrimp) MOS 1 N omnivore -23.14 7.05 1.92 Macrobrachium lar (Tahitian prawn) TP 3 I omnivore -22.43 ± 0.37 9.02 ± 0.68 2.42±0.16 Herptofauna Palea steindachneri (Wattle-necked softshell turtle) WST 1 I omnivore -20.64 10.46 2.66 Rana catesbeiana (Bullfrog) BFG 1 I herbivore -30.93 4.23 1.92 Fish Archocentrus nigrofasciatus (Convict cichlid) CVC 4 I omnivore -23.59 ± 0.72 10.48 ± 0.38 2.98±0.15 Awaous guamensis (‘O‘opu nākea goby) ONG 1 N omnivore -22.99 9.67 2.67 Clarias fuscus (Chinese catfish) CC 4 I carnivore -23.51 ± 0.58 10.22 ± 1.41 2.89±0.39 Gambusia affinis (Mosquitofish) MQF 3 I omnivore -25.13 ± 0.91 8.95 ± 0.13 2.69±0.06 Misgurnus anguillicaudatus (Oriental weatherfish) ORW 2 I carnivore -22.79 ± 2.5 9.38 ± 0.92 2.57±0.01 Poecilia sp. Hybrid complex (Liberty/Mexican molly) MXM 4 I omnivore -24.79 ± 0.76 9.58 ± 0.45 2.84±0.18 Poecilia eticulate (Guppy) GPY 3 I omnivore -23.88 ± 0.54 8.81 ± 0.75 2.52±0.25 Xiphophorus helleri (Green swordtail) GSW 3 I omnivore -25.09 ± 1.56 9.29 ± 0.67 2.79±0.3 Kapa’a (lower) Insect/worms Cheumatopsyche pettiti (Hydropsychid caddisfly) HC 1 I herbivore -23.79 7.53 1.9997 Moderate disturbance Turbellaria sp. (Flatworm) FW 1 N carnivore -22.37 8.04 2.37 2009 Turbellaria sp. (Non-native Flatworm) FWII 1 I carnivore -23.84 5.04 1.26 Mollusks Corbicula fluminea (Asiatic freshwater clam) AFC 1 I detritivore -13.24 6.86 3.45 Melanoides tuberculata (Thiarid snail) TS 3 I herbivore -18.19 ± 0.736 4.56 ± 1.43 2±0.4 Crustaceans Atyoida bisulcata (Mountain ‘Ōpae shrimp) MOS 1 N omnivore -19.93 5.69 2.06 Hyalella sp. (Amphipod) AMP 1 N omnivore -23.75 5.02 1.27 Macrobrachium grandimanus (‘Ōpae ‘oeha’a prawn) OOP 3 N omnivore -22.34 ± 1.4 8.82 ± 0.15 2.61±0.225 Macrobrachium lar (Tahitian prawn) TP 3 I omnivore -21.61 ± 0.75 9.39 ± 1.57 2.89±0.54 Fish Awaous guamensis (‘O‘opu nākea goby) ONG 3 N omnivore -22 ± 0.788 10.52 ± 0.446 3.16±0.05 Eleotris sandwicensis ('O'opu 'akupa goby) OAG 3 N carnivore -20.7 ± 0.401 9.23 ± 0.608 2.98±0.12 Kuhlia sandvicensis (Hawaiian flagtail) HFT 4 N carnivore -22.79 ± 0.92 10.13 ± 0.343 2.92±0.04 Mugil cephalus (Striped mullet) SM 3 N omnivore -19.77 ± 0.503 4.3 ± 0.582 1.68±0.21 Poecilia sp. hybrid complex (Liberty/Mexican molly) MXM 3 I omnivore -22.9 ± 0.565 9.77 ± 0.828 2.8±0.16 Poecilia reticulata (Guppy) GPY 1 I omnivore -24.58 8.61 2.19 17

Table 1 (Continued)

Stream site (reach)/ δ13C δ15N TP Disturbance level/ Year Type of Organism Web Member ID n Status Feeding Niche (mean±SD) (mean±SD) (mean±SD) Stenogobius hawaiiensis ('O'opu naniha goby) ONAG 2 N omnivore -24.72 ± 1.16 9.42 ± 2.91 2.41±1.04 Hulē'ia (lower) Insect/worms Cheumatopsyche pettiti (Hydropsychid caddisfly) HC 1 I herbivore -26.88 4.77 1.73 Moderate disturbance Mollusks Melanoides tuberculata (Thiarid snail) TS 3 I herbivore -17.46 ± 4.27 5.31 ± 1.32 2.09±0.299 2008 Crustaceans Atyoida bisulcata (Mountain 'Ōpae shrimp) MOS 1 N omnivore -20.91 4.44 1.76 Macrobrachium grandimanus ('Ōpae 'oeha'a prawn) OOP 3 N omnivore -23.7 ± 0.513 7.34 ± 0.238 2.55±0.06 Herptofauna Chrysemys scripta elegans (Red-eared slider) RES 1 I omnivore -19.33 11.55 3.88 Fish Eleotris sandwicensis ('O'opu 'akupa goby) OAG 3 N carnivore -22.06 ± 1.12 6.49 ± 0.914 2.34±0.26 Kuhlia sandvicensis (Hawaiian flagtail) HFT 3 N carnivore -22.76 ± 1.41 9.17 ± 0.349 3.11±0.13 Micropterus salmoides (Largemouth bass) LMB 1 I carnivore -24.19 11.11 3.65 Mugil cephalus (Striped mullet) SM 3 N omnivore -22.77 ± 3.65 7.63 ± 0.547 2.66±0.14 Poecilia sp. hybrid complex (Liberty/Mexican molly) MXM 3 I omnivore -30.18±5 8.18 ± 1.47 2.66±0.38 Oreochromis/Sarotherodon sp. (Tilapia) TL 2 I herbivore -25.14 ± 0.055 8.02 ± 0.516 2.73±0.15 Stenogobius hawaiiensis ('O'opu naniha goby) ONAG 3 N omnivore -26.11 ± 0.642 8.67 ± 0.609 2.895±0.189 Hulē'ia (lower) Insect/worms Cheumatopsyche pettiti (Hydropsychid caddisfly) HC 1 I herbivore -25.59 5.9 1.94 Moderate disturbance Ischnura sp. (Damselfly) ISC 1 N carnivore -23.33 8.11 2.68 2009 Mollusks Corbicula fluminea (Asiatic freshwater clam) AFC 1 I detritivore -15.15 6.86 2.65 Melanoides tuberculata (Thiarid snail) TS 3 I herbivore -21.74 ± 2.64 5.63 ± .508 2.02±0.042 Crustaceans Atyoida bisulcata (Mountain 'Ōpae shrimp) MOS 1 N omnivore -19.1 3.74 1.57 Hyalella sp. (Amphipod) AMP 1 N omnivore -23.17 5.32 1.87 Macrobrachium grandimanus ('Ōpae 'oeha'a prawn) OOP 3 N omnivore -22.82 ± .808 8.68 ± .967 2.87±0.26 Macrobrachium lar (Tahitian prawn) TP 1 I omnivore -22.95 9.09 2.99 Fish Cichla ocellaris (Peacock bass) PKB 1 I carnivore -22.28 12.36 3.98 Clarias fuscus (Chinese catfish) CC 1 I carnivore -22.31 9.21 3.05 Eleotris sandwicensis ('O'opu 'akupa goby) OAG 3 N carnivore -22.37 ± .76 8.6 ± .582 2.87±0.1995 Gambusia affinis (Mosquitofish) MQF 1 I omnivore -27.19 7.48 2.35 Kuhlia sandvicensis (Hawaiian flagtail) HFT 2 N carnivore -21.89 ± 1.27 10.05 ± .247 3.31±0.02 Micropterus dolomieui (Smallmouth bass) SMB 1 I carnivore -23.58 12.16 3.86 Micropterus salmoides (Largemouth bass) LMB 1 I carnivore -23.58 12.14 3.86 Mugil cephalus (Striped mullet) SM 2 N omnivore -22.43 ± 2.66 8.05 ± 1.8 2.7±0.42 Poecilia sp. hybrid complex (Liberty/Mexican molly) MXM 3 I omnivore -26.75 ± 1.46 8 ± 1.58 2.51±0.47 Poecilia reticulata (Guppy) GPY 1 I omnivore -25.73 7.46 2.4 Stenogobius hawaiiensis ('O'opu naniha goby) ONAG 3 N omnivore -23.31 ± 1.67 8.57 ± 1.82 2.82±0.48 'Opaeka'a (upper) Insect/worms Cheumatopsyche pettiti (Hydropsychid caddisfly) HC 1 I herbivore -21.74 6.32 1.5 High disturbance Mollusks Corbicula fluminea (Asiatic freshwater clam) AFC 2 I detritivore -21.83 ± 4.35 7.72 ± 0.11 1.91±0.41 2008 Melanoides tuberculata (Thiarid snail) TS 1 I herbivore -23.43 10.34 2.82 Pomacea sp. (Apple snail) AS 3 I herbivore -25.96 ± 0.471 6.43 ± 0.30 1.89±0.049 Crustaceans Macrobrachium lar (Tahitian prawn) TP 3 I omnivore -24.28 ± 0.313 10.97 ± 1.04 3.08±0.28 Neocaridina denticulata sinensis (Grass shrimp) GS 3 I omnivore -26.49 ± 1.74 9.96 ± 0.43 2.98±0.07 Procambarus clarkii (Louisiana crawfish) LC 3 I omnivore -21.72 ± 2.34 8.72 ± 1.29 2.2±0.24 Herptofauna Bufo marinus (Cane toad) CT 2 I herbivore -18.2 ± 3.11 7.56 ± 0.27 1.55±0.35 Fish Clarias fuscus (Chinese catfish) CC 3 I carnivore -24.03 ± 0.872 10.69 ± 0.03 2.98±0.085 Corydoras aeneus (Bronze catfish) BC 3 I carnivore -26.5 ± 2.59 12.64 ± 0.64 3.77±0.088 Gambusia affinis (Mosquitofish) MQF 3 I omnivore -22.21 ± 2.88 11.45 ± 1.22 3.05±0.13 Micropterus dolomieui (Smallmouth bass) SMB 3 I carnivore -23.54 ± 1.65 13.29 ± 0.6 3.7±0.035 Oreochromis/Sarotherodon sp. (Tilapia) TL 3 I herbivore -25.47 ± 1.04 9.54 ± 1.1 2.77±0.33 'Opaeka'a (upper) Insect/worms Ischnura sp. (Damselfly) ISC 1 N carnivore -31.49 9.24 4.88 High disturbance Mollusks Corbicula fluminea (Asiatic freshwater clam) AFC 2 I detritivore -26.49 ± 0.333 7.97 ± 0.058 2.34±0.16 2009 Melanoides tuberculata (Thiarid snail) TS 3 I herbivore -26.61 ± 0.395 7.81 ± 0.595 2.35 Pomacea sp. (Apple snail) AS 3 I herbivore -27.1 ± 0.728 5 ± 0.739 1.73±0.53 18

Table 1 (Continued)

Stream site (reach)/ δ13C δ15N TP Disturbance level/ Year Type of Organism Web Member ID n Status Feeding Niche (mean±SD) (mean±SD) (mean±SD) Crustaceans Macrobrachium lar (Tahitian prawn) TP 5 I omnivore -24.93 ± 0.673 9.78 ± 0.885 2.2±0.4 Procambarus clarkii (Louisiana crawfish) LC 4 I omnivore -24.9 ± 1.684 8.58 ± 0.559 1.83±0.76 Herptofauna Chrysemys scripta elegans (Red-eared slider) RES 1 I omnivore -23.59 11.44 2.1 Palea steindachneri (Wattle-necked softshell turtle) WST 1 I omnivore -19.31 12.2 0.48 Rana catesbeiana (Bullfrog) BFG 1 I herbivore -20.96 10.99 0.84 Fish Clarias fuscus (Chinese catfish) CC 2 I carnivore -24.55 ± 2.4 10.58 ± 0.912 2.27±1.31 Gambusia affinis (Mosquitofish) MQF 3 I omnivore -22.93 ± 3.4 12.1 ± 0.561 2.02±1.41 Micropterus dolomieui (Smallmouth bass) SMB 1 I carnivore -24.52 13.8 3.19 Oreochromis/Sarotherodon sp. (Tilapia) TL 2 I herbivore -23.66 ± 1.92 11.8 ± 1.12 2.23±0.5 Xiphophorus helleri (Green swordtail) GSW 2 I Omnivore -30.44 ± 0.594 10.9 ± 1.43 4.92±0.68

Note. Included is each member's species ID, total number of samples collected (n), species status (native=N or invasive=I), general feeding niche, δ13C and δ15N signatures (mean ± SD), and trophic position (mean ± standard deviation).

19

Table 2. Primary producers sampled at each stream site in 2008 and 2009

Level of Stream Basal δ13C δ15N Year disturbance (reach location) Source n (mean ± SD) (mean ± SD) Filamentous 2008 Low Limahuli (upper) algae 6 -14.73 ± 0.11 1.91 ± 0.47 Periphyton 6 -28.21 ± 0.16 7.04 ± 1.09 Leaf litter 5 -29.85 ± 1.15 0.13 ± 1 Filamentous Moderate Hulē’ia (lower) algae 6 -31.68 ± 0.23 4.75 ± 0.6 Periphyton 6 -22.83 ± 0.17 4.68 ± 0.74 Leaf litter 6 -29.95 ± 1.88 2.11 ± 1.05 Filamentous High 'Opaeka'a (upper) algae 5 -43.2 ± 0.67 6.84 ± 0.99 Periphyton 6 -29.51 ± 0.44 7.52 ± 1.44 Leaf litter 6 -27.16 ± 0.43 4.23 ± 1.45 Filamentous 2009 Low Limahuli (upper) algae 3 -18.36 ± 0.37 1.73 ± 0.089 Periphyton 3 -22.39 ± 0.56 2.76 ± 0.32 Leaf litter 3 -29.72 ± 0.12 0.62 ± 0.059 Low Limahuli (lower) Periphyton 3 -26.74 ± 0.2 2.02 ± 0.92 Leaf litter 3 -31.34 ± 0.13 0.93 ± 0.18 Kapa'a (upper) Filamentous Moderate algae 3 -21.93 ± 0.67 10.3 ± 0.56 Periphyton 3 -24.82 ± 0.35 4.38 ± 0.74 Leaf litter 3 -29.63 ± 0.27 1.46 ± 0.06 Moderate Kapa'a (lower) Periphyton 3 -23.74 ± 0.11 7.51 ± 0.39 Leaf litter 3 -31.47 ± 0.296 1.54 ± 0.1 Moderate Hulē’ia (lower) Periphyton 3 -25.41 ± 0.15 4.87 ± 0.65 Leaf litter 3 -30.77 ± 0.2 2.24 ± 0.09 High 'Opaeka'a (upper) Periphyton 3 -31.94 ± 0.4 6.9 ± 0.12 Leaf litter 3 -29.49 ± 0.09 3.82 ± 0.02 Total number of samples collected (n), δ13C and δ15N signatures (mean ± standard deviation).

The moderately disturbed streams contained more non-native species (Figure 3a-d).

Kapa’a stream contained thirteen non-natives at the upper reach site, (87% non-native species), and seven non-natives at the lower reach site (53% non-native species) (Figure

3a-b). Hulē’ia stream contained six non-native species in 2008, (50% non-native species), 20 and eleven non-natives in 2009 (58% non-native species) (Figure 3c-d). The range of

δ13C signatures and trophic positions in the moderately disturbed stream food webs were larger than those in Limahuli; 12.72 (-30.18 to -17.46‰) was the largest range of consumer δ13C signatures in this study, found in Hulē’ia during 2008 sampling year

(Table 1). Non-native consumer species were the dominant consumers inhabiting the top trophic level in the moderately disturbed streams (Figure 3a-d). There were differences in the isotopic signatures among primary producers in the moderately disturbed streams compared to those similar taxa in Limahuli (Figure 4a-f). On average, filamentous algae was more δ13C-depleted and δ15N-enriched in the moderately disturbed streams, periphyton was highly δ13C-enriched in a two sites but lacked any general trends in variation between the lesser and moderately disturbed streams, and leaf litter was more

δ15N-enriched but lacked any general trend in variation for mean δ13C signatures

(Figure 4a-f).

‘Opaeka’a, the more heavily disturbed stream, contained thirteen non-natives in 2008 and was completely absent of native consumers (100% non-native species), and in 2009 contained thirteen non-natives and one native consumer, (93% non-native species), making ‘Opaeka’a, the most heavily invaded stream in this study (Figure 5a-b). The range of δ13C signatures in ‘Opaeka’a were generally larger than those in Limahuli and

‘Opaeka’a contained the largest trophic position range 4.44 (0.479 to 4.92) in this study in 2009 (Table 1). Consumers inhabiting the top trophic level in ‘Opaeka’a were all non- native species (Figure 5a-b). Isotopic signatures of the primary producers were generally different in the heavily disturbed stream compared to Limahuli (Table 2; Figure 4).

Filamentous algae was most δ13C-depleted and δ15N-enriched in ‘Opaeka’a compared to 21

a) b)

c) d)

Figure 3. Mean δ13C and TP (±standard deviation) values of consumers in the moderately disturbed streams; a) upper Kapa’a site in 2009,b) lower Kapa’a site in 2009, c) lower Hulē’ia site in 2008 and d) lower Hulē’ia site in 2009. Symbols represent type of organism; triangle=fish, square=herptofauna, diamond=crustacean, circle=mollusks, and line=aquatic insect/flatworms. Black represents s native consumer taxa and white represents non-native consumer taxa.

22

Figure 4. Differences in mean isotopic signatures of primary producers in 2008 and 2009 across sites with varying levels of disturbance including results of Kruskal-Wallis Test below. a) δ13C filamentous algae (2008, H=15.16, P=0.001), and b) δ15N filamentous algae (2008, H=15.16, P=0.001), c) δ13C periphyton (2008, H=15.16, P=0.001; 2009, H=15.83, P=0.007), d) δ15N periphyton (2008, H=10.89, P=0.004; 2009, H=15.83, P=0.007), e) δ13C leaf litter (2008, H = 7.41, P = 0.025; 2009, H=14.99, P=0.01), and f) δ15N leaf litter (2008, H=11.66, P =0.003; 2009, H=16.25, P=0.006). Box plots include mean (center line), interquartile range (box), and outliers (outer lines). Note: “d” in figures represent δ.

23 a) b)

Figure 5. Mean δ13C and TP (±standard deviation) values of consumers in the highly disturbed stream, ‘Opaeka’a, in the a) upper site in 2008 and b) upper site in 2009. Symbols represent type of organism; triangle=fish, square=herptofauna, diamond=crustacean, circle=mollusks, and line=aquatic insect/flatworms. Black represents s native consumer taxa and white represents non-native consumer taxa.

all other streams (Figure 4a-b). Periphyton was generally more δ13C-depleted and δ15N-

enriched and leaf litter was slightly δ13C-enriched and δ15N-enriched in ‘Opaeka’a

(Figure 4c-f).

Shifts in Native Species δ13C and Trophic Positioning

All seven native freshwater fish species were sampled in this study in addition to five

native invertebrates, but only three native taxa were found in multiple sites (lesser and

moderately disturbed streams) to be included in this analysis; Awaous guamensis,

Eleotris sandwicensis, and Atyoida bisulcata (Table 3). There were minimal differences

in mean δ13C signatures and mean trophic positions for each of the three native species

across streams, though; there were general trends in variation (Figure 6). Awaous

guamensis had the lowest mean trophic position (2.67) and the highest mean trophic

position (3.16) in the same stream, Kapa’a (Fig 6a). A. guamensis was most δ13C- 24

Table 3. Results of Kruskal-Wallis Test comparing native consumer species mean δ13C and mean trophic positions between stream sites with varying levels of disturbance and between sampling years Sources of Variation Sites Year Native consumers H DF P H DF P Awaous guamensis δ13C 1.56 3 0.67 0.43 1 0.513 Trophic position 2.33 3 0.506 0.43 1 0.513 Eleotris sandwicensis δ13C 5.96 2 0.051 0.01 1 0.926 Trophic position 2.49 2 0.288 6.23 1 0.013* Atyoida bisulcata δ13C 4 4 0.406 0.09 1 0.77

enriched in Limahuli (-21.99‰) and most δ13C-depleted (-22.99‰) in Kapa’a (Fig 6b).

Eleotris sandwicensis had the highest mean trophic position (2.98) in Kapa’a and lowest mean trophic position (2.74) in Limahuli (Fiure. 6c). E. sandwicensis was most δ13C- enriched in Kapa’a (-20.7‰) and the most δ13C-depleted (-22.95‰) in Limahuli (Figure

6d). Atyoida bisulcata had the highest mean trophic position in Limahuli (2.38) and the lowest (1.57) in Hulē’ia (Figure 6e). A. bisulcata was most δ13C-enriched (-19.93‰) in

Lower Kapa’a and Hulē’ia (-19.1‰) whereas the shrimp was most δ13C-depleted in

Limahuli (-23.54‰) and (-23.14‰) in Lower Kapa’a (Figure 6f).

Variation in Food Web Trophic Diversity and Trophic Redundancy

Mean centroid distance (CD), mean nearest neighbor distance (NND), and standard deviation of mean nearest neighbor distance (SDNND) varied among streams in both sampling years (Table 4). The food web with the highest CD was in ‘Opaeka’a in 2009 and the lowest CD was in Lower Limahuli in 2009 (Table 4). Highest NND was in Upper

Limahuli, 2008, and the smallest NND was in Lower Limahuli in 2009 (Table 4). Highest 25

Figure 6. Comparison of mean trophic positions and mean δ13 signatures (± standard deviation)of a-b) Awaous guamensis, c-d) Eleotris sandwicensis, and e-f) Atyoida bisulcata across sites with lesser (Limahuli sites) and moderate (Kapa’a and Hulē’ia sites) levels of disturbance in 2009. 26

Table 4. Calculated mean centroid distance (CD), mean nearest neighbor distance (NND), and standard deviation of nearest neighbor (SDNND) for all streams sites with varying levels of disturbance in both 2008 and 2009 sampling years Level of Stream Year Disturbance (reach location) CD NND SDNND 2008 Low Limahuli (upper) 1.94 1.55 1.7 Moderate Hulē’ia (lower) 2.67 1.42 0.94 High Opaeka'a (upper) 2.01 0.88 0.86 2009 Low Limahuli (upper) 2.23 0.77 0.37 Low Limahuli (lower) 0.92 0.54 0.51 Moderate Kapa'a (upper) 1.52 0.75 1.22 Moderate Kapa'a (lower) 2.13 0.73 1.24 Moderate Hulē’ia (lower) 1.89 0.699 1.05 High Opaeka'a (upper) 4.28 0.68 0.55

SDNND was in Upper Limahuli in 2008 and the lowest SDNND was at the same site

2009 (Table 4).

CHAPTER IV

Discussion

Disturbance in Hawaiian Streams

Current research suggests that there is a clear relationship between anthropogenic disturbance and the introduction of invasive species in Hawai’i’s ecosystems (Brasher

2003; Brasher et al. 2006). In Hawai’i’s freshwater ecosystems, increasing abundance and encroachment of invasive species may be related to declining native populations

(Englund 1999; Englund and Polhemus 2001; Englund et al. 2001). Still, the literature is

unclear as to the impact that invasive species have on the structure of stream food webs.

This study is the first of its kind to depict the effects of disturbance in Hawaiian stream food webs using stable isotope analysis and the first study to examine the implications of non-native biota to Hawai’i’s stream ecosystems by employing the use of stable isotopes.

The results of our study suggest that there are significant trophic and structural changes

occurring to Hawaiian stream food webs with increased levels of biotic disturbance.

Ecosystem-Level Processes Affecting Overall Food Web Structure

The forces that drive variation in the isotopic signatures of biota among different

systems are the result of a both natural processes and anthropogenic disturbance

occurring at the ecosystem level. A variety of natural biogeochemical processes can

cause differences at the base of these streams food webs, specifically, in the isotopic

makeup of primary producer taxa across systems (Coat et al. 2009; Hobson et al. 2007;

27 28

Natural variation among isotopic signatures of different primary producers of a single food web is common (Coat et al. 2009; March and Pringle 2003). Autochthonous and allochthonous instream resources have been reported to be isotopically distinct in

Hawai’i stream systems; in particular filamentous algae show enriched δ13C and δ15N signatures relative to instream leaf litter (Hobson et al. 2007). In this study, isotopic signatures of primary producers clearly varied within each site. δ13C signatures of filamentous algae and periphyton were more enriched than leaf litter in all sites, except

‘Opaeka’a, where autochthonous producers (filamentous algae and periphyton) were more depleted than allochthonous producers (instream leaf litter). δ15N signatures of filamentous algae or periphyton were most enriched across sites and δ15N of instream leaf litter were most depleted in all sites.

Anthropogenic disturbance, like input of wastewater and pollution, has been linked to changes in isotopic signatures across systems, including enrichment in δ15N signatures of stream primary producers and changes in the δ13C signatures of food web members

(deBruyn et al. 2003; Gammons et al. 2010; Mercado-Silva et al. 2008; Saito et al. 2008).

In the current study isotopic signatures of primary producers varied across stream systems. Filamentous algae was more δ13C-depleted and δ15N-enriched in disturbed sites, specifically ‘Opaeka’a stream, suggestive of altered ecosystem processes which are potentially related to higher inputs of nitrogen into this watershed. Variation in δ13C signatures of algae across freshwater ecosystems may be related to changes in flow rate in modified systems (Finlay et al. 1999). δ13C signatures of periphyton and leaf litter lacked a consistent trend in isotopic variation across stream sites. Yet leaf litter was slightly more δ15N-enriched in highly disturbed sites in both sampling years, possibly be 29 due to different terrestrial plant species occurring along stream or, alternatively, leaf litter samples could have been contaminated by algal signals. Changes in the δ13C and δ15N signatures of autochthonous resources (filamentous algae) are an indication that biogeochemical processes within stream systems are affecting the chemical makeup of aquatic primary production.

Filamentous algae stands were low in 2009; lowered density of macroalgae has been shown to be due to top-down control of basal resources via herbivore grazing or physical disturbance like flooding (Grimm and Fisher 1989; March et al. 2002; Sherwood and

Kido 2002). Changes in algal abundance are suggested to be related to changes in stream flow and periodic disturbance In Hawaiian stream ecosystems (Kido 1997). In the weeks prior to sampling in March of 2009, there was a large storm, and this disturbance may help explain changes to filamentous algae densities at four of the six stream sites that year. Algal resources are considered to be an important trophic resource in tropical stream food webs (Kido 1996; Kido 1997; Lau et al. 2008; Sherwood and Kido 2002).

The absence of filamentous algae in many of the streams in 2009 does not lessen its importance in these food webs but merely points to the reality of seasonal variation in food sources and the implications that has for isotopic studies that only capture a brief time period of community structure.

Community-Level Processes Affecting Overall Food Web Structure

Processes at the community level may cause changes in the overall structure of

Hawaiian stream food webs. Systems confounded by biotic disturbance show stark differences in the structure of food webs in comparison to systems lacking invasion 30

(Vander Zanden et al. 1999; Vander Zanden et al. 2003). In this study the overall structure of food webs with increasing levels of disturbance differed in the following consistent manner; increased numbers of invasive species, larger proportion of non- natives present, changes in the range of consumer δ13C and range of consumer trophic positions, and the origin (native/non-native) of species inhabiting the top trophic level.

Previous research suggests that more species occupy Hawaiian streams impacted by human development and typically, non-natives outnumber native species in these disturbed systems (Brasher et al. 2006). In the current study, Limahuli was the least impacted by biotic disturbance, contained four of the five native gobies with only two non-native taxa present at each site, and represents the most pristine stream. The number of non-native species inhabiting stream food webs increased dramatically in the moderately and highly disturbed streams, and the proportion of non-natives increased in disturbed food webs, including one sample composed entirely of non-natives.

As stated earlier, in stream systems, autochthonous resources are generally more δ13C- enriched then allochthonous resources (Coat et al. 2009; Hobson et al. 2007; March and

Pringle 2003). The range of consumer δ13C signatures in Limahuli, the least disturbed food web, was relatively narrow and δ13C signatures overall were relatively enriched, similar to the δ13C signature for filamentous algae. These findings suggest that autochthonous resources may act as major carbon supply in Limahuli and that algae should be considered an important trophic resource for native gobies and other Hawaiian freshwater biota (Kido 1996; Kido 1997; Lau et al. 2008; Sherwood and Kido 2002).

Comparisons of native and non-native herbivores in Limahuli suggest that consumers are utilizing different basal resources in Hawaiian streams. The native snail, Neritina 31 granosa, has a similar trophic position to the non-native caddisfly, Cheumatopsyche pettiti, but the caddisfly was more δ13C-depleted, inferring that these two species feed on sources with similar trophic positions but with distinct δ13C. In Hawaiian streams, C. pettiti is reported to rely equally on instream leaf litter and algae (Kondratieff et al.

1997). In Limahuli, the relatively depleted δ13C signatures of C. pettiti suggests a heavy reliance on δ13C-depleted leaf litter, and suggest that non-native and native herbivores utilize different basal resources in relatively pristine conditions.

Increasing ranges of consumer δ13C signatures between systems with varying levels of invasive species is an indication that, as a whole, food web members are utilizing a larger breadth of basal food resources (Mercado-Silva et al. 2008). For example, Hulē’ia, one of the moderately disturbed streams, exhibited the largest range in consumer δ13C signatures. Interestingly, Hulē’ia contained one of the highest total number of consumers and the most evenly proportioned food web in terms of native and non-native species present, suggesting that the moderately disturbed systems contain the most species and exploit the largest breadth of resources. In the heavily disturbed stream, ‘Opaeka’a, the range of consumer δ13C signatures was similar to Limahuli, suggestive that the food web structure of ‘Opaeka’a mirrors the structure of pristine Hawaiian stream food web, and in the absence of native stream species, non-native aquatic species may in fact be utilizing similar resources and replacing the trophic roles of native Hawaiian stream species. To be more confident in these results we would need to examine a greater spatial and temporal range of the food webs within this watershed.

An increase in the overall range of consumer trophic positions are an indication that more trophic levels are being inhabited (Mercado-Silva et al. 2008). Differences in the 32 trophic positioning of a single species in different systems is an indication that the organism is consuming at different trophic levels, for example, an omnivore with a relatively low trophic positions is possibly indicative of a reliance on primary producers as a dominant food source (Mercado-Silva et al. 2008). The low and narrow trophic range found in Limahuli stream suggests short food chains, minimal trophic levels, and heavy reliance on primary production as sources of food. The streams with greater levels of disturbance and invasion had correspondingly larger ranges of consumer trophic positions compared to Limahuli, due to more non-native species present, inferring increased trophic inhabitance. In addition, species inhabiting the highest trophic positions in the more disturbed streams were primarily non-native species, filling predatory roles. Hulē’ia contained an additional trophic level not present in Limahuli which included three predatory bass, Cichla ocellaris, Micropterus salmoides, Micropterus dolomieu, and one non-native omnivorous turtle, Chrysemys scripta elegans. The presence of this many novel species inhabiting top trophic levels most likely increases the level of predator-prey interactions.

The most highly disturbed stream, ‘Opaeka’a held the largest range in trophic positions in 2009 due to the extremely high trophic positions of a native Ishnura spp. and the non- native Xiphophorus helleri in addition to the extremely low trophic positions of the non- native Palea steindachneri. The non-native clam, Corbicula fluminea in Lower Kapa’a also had an extremely enriched trophic position like those found in ‘Opaeka’a. Ishnura spp., C. fluminea, and X. helleri have relatively low δ15N signatures, not suggestive of holding high trophic positions. Large calculated trophic positions with low δ15N are probably due to their extremely enriched δ13C signature, which may affect the 33 calculation of δ15Nbaseline and trophic positioning (Sudeep Chandra pers. comm.). A

similar explanation may explain the extremely depleted trophic position of the Palea

steindachneri which are known to be carnivorous (Yamamoto et al. 2000).

Shifts in Native Species Trophic Space in Variably Disturbed Streams

A change in the isotopic signatures and trophic positioning of a single species across

different systems is an indication that the organism is potentially altering its feeding

habits (Mercado-Silva et al. 2008). The presence of invasive species in aquatic food webs

can change the trophic positioning of native species, by out-competing natives for their

preferred food sources and displacing natives in trophic space (Vander Zanden et al.

1999). In this study, three native stream consumers, Awaous guamensis, Eleotris

sandwicensis, and Atyoida bisulcata inhabited multiple sites with lesser and moderate

levels of disturbance, and were found to have slight variations in carbon sourcing and

trophic positioning.

Awaous guamensis is omnivorous and is known to feed on filamentous algae and

various aquatic invertebrates (Yamamoto et al. 2000). δ13C-enriched and δ15N-enriched

signatures of A. guamensis in Limahuli generally reflects isotopic signatures of this

species in other published studies and suggests a heavy reliance on algal food sources

(Hobson et al. 2007). In the more disturbed stream, Lower Kapa’a, A. guamensis had the

highest mean trophic position indicative of a reliance on food sources with higher trophic

positioning, like, aquatic invertebrates and crustacean species. Many invertebrate species

were present in Kapa’a compared to Limahuli; nine species in Kapa’a and only four

species in Upper Limahuli in the same year. With more food sources available in Kapa’a, 34

A. guamensis may utilize various food items not available to the goby in more pristine stream food webs. A. guamensis was most δ13C-depleted (allochthonous reliance) in

Upper Kapa’a and more omnivores inhabiting similar trophic space may be displacing A. guamensis. Seven non-native omnivorous fish and crustacean species with similar feeding habits were present in Upper Kapa’a, compared to only one non-native omnivore in Upper Limahuli.

Eleotris sandwicensis is the islands only amphidromous freshwater predator species and consumes various fish and aquatic invertebrates (Yamamoto et al. 2000). The δ15N signatures of E. sandwicensis in Limahuli generally reflect the signatures of this species in other published studies (Hobson et al. 2007). δ13C signatures of E. sandwicensis in

Limahuli were more depleted than results in similar studies, possibly due to inherent differences in δ13C of basal resources across different systems or differences in particular food sources the predatory goby is utilizing in Limahuli compared to other studies (deBruyn et al. 2003; Gammons et al. 2010; Hobson et al. 2007; Vander Zanden et al. 1999). The predatory native goby was most δ13C-enriched in Lower Kapa’a suggestive that the gobies food sources have relatively more enriched δ13C signatures or the goby is relying on different food sources in Kapa’a relative to Limahuli, including various invertebrates and small fish species not present in Limahuli. E. sandwicensis had the highest mean trophic position in Lower Kapa’a perhaps because more possible food sources for the predatory goby had higher trophic positions themselves than possible food sources in Limahuli. E. sandwicensis shared similar δ13C and trophic space with Kuhlia sandvicensis, Macrobrachium grandimanus, Macrobrachium lar, and Poecilia sp. hybrid complex, and is possibly predating on these species. 35

The final native species found at numerous streams in this study was Atyoida bisulcata; a small shrimp that uses pincers to pluck food off the surface of benthic substrate and feeds on suspended food particles by filtering them out of the water column

(Yamamoto et al. 2000). The native shrimp had the highest mean trophic position in the upper reach of Limahuli and the most enriched δ13C in the in the lower reaches of

Kapa’a and Hulē’ia. Differences in trophic positioning of the native shrimp may be due to inherent differences in the isotopic signatures of food sources found on substrate or in water column in different streams and more δ13C-enriched signatures in Hulē’ia may suggest a heavier reliance on algal sources (deBruyn et al. 2003; Gammons et al. 2010;

Hobson et al. 2007; March et al. 2002).

Trophic Diversity and Redundancy in Hawai’i’s Disturbed Stream Food Webs

Food web dispersion, or mean centroid distance, mean nearest neighbor distance, and standard deviation of nearest neighbor distance, together suggest the degree of interspecific interactions occurring in a given food web (Layman et al. 2007; Mercado-

Silva et al. 2008). ‘Opaeka’a had the largest centroid distance in 2009, but the centroid distance for the same stream in the previous year was two units lower. The high centroid distance in 2009 was most likely due to the extremely enriched and depleted trophic positions of those mentioned above, Ishnura spp. Xiphophorus helleri, and Palea steindachneri. The next highest centroid distance was found in the moderately disturbed stream, Hulē’ia, and is the most likely food web with the greatest trophic diversity. The native species in Hulē’ia may be confounded with higher levels of interspecific interactions, like competition and predation, since this stream contained the largest 36 centroid distance and the shortest nearest neighbor distance and some of the highest number of total consumer present. A large clustering of native and non-native consumers were seen in the food web of Hulē’ia in both sampling years sharing similar δ13C signatures and similar trophic positions including the Eleotris sandwicensis, Stenogobius hawaiiensis, Kuhlia sandvicensis, Mugil cephalus, Macrobrachium grandimanus,

Ischnura spp., in addition to the non-native Oreochromis/Sarotherodon spp. and

Macrobrachium lar suggesting that this cluster of species are competing for the same food sources or possibly predating on one another.

Lower Limahuli had the least trophic diversity and the highest trophic redundancy.

Interestingly, Upper and Lower Limahuli contained the largest and smallest standard deviation of nearest neighbor distance, meaning two spatially explicit food webs in the least disturbed stream have the least and most evenly concentrations of species, suggesting that the community structure of native Hawaiian streams are spatially different throughout a single watershed (Kinzie 1988; Yamamoto et al. 2000).

Interestingly, the lower and mid reach sites on Kapa’a showed similar variation in community makeup within the single watershed, where the upper site on Kapa’a had far more non-native consumers than the lower site. In all, calculations of trophic diversity and redundancy suggests that the trophic structure of Limahuli stream is the simplest, characterized by low species diversity and represents characteristics of native Hawaiian stream communities reported in the literature (Brasher 2003).

37

Implications for Hawai’i’s Native Freshwater Communities

By employing the use of stable isotope analysis we were able to detect differences in the structure of and interactions within stream food webs among varying levels of disturbance. In this study, the isotopic signatures of autochthonous resources (filamentous algae) varied considerably across levels of both disturbance and invasion in a manner consistent with alterations to instream processes. It may be that increased levels of agricultural runoff and urban waste in the moderate and highly disturbed systems are changing the isotopic structure of the food web’s primary producers.

In addition large numbers of invasive species are changing the structure of food webs and altering species interactions in the disturbed streams. The streams with increased levels of disturbance (Kapa’a, Hulē’ia, and ‘Opaeka’a) have more non-native species than Limahuli, but at the same time these streams also have increased trophic diversity and less niche redundancy. It appears that non-native consumers have filled the vacant niches previously utilized by native taxa, thereby increasing niche inhabitance and overall community trophic diversity. Yet at the same time, non-native predatory fish and herptofauna are generating entirely novel predator/prey interactions in these historically structurally simple food webs. Furthermore, non-native omnivorous consumers are increasing competitive interactions in moderately and highly disturbed Hawaiian streams.

The minimal changes in the isotopic signatures and trophic positions of the three native species examined illustrates that native Hawaiian stream consumers may be maintaining their preferred niche space in the presence of non-native species with similar feeding habits. This suggests that some level of invasion disturbance may be tolerated by the native taxa, without comprising the niche integrity of the native fishes. At the same time 38 we did find minor shifts in the isotopic structuring of native species which may be due to their utilizing novel (and invasive) food sources not present in more pristine Hawaiian streams where the majority of their omnivorous diet is derived primarily from algal- resources.

Landscape level forces of disturbance (i.e. agriculture and urban development) within the Kapa’a, Hulē’ia, and ‘Opaeka’a watersheds, may be altering the conditions of these stream ecosystems and facilitating the establishment of non-native aquatic species

(Brasher 2003; HDAR and Bishop Museum 2008b, 2008c, 2008d). In contrast, the

Limahuli watershed is managed completely for conservation and indigenous land management purposes. Although this study and other published accounts, have shown some level of species invasion in the watershed, the native freshwater community remains intact, as all five native freshwater gobies currently reside in this watershed, and the entire watershed is managed for conservation purposes (HDAR and Bishop Museum

2008a; Kido 2007). As a result, it seems fairly intuitive that in order to maintain Kaua’i’s indigenous freshwater communities, landscape-level rehabilitation and restoration practices should be implemented.

It is interesting to note that within a number of the streams we sampled in Kaua’i, we found established and self reproducing populations of the wattle-necked softshell turtle,

Palea steindachneri (Tag Engstrom pers. comm.) which appears to be relatively abundant in certain drainages but is currently in decline and protected in its native range of Asia

(Ernst and Lovich 2009; McKeown and Webb 1982; Yamamoto et al. 2000). This presents an interesting conservation challenge as the turtle is clearly a significant player in the invaded food webs and is therefore a potential threat to Kaua’i’s native taxa. Yet at 39 the same time, the turtle species is threatened in its native habitat. Increasingly situations like this with species that are both possibly endangered and invasive are likely to become more common. In future studies it would seem advisable to combine population data (i.e. abundance and distribution of P. steindachneri) with the results of stable isotope food webs, as this may help us to better understand the effects the turtles have on the native stream food webs while at the same time it may also aid in our ability to manage the species in its native range.

In conclusion, stable isotope analysis allowed us to examine the structure of Hawaiian stream food webs across varying levels of biotic disturbance. Although natives are nearly absent in highly invaded streams like ‘Opaeka’a, this study suggests that where present, native aquatic species are able to take advantage of the presence of non-native species and potentially expand their niche breadth. In addition, thoughtful ecological management and restoration of the island’s disturbed streams may allow native species and trophic relationships to return.

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