REPRODUCTIVE SCHEDULE AND TRAITS OF AMBLOPS () IN THE FLINT RIVER SYSTEM, HUNTSVILLE,

by

CRISSY L. TARVER

A THESIS

Submitted in partial fulfillment of the requirements for the degree of Masters of Science in The Department of Biological Sciences The School of Graduate Studies of The University of Alabama in Huntsville

Huntsville, Alabama 2015

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ABSTRACT The School of Graduate Studies The University of Alabama in Huntsville

Degree Master of Science College/Dept. Science/Biological Sciences

Name of Candidate Crissy L. Tarver

Title Reproductive Schedule and Traits of Hybopsis amblops (Bigeye Chub) in the Flint River System in Huntsville, Alabama

The goal of this study was to ascertain a reproductive schedule and describe reproductive traits of Hybopsis amblops in the Flint River system in Huntsville, AL.

Members of this species were collected from the Flint River from May, 2013 to

July, 2014. The standard length (SL) of each was measured using digital slide calipers. Gross body mass (GBM) and gonadal mass (GM) were obtained using a digital balance. In adult fish, gonadosomatic indices (GSI) were calculated to aid in determining reproductive timing. Ovaries and oocytes were categorized into developmental stages.

Total fecundity (TF) of each female was calculated from the number of oocytes found in each ovary. The average size of oocytes in each stage of development was defined by diameter measurements. A spawning season from March to June with peaks in April and

May were indicated by mean monthly GSI values. The primary reproductive month was

April as evidenced by a peak GSI value with the retrieval of more than 800 stage III mature and stage IV ripe ova from females collected that month. An absolute fecundity ranged from 90 to 2,566 oocytes. This study examined multiple relationships between

SL, GBM, GSI values, GM, TF, and water temperature. GSI values were found to be independent of SL and GBM in both sexes. Total fecundity not strongly correlated to

GSI values (R2=0.306; p < .0001). The strongest relationship was seen between TF and

GM (R2 = 0.621; p < .0001). Similar trends were noted for the mean monthly water

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ACKNOWLEDGMENTS

This project was made possible by the assistance and support of my mentors, peers, and family. I would like to express my appreciation to Dr. Bruce Stallsmith for his assistance, guidance, and persistence on collection trips during this project. My gratitude extends to Dr. Yong Wang for expanding my knowledge on the application of statistical methods in a biological context. I am grateful to Dr. Luciano Matzkin for all of his lectures on evolution, and for introducing me to JMP 10 statistical software. The completion of this research would not have been possible without the assistance of Kelly

Hodgskins, Joshua Mann, Kara Million, Breann Roberts, Ethan Tarver, Roy Tarver Jr., and Jeffrey Warner who helped with the collection of data and fish. Finally, I would like to thank Roy, Ethan, and Victoria Tarver for their assistance and unrelenting support.

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TABLE OF CONTENTS

Page

List of Figures………………………………………………………………………..….viii

List of Tables……………………………………………………………………………...x

Chapter One: Introduction

1.1 Rationale...... 1 1.2 Cyprinids ...... 6 1.3 Reproductive schedules and strategies of cyprinids ...... 10 1.4 Hybopsis amblops, Bigeye chub ...... 15 1.5 Statement of purpose and hypothesis ...... 19

Chapter Two: Material and Methods

2.1 Study site and sampling...... 20 2.2 Laboratory analysis ...... 22 2.3 Statistical analysis ...... 25

Chapter Three: Results

3.1 Study site data ...... 27 3.2. Sex bias and size structure ...... 28 3.3 Reproductive schedule ...... 32 3.4 Ovarian development, oocyte counts, and oocyte diameters ...... 33 3.5 Relationships………………………………………………………………………39

Chapter Four: Discussion

4.1 Study goals and limitations…………………………………………………..…....42 4.2 Size and reproductive schedule ...... 43 4.3 Fecundity and maturation………...………………………………………….…….44

Appendix……………………………………………………………………..…………..46

Works Cited ...... 72

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LIST OF FIGURES

Figure Page

1.1 Triangular life history continuum model ...... 5

1.2 Deep phylogeny of ray-finned fish () ...... 7

1.3 Distribution map for Hybopsis amblops ...... 16

1.4 Hybopsis amblops , ...... 17

2.1 Flint River location on Oscar Patterson Road in Huntsville, Alabama according to

Google maps ...... 21

2.2 Flint River collection site at Oscar Patterson Road access point in

Huntsville, Alabama ...... 21

2.3 Stages of ovarian development ...... 23

2.4 Stages of oocyte development ...... 24

3.1 Mean (±SD) monthly standard length for female, juvenile, and male

Hybopsis amblops collected from May, 2013 to July, 2014. No females were

collected in January, 2014; no juveniles were collected in July, 2013,

January or June, 2014...... 29

3.2 Mean (±SD) monthly body mass for female, juvenile, and male

Hybopsis amblops collected from May, 2013 to July, 2014. No females were

collected in January, 2014; no juveniles were collected in July, 2013,

January or June, 2014...... 30

3.3 Standard length vs gross body mass relationship in female Hybopsis amblops

collected between May, 2013 and July, 2014...... 30

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3.4 Standard length vs gross body mass relationship in male Hybopsis amblops collected

between May, 2013 and July, 2014...... 31

3.5 Standard length vs gross body mass relationship in juvenile Hybopsis amblops

collected between May, 2013 and July, 2014...... 31

3.6 Mean monthly gonadosomatic indices (GSI) for adult male and female

Hybopsis amblops collected from May, 2013 to July, 2014. No females were

collected in January, 2014. Levels not connected by the same letter are

significantly different...... 32

3.7 Mean (±SE) monthly number of oocytes per stages I, II, III, and IV for

Hybopsis amblops collected from May to June, 2013, and February to July, 2014. . 35

3.8 Standard length vs oocyte counts in developmental stages I-IV for

Hybopsis amblops collected from May, 2013 to July, 2014...... 38

3.9 Total fecundity vs GSI values in female Hybopsis amblops collected from

May, 2013 to July, 2014...... 39

3.10 Total fecundity vs gonadal mass in female Hybopsis amblops collected from

May, 2013 to July, 2014...... 39

3.11 Total fecundity vs GBM in female Hybopsis amblops collected from

May, 2013 to July, 2014...... 40

3.12 Common trends for mean monthly fecundity values vs mean water

temperatures (ºC) collected from May, 2013 to July, 2014...... 40

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LIST OF TABLES

Table Page

3.1 Monthly water temperatures and average rate of river discharge (m3/sec) from the

U.S. Geological Survey database 03575100 at Brownsboro, AL from

May, 2013 to July, 2014...... 27

3.2 Total number of male, female, and juvenile Hybopsis amblops collected with monthly

totals from May, 2013 to July, 2014. No females were collected in January, 2014;

no juveniles were collected in July, 2013, January or June, 2014………………….29

3.3 Number of ovaries per month categorized into developmental stages I-V for

Hybopsis amblops collected from May, 2013 to July, 2014 ...... 34

3.4 Mean (±SE) monthly oocytes per stages I, II, III, and IV for Hybopsis amblops

collected from May to June, 2013, and February to July, 2014...... 35

3.5 Significance of variations in the number of stage III mature oocytes during

reproductive months for female Hybopsis amblops. Levels not connected by the

same letter are significantly different...... 36

3.6 Mean (±SE) monthly oocyte diameters at each developmental stage I-IV for female

Hybopsis amblops collected during spawning months May to June, 2013, and

February to July, 2014……………………………………………………………... 36

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CHAPTER ONE

INTRODUCTION

1.1 Rationale

Life history research is performed to study patterns of variation in key traits of the life cycle due to selection for traits that increase an organism’s level of fitness[1]. Life history traits are associated with reproduction, growth/maturation, and survival. They are often defined as demographic factors such as size/age at sexual maturity, fecundity, clutch size, reproductive schedule, life span, senescence, and quantity of offspring.

According to Stearns (2000), deviations in these strategies can emerge in response to intrinsic and extrinsic factors[2]. Intrinsic factors involve the interactions of various traits, and lead to tradeoffs between them. While the level of fitness decreases for one set of traits in response to extrinsic factors, it increases for other traits. This negative correlation generates constraints, placing limits on phenotypic expression of both traits[3,

4]. Inadequate food sources, predators, temperature, competition, and environmental changes are examples of external influences that affect fitness[5]. The relationship between changes in functional traits, environmental factors, and mechanisms have become a renewed focus in biology.

Many freshwater have acclimated to their ecological niches through phenotypic plasticity and genetic variation. Phenotypic changes, such as body size, are

1 shaped by selection pressures, and increase the fish’s level of fitness[5]. Patterns of variation are commonly seen in traits associated with reproduction, growth/maturation, and survival in freshwater fishes. Trade-offs between growth and breeding effort, number and size of young, and maturation and life-span are believed to be partially due to limited resources[3]. One limited resource required by these functional traits is energy.

A fish’s metabolic rate is steady under unchanging environmental conditions. Ecological variation such as increased temperature, predator density, and alteration of water flow can quickly change energy requirements[6]. Growth rate changes are phenotypic responses to intrinsic and extrinsic influences. Before a juvenile fish achieves reproductive maturity, resources are directed to somatic tissue growth. Adult fishes distribute energy primarily to reproduction[7]. For example, a semelparous species allocates all resources and energy into one “big bang” reproductive event. This depletes the individual of resources and results in death. An iteroparous species saves energy for continuation of life by consuming less energy per spawning season, and reproduces more than once.

Pacific salmon are semelparous, and Atlantic salmon are iteroparous[8].

Maturity is the age of first reproduction in fishes. Sexual maturity can occur at an early or later age, with contrasting benefits and costs. One of the obvious benefits of early maturity is the increased chance of survival to reproduce, but the cost is lower overall fecundity and quality of offspring. This strategy is advantageous for species that produce large clutches of and semelparous species[3]. Benefits of maturing later include a longer growth time and higher overall fecundity. The cost is a decreased chance of survival to adulthood. Postponed development is beneficial when reproductive

2 success is contingent upon age, size, or social status. It is also advantageous for species that invest heavily in reproduction[3].

A wide range of variation in reproductive efforts, age, sizes of eggs produced, fecundity, frequency of spawning, and spawning have been recognized in freshwater fishes. Balon’s [10] grouping of fishes into “reproductive guilds” identified similarities and dissimilarities in reproductive strategies between different species.

Fishes were grouped by ethological and ecological behaviours. These groups ranged from scatterers to live-bearers, and ecological assemblages were identified by spawning substrate preferences[9, 10]. Some fish species expel their gametes freely into the water for fertilization, egg scatterers or broadcast spawners, and produce a large number of small eggs. Several fishes build nests for their eggs, and they are found in the genera Luxilus, Campostoma, Semotilus, and . Mouthbrooding cichlids, lay few eggs but carry fertilized ova in their mouths for protection until hatching[11]. This behaviour is an example of a compromise in the number of offspring produced and parental investment.

Trade-offs between the number and size of eggs produced due to energy allotment were established with respect to maturation time. Heins [12] detected a significant decrease in reproductive competence as a response to parasitism in Gasterosteus aculeatus, three-spined stickleback. Trade-offs were discovered between the number of eggs produced and egg sizes. The non-parasitized females contained a smaller number of larger eggs than the parasitized females. Results of this study verified a correlation between host body condition and parasitic infection. The body mass of these fish decreased with increased parasite loads[12].

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Several models, such as the r-K continuum, were developed to compare variation in life-history strategies in response to fluctuating environments[3]. The principal idea of this theory was selective pressures result in trade-offs (genetic constraints) between life strategies such as age of maturity, fecundity, survivorship, life expectancy, and senescence. For example, individuals on the r end of the spectrum reach maturity at a young age allocating more energy to breeding, reproduce more frequently, have shorter life spans, and usually experience faster rates of senescence. Individuals on the K end mature slower, defer reproduction, have longer life spans, and experience slower rates of senescence. Natural selection is directional for r-selected species in unstable settings with low population densities, and directional for K-selected species in more stable environments with higher population densities[13].

A more comprehensive model using three fundamental strategies to illustrate common patterns of diversity was proposed by Winemiller and Rose (1992) and later revised by Winemiller (2005)[1, 14]. This model forms a triangle using three fundamental strategies: opportunistic, periodic, and equilibrium strategies. Winemiller

(2005) described opportunistic strategists as “…short generation time, high reproductive effort, small body size, low batch fecundity, and low investment per offspring.” He outlined the traits of a periodic strategists as “…long generation time, moderate reproductive effort, large body size, high batch fecundity, and low investment per offspring.” For equilibrium strategists, Winemiller depicted traits as “…moderate to long generation time, low reproductive effort, variable body size, low batch fecundity, and high investment in offspring.” While the internal angles of the triangle represent these strategies, each external side of the triangle represents how specific strategies correlate to

4 certain ecological conditions (Figure 1.1)[1]. In addition to its theoretical value, application of this model can be used to forecast the response of numerous species and populations to certain environmental influences.

Figure 1.1 Triangular life history continuum model[1]

Trait-based studies have been frequently used to group fishes into categories and examine their response to fluctuating environments. However, this method of evaluating the response of functional traits to certain environmental alterations has been constrained by the limited availability of data. Pauly [15] suggested the concept of a database containing biological facts on fishes. In 2005, FishBase listed a total of 28,900 fish species, comprising of 13,000 freshwater fishes, and 1,741 species located in North

America. FishBase currently contains a list of all fishes identified, but fundamental data are needed for many species[15]. In an attempt to encourage ecological and conservation studies, Frimpong and Angermeier [16] compiled the life history findings of many fishes into a database called FishTraits. This database uses features such as trophic ecology, habitation, reproductive strategy, body size, and other attributes classified at an individual

5 species level. It displays areas of dispersal for each species, but a map of trait distribution is not exhibited[16]. Over the last decade, an abundance of open-access databases have been developed to manage data of freshwater fishes. However, these databases lack certain life history data for several species, including H. amblops. Trait- based studies and open-access databases will become progressively beneficial as information is enhanced from multiple localities.

The application of trait-based studies in freshwater fish ecology has quickly expanded with improvements in technology. Immediate data can be retrieved from several open-access databases, but data deficient species remain a challenge. Frimpong and Angermeier [16] stated that the database FishTraits included “informational gaps” on several species, but it would be updated as data became available[16]. In order to understand patterns of phenotypic variation in life history traits and adaptive evolution of a species, acquiring trait data is essential. Once individual and population responses to intrinsic and extrinsic influences can be anticipated, the destruction of some fish species will be more preventable. Trait-based research aids in maintaining biodiversity, increases the understanding of evolution, and helps expand our knowledge on the underlying ecology of populations[10].

1.2 Cyprinids

The class Actinopterygii consists of the ray finned fishes, and it is one of the two major clades of bony fish. This class contains approximately 42 orders, 431 families, and

24,000 species. The teleosts is found to be the leading form of vertebrate existence.

They encompass more than 20,000 species of fishes among 40 orders. Euteleostei is the

6 largest subgroup of teleosts, and contains three groups including Ostariophysi. This superorder, Ostariophysi, contains the order . Cyprinids belong to the order cypriniformes (Figure 1.2)[17].

Figure 1.2 Deep phylogeny of ray-finned fish (Actinopterygii)[18]

There are approximately 32,000 known fish species. The family

(members of this family are commonly referred to as cyprinids) contains 2,700 species.

Difficulty has surrounded the description of phylogenetic relationships in North

American cyprinids. They were first divided into two major clades, the leucisins and the phoxinins. Later, Mayden [11] analyzed relationships in Cyprinella based on morphological traits. While examining cyprinid outgroups, he recognized that one group shared a single trait of an opening in the bottom of the posterior myodome (OPM).

Mayden placed this group into their own clade. Coburn and Cavender [19] produced the

7 western clade, the chub clade, and the shiner clade based on morphology. The identification of three clades (the western clade, the Creek chub + plagopterin clade, and the OPM clade) was later confirmed by gene sequencing. The first molecular studies used mitochondrial cytochrome b and 16S rRNA sequences. In order to reexamine these relationships, recent studies use nuclear DNA to build phylogenetic trees[11].

The Cyprinidae family consists of a large number of species with conserved morphological characteristics. Cyprinids have a single dorsal fin midway down their back and an anal fin between the pelvic fins. Their caudal fin is usually rounded with wide lobes, and all species have cycloid scales covering their body. Most of these fishes are small in size, and their mouth position varies between species. All cypriniform fishes have a distinctive method of premaxillary jaw protrusion due to the kinethmoid.

Cypriniform families have shown variation in the shape of the kinethmoid suggesting adaptive radiation. All cyprinids possess a palatal organ on the top of the pharynx and pharyngeal teeth for the crushing, cutting, and shredding of food[11].

An example of cyprinid diversity can be seen in the genus Cyprinella, which contains approximately 30 species[11]. Cyprinella lutrensis, Red Shiners, reach maturation age between 24-30 mm in standard length and obtain a maximum length of 90 mm. Males establish areas around crevices for spawning, and females release their eggs into the crevices. Females can expel eggs up to 16 times a day, and an average clutch size is 585 eggs. The Red Shiner has a life span of approximately 3 years. Cyprinella galactura,

Whitetail Shiners, are sexually mature between 50 to100 mm in standard length and have a maximum length of 150 mm. They are crevice spawners, but female fecundity ranges

8 between 400 and 1,800 eggs. Life expectancy for Whitetail Shiners is approximately 4 years[20].

Cyprinid species have demonstrated inter- and intraspecific diversity with different levels of phenotypic plasticity. Gotelli and Pyron [20] explored the relationship between interspecific variation, phylogeny, and the environment in several species of , Hybopsis, and

Cyprinella. They compared six life history traits at numerous latitudes using linear regression analysis. They used the same six traits in a correlation test to evaluate the association between these traits and phylogeny. A strong correlation was revealed between higher latitudes and the duration of spawning season. Phylogeny played a role in similar body sizes for more closely related species. Gotelli and Pyron [20] concluded that inter- and intraspecific diversity results from genetic variation and phenotypic changes in response to ecological fluctuation[21].

Phenotypically plastic responses to low levels of dissolved oxygen and effects on swimming abilities were assessed in 12 cyprinids from different . Interspecific diversity and habitat variation made members of the Cyprinidae family ideal for this study. Fu et al. [21] observed a fish’s tolerance for hypoxia (low levels of oxygen), and inspected each fish for vivid modifications in gill morphology. Cyprinids from a rapid-flow environment exhibited an improvement in swimming, but a lower endurance for decreased levels of oxygen. Fishes from fast-flowing systems did not experience gill tissue changes in response to attempts at acclimation.

Species from water systems with a slower discharge presented improvements in swimming abilities, and increased endurance for lower levels of dissolved oxygen. These fish underwent gill tissue transformations in response to hypoxia acclimation. Ctenopharyngodon idellus, Grass

Carp, and Parabramis pekinensis, Chinese Bream, are two closely related species that

9 experienced alternative changes in gill morphology. These results indicated that gill plasticity in cyprinids were habitat dependent[22].

1.3 Reproductive schedules and strategies of cyprinids

Members of the Cyprinidae family share certain reproductive characteristics. Most cyprinids are not sexually dimorphic. Some fishes may develop prominent colors of red, orange, yellow, blue, and iridescent during breeding season. The males (and some females) develop nuptial tubercles on their head, fins, and body. Tubercles on the head are used for defense, to stimulate females, and push aside pebbles to build nests.

Abundant tubercles on the body and fins are thought to be used in the act of spawning[11].

Most North American cyprinids are broadcast spawners or egg scatterers. A male and female will break away from other fishes, and the male will clasps the female. This method involves the expelling of gametes freely into the water for fertilization.

Ptychocheilus oregonensis, Northern Pikeminnow, spawn over coarse gravel.

Rhinichthys atratulus, Eastern Blacknose Dace, indirectly bury their eggs by vibrating on substrate while reproducing. Numerous species of cyprinids prime substrate or build nests for breeding[11]. Luxilus chrysocephalus (Striped Shiners), Nocomis biguttatus

(Hornyhead Chub), and Lythrurus umbratilis (Redfin Shiner) will spawn in nests built by other species. The mating activities of Dionda diaboli, the Devils River , in a laboratory setting indicated a preference for gravel substrate. An observatory study performed in the wild by Phillips et al. [22], revealed similar results with spawning taking place over active and inactive centrarchid nests[23].

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In contrast with the “big bang” strategy of semelparous species, some fishes spawn annually for several years. Annual single spawners can be found in the genus

Prochilodus or the genus Pseudoplatystoma. Other fishes spawn multiple clutches within a reproductive season (multiple spawners), with either yearly reproductive activity or reproduction every two to three years[11]. Rinchard and Kestemont [23] examined the reproductive strategies of Rutilus rutilus (Roach), Alburnus alburnus (Bleak), and Blicca bjoerkna (White Bream). Rutilus rutilus were found to be single spawners, while

A.alburnus and B.bjoerkna were multiple-spawners[24]. Acanthobrama telavivensis, the

Yarqon Bleak, are multiple-spawners. This fish faces potential reproductive output due to its endemic environment in a mediterranean-type stream. The Yarqon Bleak compensates for this possible decrease by spawning multiple clutches each season[25].

North American cyprinids are spring and summer spawners. Their fertilized eggs mature and hatch in the same summer due to the warm temperature of the water. This provides an opportunity for the young to become self-sufficient in time for the following summer. However, there is variation in this timing due to niche partitioning and environmental factors[5]. DeHaven et al. [25] investigated the reproductive timing of three species collected from Coweeta Creek in North Carolina. The three species were

Cottus bairdi (Mottled Sculpin), funduloides (Rosyside Dace), and

Rhinichthys cataractae (Longnose Dace). Reproductive timing had been determined by fluctuations in a fish’s gonadosomatic index (GSI). This value is calculated using the mass of the gonadal tissue. The mass of the gonadal tissue was divided by body mass and multiplied by 100. Due to the fact individuals have different body sizes, accuracy of this method was questioned. In this study, the authors remodeled the equation to

11 calculate relative gonadosomatic indices. These values provided a means of measuring the spawning condition of different sized fishes. Relative GSIs were calculated to determine the reproductive timing of each species. Results indicated C.bairdi spawned between March and May, and the reproductive season for C.funduloides was from May to

August. R.cataractae spawned from June to July. Earlier spawning in C.bairdi revealed a proximate response to temperature and photoperiod[26]. Hamed et al. [26] examined the life history traits of Phoxinus tennesseensis (Tennessee Dace) and found that spawning transpired from April to June. Female fecundity was noted to be substantially less than fecundity of other Phoxinus species. A small population size and low female fecundity causes susceptibility to environmental fluctuations in this population[27].

Another North American cyprinid, Notropis topeka (Topeka Shiner), was studied in southeastern . Dahle [27] used ovarian classification according to level of maturity to establish a spawning time for this species. The GSI value was computed for comparison. Both analyses established a breeding timeline from May to August.

N.topeka were established as spring time, multiple spawners[28].

Mims et al. [28] conducted a study of eleven major fish families, including

Cyprinidae, and 603 fish species according to seven life history traits. This investigation revealed that cyprinids exhibit a wide variety of trait composition and all three strategies on the triangular continuum. In the same study, distinct geographic patterns of the three strategies were displayed in 350 watersheds across North America. In the southeastern

United States, where Cyprinidae and Percidae are the major families, the opportunistic strategy dominated watersheds. This dominance declined in northern and western watersheds where periodic strategists became more abundant. Along the Pacific coast

12 and in northern latitudes, the equilibrium strategy was primarily expressed in fish species.

This assessment did not include several North American watersheds due to a lack of information concerning species traits[29].

The diverse Cyprinidae family contains species considered to be opportunistic, periodic, and equilibrium strategists[29]. One species that is regarded as opportunistic strategist is Hemitremia flammea, the Flame Chubs. Life history research performed by

Sossamon [29] revealed various characteristics of an opportunistic strategist. Fish in this study fluctuated in size from 27 to 68 mm in total length. Data indicated that Flame

Chubs experience early maturation as evidenced by reproduction in their first year of life[30]. Members of this species are broadcast spawners over gravel/sandy substrates, do not guard their eggs, and have low parental investment in their offspring. According to the FishTraits database, females release eggs multiple times during seasonal spawning which takes place between March and June. Flame Chubs have a life span of approximately 2 years[20].

Another cyprinid that is considered an opportunistic strategists is Notropis chalybaeus, Ironcolor Shiner. Perkins et al. [30] examined life history aspects of an

Ironcolor Shiner population in a spring environment. This cyprinid was found to have a mean standard length of 41.9 mm with a range of 31-52 mm. Minimum standard length at sexual maturity was 36 mm, indicating a maturation age of 1 year. Ironcolor Shiners spawn adhesive egg cells that cling to gravel or sand substrate, and no gamete guarding takes place. The number of adhesive eggs released ranged from 46 to 326 eggs. A reproductive schedule, according to GSI values, indicated a spawning season extending

13 from March to December. Multiple clutches were produced during this period. The fish within this study exhibited a maximum life span of 2.5 years[31].

Ptychocheilus lucius, Colorado Pikeminnow, is a cyprinid thought to be a periodic strategists[29]. The NatureServe status of this species was last reviewed in April, 2012, and listed as G1/N1-critically imperiled. This status resulted from a direct loss of habitat, environmental changes in water flow and temperature, and obstruction of migration channels by the building of large reservoirs. This species is one of the largest cyprinids of North America with individuals reaching 1.8 m long and weighing 45 kg. Colorado

Pikeminnows experience a later age of maturation reaching reproductive maturity in 5-7 years, at a total body length of 50 cm[32]. These fish are broadcast spawners over rock/gravel substrates with the female releasing up to 100,000 eggs. Spawning has been documented as early as mid-June and as late as August. Members of this species may live 30 years or more[20].

An intermediate strategists, falling between periodic and equilibrium strategies, is

Cyprinus carpio, Common Carp[29]. This species is regarded as a “pest fish” in North

America because they damage vegetation and diminish habitat heterogeneity for indigenous species. Members of this species are well known for their tolerance of temperature variation, diverse salinities, and fluctuating oxygen levels[33]. Their vast abundance could be attributed to their longevity, mobility, and high levels of fecundity.

Bajer and Sorensen [33] studied two populations of Common Carp in Lake Susan and

Lake Echo in Minnesota. Fish in these populations ranged from 340 to 820 mm in total length, demonstrating variability in body size (equilibrium strategy). The average female in Lake Susan contained approximately 750,000 ova while the average female in Lake

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Echo contained 560,000 ova. This high batch fecundity is a periodic strategy. Parental investment in offspring was revealed in this study in that adults did pursue access to habitats where their young would have greater probabilities of survival[34].

Gila atraria, Utah Chub, are another example of intermediate strategists.

Members of this species are natives of the Lake Bonneville basin and upper Snake River.

Oncorhynchus clarki, Cutthroat Trout, are natural predators of these fish. Johnson and

Belk [34] studied the effects of predation on life traits in eight populations of G.atraria.

Populations that coexisted with their predator displayed outcomes that followed size- selective predation models: larger juvenile growth rates, deferred maturation, increased body sizes at maturity, and lower female reproductive effort. Predation risk compels prey species to distribute their resources to survival. The prey in populations that coexisted with O.clarki obtained the ability to redirect their resources to growth and longevity[35].

1.4 Hybopsis amblops, Bigeye chub

Hybopsis amblops, the Bigeye Chub, are freshwater fish found in Lake Ontario,

Lake Erie drainage, River basin, and south to the drainage in northern Alabama (Figure 1.3)[32]. Bigeye Chubs were originally widespread throughout the River and the river systems in . They are currently thought to be extirpated in most of the River drainage, and rarely found in segments of the drainage. Few members of this species have been reported in the Kaskaskia and Wabash River drainages and branches of the [36].

National conservation status for this species was last reviewed in 1996 and listed as N5-

15 secure species. The conservation status of H. amblops differs across several North

American states (Figure 1.4)[32].

Figure 1.3 Distribution map for Hybopsis amblops[32]

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Figure 1.4 Hybopsis amblops conservation status, United States[32]

This species, like many cypinids, have underwent misidentification and confusion with phylogenetic relationships. These fish were mistaken for related species and renamed several times. Cyprinids were once placed in the genus Hybopsis based on the presence or absence of maxillary barbels. Gilbert and Bailey [11] pointed out that barbels could not be the only characteristic used in species classification. Hybopsis amnis (), H.hypsinotus (), H.lineapunctata (),

H.rubrifrons (), and H.winchelli () are other species in the genus[37, 38].

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H. amblops are in the Cyprinidae family. They have a cylindrical, slightly tapering body and a blunt snout. Their large eyes are 7.1 to 9.8% of their standard body length. The interorbital distance is narrow. Mouth position is inferior and horizontal with a maxillary barbel. These fish have pectoral, pelvic, and dorsal fins that are more posterior than other Hybopsis species. Pectoral fin rays range from 13-17, and they have approximately 8 pectoral and pelvic fin rays. A lateral line is complete and has 35-38 rounded scales. Their body color is straw yellow in the dorsal region with a silver, white ventral region. Most members of this species have a well-developed pre-dorsal line that darkens at the base of the dorsal fin[37].

H. amblops are frequently collected over hard sand and gravel substrates in areas of low current. The fact that these fish have a low tolerance to siltation and pollution is well documented. H. amblops reach approximately 58 mm in length after one year, and attain a maximum length of 80 mm according to previous work[37]. The FishBase and

FishTraits databases display a maximum length of 100 mm[15, 20]. Boschung and

Mayden [36] indicate that spawning occurs during the late spring and early summer[37].

FishTraits states a more specific time frame of May and June[20] while FishBase contains no reproductive information for this species[15]. Age of sexual maturity is listed as 1 year on FishTraits[20]. Boschung and Mayden[37] suggest adulthood at a size greater than 58 mm of standard length[37]. During breeding season, males develop fine tubercles scattered over their head and concentrated over the orbital region. The heads of breeding females are covered with fine projections that are smaller and less numerous than the males[37]. Members of this species are lithophilic spawners, they utilize gravel and mineral substrates in spawning instead of simply releasing eggs into the

18 sediment[20]. Data concerning female fecundity could not be found in literature or

FishBase, but FishTraits shows a value of 1,000 eggs for maximum fecundity. A life expectancy of three years is provided on FishTraits[20].

1.5 Statement of purpose and hypothesis

The purpose of this study was to establish a reproductive schedule and examine reproductive traits of H. amblops in the Flint River system. Available resources contain limited information about this species. FishTraits presents more information, but confirmation is required for this species. The collection of this information is important for future studies examining this population’s response to environmental changes. The data collected can be used in future studies comparing allopatric populations. Finally, this information is essential to any conservation effort based on scientific data.

Early reproductive maturity was expected in this collection of H. amblops due to the age of sexual maturity listed on FishTraits[20]. A significant correlation was predictable between standard length and body weight for both sexes. Spawning season for both sexes was expected to peak in April or May. A minimum of 1,000 oocytes were anticipated from each mature female. A strong relationship between GSI values and oocyte counts was expected during months of active spawning. Linear regression analysis was expected to reveal a strong relationship between standard lengths and oocyte counts in females. It was anticipated that members of this species would display characteristics of an opportunistic strategist in comparison to other cyprinids.

19

CHAPTER TWO

MATERIAL AND METHODS

2.1 Study site and sampling

All fish were collected from a 14 km section of the Flint River in northern

Alabama. The Flint River system includes approximately 141,640 hectares in Madison

County, AL, and Lincoln County, TN[39]. The Flint River is 562 km long. The location on Oscar Patterson Road (34˚48’24” N, 86˚28’21” W) was the site used in this study

(Figure 2.1 and 2.2). Mean monthly discharges in cubic meters per second for each month of collection was obtained from the U.S. Geological Survey database[40]. The river is clear to moderately turbid over exposed Tuscumbia limestone bedrock with a substrate of boulders, large cobble, small cobble, sand, silt, and mixtures of each.

H. amblops were collected monthly from May, 2013, through July, 2014. Fish were netted using a seine and cast net with the following dimensions respectively: 3.5 m length, 1.2 m depth, and a 3 mm mesh; a radius of 1.37 m and a 6 mm mesh. All fish were euthanized on site using (1:10) clove oil: 95% ethanol diluted with 350 mL river water[41]. Samples were placed in 10% phosphate buffered formalin for tissue fixation and storage[42].

20

Figure 2.1 Flint River location on Oscar Patterson Road in Huntsville, Alabama according to Google maps

Figure 2.2 Flint River collection site at Oscar Patterson Road access point in Huntsville, Alabama

21

2.2 Laboratory analysis

Standard length (SL), the length measured from the tip of the snout to the base of the caudal peduncle, was measured with digital calipers to the nearest 0.01 millimeters.

Gross body mass was obtained to the nearest 0.0001 grams using an Explorer OHAUS digital balance after excess fluid was blotted from the fish’s body. The sex of each fish was determined by excision and examination of gonadal tissue using an Olympus dissecting microscope. After excess surface fluid was blotted away, gonadal mass was obtained to the nearest .00001 grams. All gonadal tissue was stored in 10% phosphate buffered formalin.

The gonadal tissue of each fish was imaged using an Olympus SZX7 camera- scope with Galilean optical system and computer installed with CellSens standard software package. Ovarian maturation was assessed for each female using a modified scheme described by Nunez and Duponchelle[43]. Based on maturation, ovaries were divided into five stages (Figure 2.3). Immature (stage I) ovaries are small in size, usually opaque, and contained only latent oocytes. Maturing (stage II) ovaries are larger, inhabiting a larger portion of the abdominal cavity. Maturing ovaries contain various sizes of white and cream colored oocytes. Advanced maturation (stage III) ovaries are bulkier and loaded with oocytes. The oocytes are yellow to orange, and various sizes of vitellogenic ova are visible and ready to be released during spawning.

Ripe (stage IV) ovaries are partially ovulated and oocytes are released when squeezing the fish’s sides. In the ripe stage (IV), the ovary has obtained maximal development, but vitellogenic oocytes of several sizes are present in between the mature ova due to multiple spawning. Spawned and recovering (stage V) ovaries are still relatively large

22 and flaccid with remaining empty spaces, but contains different sizes of developing vitellogenic oocytes. This stage occurs in between spawning cycles until the end of spawning season.

Stage I

Stage II

Stage IV

Stage III

Stage V

Figure 2.3 Stages of ovarian development

Both ovaries from each female were teased apart using 21-gauge hypodermic needles to liberate developing oocytes from the ovarian tissue. All oocytes were arranged into a single layer on a Syracuse watch glass. Using the Olympus SZX7 camera-scope and CellSens standard software package, images were taken at a total magnification of

1.6X. When the number of oocytes exceeded one frame, multiple frames were imaged.

23

Digital images were used to categorize oocytes into stages of maturation (Figure 2.4) using the same schematic described by Nunez and Duponchelle [42]. Latent oocytes were not included in this research. Early maturing (stage I) oocytes are previtellogenic oocytes and are distinguished by their small size which is half the diameter of a ripe oocyte. Late maturing (stage II) oocytes are in early vitellogenesis and contain small yolk granules. The diameter size increases, and the formation of a nuclear envelope can be seen. Mature (stage III) oocytes are in late vitellogenesis. They are yellow in color and filled with yolk globules. The vitelline membrane is obviously divided from the yolk. Ripe (stage IV) oocytes are larger than all other oocytes, yellow to dark yellow brown in color. Ripe ova have vitelline membranes that are completely separated from the yolk mass.

Early Maturing

Late Maturing

Mature

Ripe

Figure 2.4 Stages of oocyte development

All oocytes (excluding latent oocytes) were counted by stages and the total number of eggs were calculated for each female. Oocyte counts were performed using

EggHelper[44], a customized program developed in Microsoft Visual Studio 2013, and confirmed using CellSens software. The diameter of ten oocytes per developmental stage

24 per ovary were measured, and the monthly average diameter for each stage was calculated for each female.

2.3 Statistical analysis

The total number of male, female, and juvenile (unsexed) fish were calculated for each monthly collection. A paired t-test was used to assess for a significant difference between the number of males and females in monthly collections. The equation

W = aLb is frequently used to express the relationship between length (L) and body weight (W) of fishes. This equation was modified to use standard length (SL) and gross body mass (GBM) in this study. A log linear regression model using log10 GBM = log10 a + b·log10 SL (GBM is the gross body mass of each fish in g, SL is each fish’s standard length in mm, log “a” is the y-intercept, and b is the coefficient) was used to determine the relationship between SL and GBM for this species. A linear regression analysis displaying log10 GBM against log10 SL produces a line with slope b and a Y-intercept of log10 a[45].

Average monthly gonadosomatic index values (GSI) were evaluated for adult males and females from May, 2013 to July, 2014. These values were calculated for each

퐺표푛푎푑푎푙 푚푎푠푠 (푔) adult fish using the equation GSI = X 100. In order to determine if 퐺푟표푠푠 퐵표푑푦 푚푎푠푠 (푔) statistically significant differences between monthly GSI values existed between males or females, one-way ANOVAs with post hoc Tukey-Kramer tests were performed. After oocyte counts were completed for every female, ANOVAs with post hoc Tukey-Kramer tests were once again used to investigate significance of variation in stage III mature and stage IV ripe oocyte counts during reproductive months. Average diameter

25 measurements were calculated for each developmental stage I-IV during spawning months. Linear regression analysis was performed to investigate the relationships between SL vs oocyte counts in each developmental stage I-IV, SL and GSI values, total fecundity vs GSI, total fecundity vs gonadal mass, total fecundity vs GBM. Linear regression was also used to determine if a relationship exists between fecundity vs water temperature (ºC), and the flow rate of the river vs oocyte counts.

26

CHAPTER THREE

RESULTS

3.1 Study site data

The water temperature was taken during each collection. The average rate of discharge for each month of collection was obtained from the U.S. Geological Survey database 03575100 at Brownsboro, AL (Table 3.1)[40].

Month/Yr River Discharge Water Temp (ºC) Monthly Mean in m3/s May, 2013 21.9 33.3 June, 2013 25.7 11.8 July, 2013 26.4 26.4 August, 2013 25.1 23.1 September, 2013 22 7.4 October, 2013 14.4 4.5 November, 2013 15.3 7.1 December, 2013 13 32.3 January, 2014 8 25.4 February, 2014 10.2 35.3 March, 2014 15.6 15.3 April, 2014 17.2 30.9 May, 2014 21.1 15.4 June, 2014 24.1 21.7 July, 2014 24.5 6.2

Table 3.1 Monthly water temperatures and average rate of river discharge (m3/sec) from the U.S. Geological Survey database 03575100 at Brownsboro, AL from May, 2013 to July, 2014.

27

3.2. Sex bias and size structure

The number of males (N=137), females (N=94), and juveniles (N=84) collected monthly were calculated with a monthly total from May, 2013 to July, 2014 (Table 3.2).

A paired t-test was performed on the 15 months of collections to assess for a statistically significant difference between the number of males and females collected for this study.

A statistically significant difference between the number of males and females was not indicated in the results, t(14) = 2.1431, p = 0.0502. SL in females (N=94) varied from

47.74 to 77.16 mm with calculated mean ± SD as 63.22 ± 0.64 mm (Figure 3.1); an average GBM of 3.00 ± 0.10 g was determined (Figure 3.2). Male fish (N=137) displayed a range between 49.33 to 74.35 mm in SL, with a mean of 61.58 ± 0.49 mm

(Figure 3.1); an average GBM of 2.80 ± 0.06 g was calculated (Figure 3.2). SL for juveniles (N=84) differed between 29.43 to 57.94 mm with a mean of 46.86 ± 0.99 mm

(Figure 3.1); a mean GBM of 1.20 ± 0.07 g was assessed (Figure 3.2). In order to examine the relationship between GBM and SL, a linear regression analysis displaying log10 GBM against log10 SL was completed for female, male, and juvenile H. amblops.

According to the coefficient of determination (R2=0.65) and p < .0001, a strong relationship was revealed between GBM and SL in the females (Figure 3.3). A strong relationship (R2=0.54, p < .0001) between GBM and SL was indicated for male

H. amblops (Figure 3.4). In juveniles, a very strong and dependable relationship was shown by the coefficient of determination, R2=0.83, and p < .0001 (Figure 3.5).

28

Month/Yr Females Juveniles Males Total May, 2013 2 1 6 9 June, 2013 10 5 20 35 July, 2013 1 0 1 2 August, 2013 7 26 17 50 September, 2013 5 1 10 16 October, 2013 4 2 5 11 November, 2013 6 6 15 27 December, 2013 11 10 5 26 January, 2014 0 0 2 2 February, 2014 1 13 2 16 March, 2014 7 5 2 14 April, 2014 5 9 6 20 May, 2014 16 3 24 43 June, 2014 9 0 15 24 July, 2014 10 3 7 20

Table 3.2 Total number of male, female, and juvenile Hybopsis amblops collected with monthly totals from May, 2013 to July, 2014. No females were collected in January, 2014; no juveniles were collected in July, 2013, January or June, 2014.

Figure 3.1 Mean (±SD) monthly standard length for female, juvenile, and male Hybopsis amblops collected from May, 2013 to July, 2014. No females were collected in January, 2014; no juveniles were collected in July, 2013, January or June, 2014.

29

Figure 3.2 Mean (±SD) monthly body mass for female, juvenile, and male Hybopsis amblops collected from May, 2013 to July, 2014. No females were collected in January, 2014; no juveniles were collected in July, 2013, January or June, 2014.

Figure 3.3 Standard length vs gross body mass relationship in female Hybopsis amblops collected between May, 2013 and July, 2014.

30

Figure 3.4 Standard length vs gross body mass relationship in male Hybopsis amblops collected between May, 2013 and July, 2014.

Figure 3.5 Standard length vs gross body mass relationship in juvenile Hybopsis amblops collected between May, 2013 and July, 2014.

31

3.3 Reproductive schedule

The average monthly gonadosomatic indices (GSI) were evaluated for male and female H. amblops collected from May, 2013 to July, 2014. GSI values began to rise in

February, peaked in April and May, and showed a decline from May to June for both females and males (Figure 3.6). Female values peaked in May, 2013 with an average

GSI of 13 ± 1.8%, and again in May, 2014 with an average GSI of 12.7 ± 0.65%. In

April, 2014 a GSI value of 12.3 ± 1.1% was revealed (Figure 3.6). Male GSI values peaked in April, 2014 with a mean of 1.73 ± 0.14%, and again in May, 2014 with a value of 1.58 ± 0.07% (Figure 3.6). Significant differences between and within groups of both sexes was examined by performing ANOVAs with post hoc Tukey-Kramer tests. A statistically significant difference between female groups was revealed by ANOVA results, F13, 80 = 19.23, p < .0001 (Figure 3.6). There were statistically significant difference between male groups as well with F14, 122 = 17.24, p < .0001 (Figure 3.6).

Figure 3.6 Mean monthly gonadosomatic indices (GSI) for adult male and female Hybopsis amblops collected from May, 2013 to July, 2014. No females were collected in January, 2014. Levels not connected by the same letter are significantly different.

32

3.4 Ovarian development, oocyte counts, and oocyte diameters

Once the ovaries were excised from each female fish, the developmental stages of the ovaries were assessed and categorized into developmental stages I-V. Latent ovaries were not included in this study. Stage I ovaries were most abundant (10) in samples collected in December, 2014. Only one stage II ovary was recovered from a fish collected in February, 2014. In March, 2014, 5 females contained ovaries in developmental stage III. The month of May, 2014 contained the most females with ovaries in developmental stage IV; while in June, 2014 only 6 females with ovaries in developmental stage V were uncovered. Results were recorded for each monthly samples

(Table 3.3).

A total of 94 females were analyzed, and 65 contained oocytes. The smallest fecund female with 550 oocytes measured 54.20 mm SL, 2.89 g GBM, and had a gonadal mass of 0.23 g. The largest fecund female contained 1,320 oocytes and was 77.16 mm

SL, 5.80 g GBM, and had a gonadal mass of 0.32 g. Approximately 90 oocytes, the smallest number of total oocytes, were removed from a fish 68.59 mm in SL, 3.45 g

GBM, and 0.09 g in gonadal mass. A female that measured 76.17 mm SL, 5.88 g GBM, and 0.78 g in gonadal mass contained the largest amount of oocytes (2,566). This data illustrates that the biggest female did not produce the greatest number of oocytes.

The average number of stage I oocytes peaked in February, 2014 at 1,083, while most stage II oocytes were found in March, 2014 at 630 ± 123. An average of 754 ± 120 stage III oocytes were present in ovaries in April, 2014; and the most stage IV oocytes

(58 ± 21) were displayed in April, 2014 (Table 3.4 and Figure 3.7). The largest monthly average was 1,271 oocytes recovered in March, 2014, but more than 800 stage III and IV

33 oocytes were removed from females collected in April, 2014 (Table 3.4). A statistically significant difference in the amount of stage III mature oocytes was found between reproductive months, F6, 36 = 7.67, p < .0001 (Table 3.5). In stage IV ripe oocytes, a statistically significant difference was not indicated between reproductive months,

F6, 31 = 0.47, p = 0.8232.

The diameter of approximately twenty oocytes per stage of maturation per ovary were measured for comparison. Stage I early maturing oocyte diameters ranged from

0.317 to 0.499 mm; stage II late maturing diameters fluctuated from 0.382 to 0.675 mm; stage III mature fluctuated from 0.501 to 1.05 mm; and stage IV ripe varied 0.785 to 1.19 mm. An average diameter for each developmental stage I-IV was determined during reproductive months (Table 3.6).

Month/Yr # of Stage I # of Stage II # of Stage III # of Stage IV # of Stage V May, 2013 0 0 0 2 0 June, 2013 0 0 0 2 4 July, 2013 0 0 0 0 1 August, 2013 0 0 0 0 3 September, 2013 0 0 0 0 0 October, 2013 0 0 0 0 0 November, 2013 3 0 0 0 0 December, 2013 10 0 0 0 0 February, 2014 0 1 0 0 0 March, 2014 0 0 5 2 0 April, 2014 0 0 1 4 0 May, 2014 0 0 1 10 3 June, 2014 0 0 0 1 6 July, 2014 0 0 0 0 3

Table 3.3 Number of ovaries per month categorized into developmental stages I-V for Hybopsis amblops collected from May, 2013 to July, 2014

34

Stage I Stage II Stage III Stage IV Total Avg Month/Yr May, 2013 44 ± 17 388 ± 52 422 ± 140 8 ± 4 861 June, 2013 184 ± 65 392 ± 116 257± 64 30 ± 9 832 February, 2014 1083 164 1247 March, 2014 404 ± 63 630 ± 123 219 ± 54 32 ± 10 1271 April, 2014 253 ± 90 164 ± 29 754 ± 120 58 ± 21 1230 May, 2014 240 ± 30 359 ± 41 425 ± 73 49 ± 12 1073 June, 2014 236 ± 51 275 ± 76 131 ± 48 41 ± 20 669 July, 2014 72 129 ± 64 574 ± 127 39 ± 11 796

Table 3.4 Mean (±SE) monthly oocytes per stages I, II, III, and IV for Hybopsis amblops collected from May to June, 2013, and February to July, 2014.

Figure 3.7 Mean (±SE) monthly number of oocytes per stages I, II, III, and IV for Hybopsis amblops collected from May to June, 2013, and February to July, 2014.

35

Month/Yr Level Level Level Mean April, 2014 A 754 July, 2014 A B 574 May, 2013 A B C 422 May, 2014 B C 367 June, 2013 B C 198 March, 2014 C 191 June, 2014 C 131

Table 3.5 Significance of variations in the number of stage III mature oocytes during reproductive months for female Hybopsis amblops. Levels not connected by the same letter are significantly different.

Month/Yr Stage I Stage II Stage III Stage IV May, 2013 0.45 ± 0.04 0.53 ± 0.03 0.79 ± 0.08 1.01 ± 0.01 June, 2013 0.43 ± 0.02 0.55 ± 0.03 0.74 ± 0.05 0.91 ± 0.03 February, 2014 0.44 0.50 March, 2014 0.45 ± 0.02 0.58 ± 0.03 0.77 ± 0.07 1.00 ± 0.07 April, 2014 0.46 ± 0.01 0.61 ± 0.01 0.91 ± 0.06 1.05 ± 0.05 May, 2014 0.47 ± 0.01 0.61 ± 0.01 0.92 ± 0.01 1.04 ± 0.01 June, 2014 0.44 ± 0.02 0.57 ± 0.04 0.74 ± 0.03 0.95 ± 0.05 July, 2014 0.40 ± 0.04 0.46 ± 0.04 0.71 ± 0.08 0.93 ± 0.05

Table 3.6 Mean (±SE) monthly oocyte diameters at each developmental stage I-IV for female Hybopsis amblops collected during spawning months May to June, 2013, and February to July, 2014.

3.5 Relationships

Linear regression analysis was performed to investigate the relationships between

SL vs oocyte counts in each developmental stage I-IV, SL vs GSI, total fecundity vs GSI, fecundity vs gonadal mass, total fecundity vs GBM. The relationship between oocyte counts vs SL exhibited a stronger correlation for stage III mature (R2 = 0.129) and stage

IV ripe (R2 = 0.095) oocytes than for stage I early maturing (R2 = 0.007) and stage II late 36 maturing oocytes (R2 = 0.068) (Figures 3.8). A strong relationship was not indicated between SL and GSI values in females (R2 = 0.004) or in males (R2 = 0.038). Total fecundity was positively but not strongly correlated to GSI values (R2=0.306; p < .0001)

(Figure 3.9). Total fecundity was more strongly correlated with gonadal mass

(R2 = 0.621; p < .0001) (Figure 3.10) than GBM (R2 = 0.142; p = 0.0022) (Figure 3.11).

Linear regression analysis was once again used to determine if a relationship existed between total fecundity vs water temperature (ºC), and fecundity vs the flow rate of the river. Results did not indicate a strong correlation between total fecundity vs water temperature (R2 = 0.101), or between fecundity vs flow rate (R2 = 0.052). Monthly average fecundity values and water temperatures (ºC) were applied to the same graph.

The graph revealed a trend: during months with increased water temperatures, oocyte counts were low. Water temperatures were lower in November when egg counts began increasing. February to May of 2014, water temperatures were low but rising as oocyte counts were cresting. In June of 2013 and 2014, the number of eggs in each female decreased as water temperatures climaxed (Figure 3.12).

37

Figure 3.8 Standard length vs oocyte counts in developmental stages I-IV for Hybopsis amblops collected from May, 2013 to July, 2014.

38

Figure 3.9 Total fecundity vs GSI values in female Hybopsis amblops collected from May, 2013 to July, 2014.

Figure 3.10 Total fecundity vs gonadal mass in female Hybopsis amblops collected from May, 2013 to July, 2014.

39

Figure 3.11 Total fecundity vs GBM in female Hybopsis amblops collected from May, 2013 to July, 2014.

Figure 3.12 Common trends for mean monthly fecundity values vs mean water temperatures (ºC) collected from May, 2013 to July, 2014.

40

CHAPTER FOUR

DISCUSSION

4.1 Study goals and limitations

The goal of this study was to show a reproductive schedule and describe reproductive traits of H. amblops in the Flint River system. Previous biological information of H. amblops from this river system has been inadequate. This study was successful in obtaining growth and reproductive data for these fish. While shortcomings were encountered with small sample sizes, the results coincided with the scarce information found in literature and databases[15, 20, 37]. In May, 2013 only 9 fish were collected for examination and 2 were female. Only 1 female was collected in July, 2013 and February, 2014. There were no females collected in January, 2014. These small sample sizes were due to heavy rainfall leading to rapid waters making fish collecting from the river problematic. The issue with small sample sizes is low significance in statistical analyses. Difficulties in collecting adequate sample sizes occurred primarily during the winter month, May and July, 2013. Adequate sample sizes were collected in

May, June, and July of 2014 for 15 months of data. Literature reveals that spawning season takes place during spring and summer months for most cyprinids[11, 37].

Reproductive timing and a description of traits was obtained for H. amblops from the data collected in this study.

41

4.2 Size and reproductive schedule

A variety of attributes including SL, GBM, size at first sexual maturity, duration of spawning season, gonadosomatic index, ovarian mass, and fecundity are frequently examined in reproductive examinations[46, 47]. These attributes were evaluated for

H. amblops in this study. SL in females ranged from 47.74 to 77.16 mm, while males ranged from 49.33 mm to 74.35 mm. Juveniles displayed a growth pattern in standard length with a monthly increase from 29.43 to 57.94 mm from May to November, 2014.

This reinforces the idea of springtime spawning to provide an opportunity for the young to become self-sufficient and sexually mature by the following summer. Boschung and

Mayden[37] showed reproductive maturity was reached at a SL greater than 58 mm[37].

After examining 65 females, the smallest fecund female was found to be 54.20 mm in SL,

2.89 gm in GBM, and contained 550 oocytes. GSI values were found to be independent of SL for both females and males.

Spawning season for H. amblops was determined to begin in March and wind down in June. April was revealed as the primary reproductive month as evidenced by the climax of GSI values, along with a peak in the number of stage III mature and stage IV ripe ova. Significant decreases were seen in GSI values between May and June, 2014.

These values were low from July to November, displayed a steady increase from

December to February, and a significant increase was seen in March, 2014 for both sexes.

A significant difference was not seen between February, March, April, and May of 2014 while June and July displayed regression. The GSI values were significantly different for

March, April, and May from all other months. Trends in GSI values and ovarian maturation indicated a reproductive schedule of March to May, with a peak in April, and

42 ending in June for H. amblops. This information differs from the FishTraits database which lists May and June as primary reproductive months for this species. These results are not surprising due to Gotelli and Pyron’s [20] results from a previous study indicating a strong correlation between higher latitudes and an increase in the duration of spawning season[21].

Additional evidence to support April and May as the primary reproductive months resides in the development of female ovaries corresponding well with GSI values.

In April and May, stage IV ripe ovaries containing numerous stage III and stage IV ova showing ongoing vitellogenesis were removed from female fish. However, more than

800 stage III and stage IV ova were found in the April collection while approximately

500 mature to ripe ova were found in the May collection. The various stages of oocyte development in the same ovary was indicative of multiple episodes of spawning[11].

Furthermore, GSI values attained indicated that females had greater GSI values than males. This result was due to the increase in female gonadal mass (ovaries which contain oocytes) that requires more energy to produce. While mature male gonads attribute to approximately 12% of body mass, mature female ovaries account for approximately 70% of body mass[11].

4.3 Fecundity and maturation

The absolute fecundity ranged from 90 to 2,566 oocytes. The largest amount of oocytes were not removed from the biggest fish, nor were the smallest amount of oocytes recovered from the smallest fish. The only information from previous studies on the fecundity of H. amblops available for comparison was listed on the FishTraits database as

43 a maximum of 1,000 oocytes. Many individuals in the same freshwater species are known to contain broad variations in fecundity and oocyte sizes. A difference in resources and nutrition within a population prior to spawning may be responsible for such deviations[11]. Another explanation for fecundity variation is multiple spawners release oocytes in batches as evidenced by ovaries containing oocytes in different stages of development. Individuals in unlike stages of ovarian maturation may have released a batch of oocytes resulting in fecundity variation.

Oocyte size distributions are additional evidence of multiple spawning. Oocyte diameters at each developmental stage I-IV was measured for female H. amblops collected during spawning months. The average size of stage I oocytes was 0.42 mm; stage II oocytes averaged 0.55 mm; stage III ova averaged 0.81 mm; and an average size of 1.00 mm was determined for stage IV ova. No information was available from previous studies for oocyte size comparison.

Fecundity was independent of fish sizes as evidenced by weak correlations between fecundity vs SL and fecundity vs GBM. The R2 value was highest (R2 = 0.621) for total fecundity vs gonadal mass relationship. Linear regression results did not indicate a strong correlation between total fecundity vs water temperature (R2 = 0.101), but changes in reproductive timing and fecundity have been associated with changes in temperature. Linear regression analysis is indicative of a population’s immediate phenotypic response to environmental fluctuations. Many fish species have to adapt to temperature trends, as indicated in Figure 3.12[48].

This study established baseline information concerning a reproductive schedule and traits of H. amblops in the Flint River in Huntsville, AL. This study is important for

44 understanding population dynamics, and for future analyses of this population’s response to environmental changes. The data collected can be used in comparing allopatric populations. Finally, this knowledge is crucial to any conservation effort centered on scientific data and could be used to advocate the managing of the species.

45

Appendix

Body Mass Gonadal Month/Yr Fish ID Sex SL (mm) (g) Mass (g) GSI 13-May HAEE01 Male 62.02 3.8067 0.0558 1.47 13-May HAEE02 Male 49.33 1.8073 0.0166 0.92 13-May HAEE03 Male 54.91 2.5963 0.043 1.66 13-May HAEE04 Female 57.74 1.8017 0.231 12.82 13-May HAEE05 Female 60.17 2.1667 0.284 13.1 13-May HAEE06 Male 55.23 2.381 0.0217 0.91 13-May HAEE07 Male 52.64 1.9907 0.0522 2.62 13-May HAEE08 Male 62.75 3.3933 0.0504 1.49 13-May HAEE09 Juvenile 29.43 1.4905 13-Jun HAFF01 Male 53.58 2.3205 0.0181 0.78 13-Jun HAFF02 Male 52.65 2.4722 0.023 0.93 13-Jun HAFF03 Male 54.27 2.7304 0.0153 0.56 13-Jun HAFF04 Female 54.2 2.8917 0.2257 7.81 13-Jun HAFF05 Male 55.76 2.7195 0.0247 0.91 13-Jun HAFF06 Juvenile 32.43 0.3843 13-Jun HAFF07 Juvenile 30.77 0.2796 13-Jun HAFF08 Juvenile 31.93 0.3955 13-Jun HAFF09 Juvenile 32.94 0.3616 13-Jun HAFF10 Juvenile 31.93 0.3769 13-Jun HAFF11 Female 61.86 3.8393 0.2542 6.62 13-Jun HAFF12 Female 57.37 3.1861 0.1723 5.41 13-Jun HAFF13 Male 71.25 3.7971 0.0803 2.11 13-Jun HAFF14 Male 63.24 2.6998 0.0281 1.04 13-Jun HAFF15 Male 60.64 2.405 0.0236 0.98 13-Jun HAFF16 Male 63.66 2.8444 0.0219 0.77 13-Jun HAFF17 Female 56.53 2.4073 0.0921 3.83 13-Jun HAFF18 Female 55.13 2.8328 0.0678 2.39 13-Jun HAFF19 Female 52.85 2.5263 0.0163 0.65 13-Jun HAFF20 Male 60.97 2.2442 0.0131 0.58 13-Jun HAFF21 Male 66.24 2.9993 0.0353 1.18 13-Jun HAFF22 Male 66.73 2.77 0.0209 0.75 13-Jun HAFF23 Female 68.13 4.0918 0.2457 6 13-Jun HAFF24 Female 59.91 2.17 0.0324 1.49 13-Jun HAFF25 Male 60.62 2.0168 0.0133 0.66 13-Jun HAFF26 Male 68.76 3.4005 0.019 0.56 13-Jun HAFF27 Male 59.94 2.1676 0.0099 0.46 13-Jun HAFF28 Male 62.92 2.4026 0.0122 0.51 13-Jun HAFF29 Male 64.02 2.5756 0.0198 0.77

46

Body Mass Gonadal Month/Yr Fish ID Sex SL (mm) (g) Mass (g) GSI 13-Jun HAFF30 Male 63.88 2.9788 0.0212 0.71 13-Jun HAFF31 Male 60.92 2.2561 0.0141 0.63 13-Jun HAFF32 Male 59.66 2.2747 0.0167 0.73 13-Jun HAFF33 Male 58.35 2.1702 0.0101 0.47 13-Jun HAFF34 Female 55.51 1.5226 0.0225 1.48 13-Jun HAFF35 Female 55.83 1.737 0.1631 9.39 13-Jul HAGG01 Male 51.56 1.9798 0.016 0.81 13-Jul HAGG02 Female 61.18 3.0009 0.0495 1.65 13-Aug HAH01 Female 55.03 2.6984 0.0534 1.98 13-Aug HAH02 Male 53.32 2.4545 0.0215 0.88 13-Aug HAH03 Male 56.2 2.5661 0.0038 0.15 13-Aug HAH04 Male 51.5 2.2673 0.0043 0.19 13-Aug HAH05 Male 55.09 2.9754 0.011 0.37 13-Aug HAH06 Female 57.82 3.1789 0.0579 1.82 13-Aug HAH07 Male 53.25 2.3524 0.0187 0.79 13-Aug HAH08 Male 53.69 2.4003 0.009 0.38 13-Aug HAH09 Juvenile 53.51 2.5571 13-Aug HAH10 Juvenile 54.65 2.7077 13-Aug HAH11 Male 57.12 2.967 0.038 1.28 13-Aug HAH12 Male 53.45 2.4416 0.0074 0.3 13-Aug HAH13 Juvenile 53.73 2.5896 13-Aug HAH14 Juvenile 54.6 2.4944 13-Aug HAH15 Male 55.66 2.8321 0.0134 0.47 13-Aug HAH16 Female 57.14 3.1113 0.057 1.83 13-Aug HAH17 Male 52.72 2.51 0.0439 1.75 13-Aug HAH18 Female 57.62 3.2788 0.0686 2.09 13-Aug HAH19 Female 66.78 4.4453 0.1168 2.63 13-Aug HAH20 Male 59.46 3.4309 0.0071 0.21 13-Aug HAH21 Male 53.55 2.6542 0.0081 0.31 13-Aug HAH22 Male 54.75 2.6183 0.0097 0.37 13-Aug HAH23 Female 57.44 3.0866 0.0487 1.58 13-Aug HAH24 Male 54.17 2.5218 0.0094 0.37 13-Aug HAH25 Female 58.33 3.2416 0.0651 2.01 13-Aug HAH26 Male 55.31 2.7739 0.0215 0.78 13-Aug HAH27 Male 54.77 2.5767 0.0127 0.49 13-Aug HAH28 Male 54.61 2.6483 0.0012 0.05 13-Aug HAH29 Juvenile 39.08 0.7732 13-Aug HAH30 Juvenile 36.47 0.5893 13-Aug HAH31 Juvenile 38.96 0.7025

47

Body Mass Gonadal Month/Yr Fish ID Sex SL (mm) (g) Mass (g) GSI 13-Aug HAH32 Juvenile 38.73 0.627 13-Aug HAH33 Juvenile 35.62 0.5829 13-Aug HAH34 Juvenile 38.36 0.7168 13-Aug HAH35 Juvenile 32.03 0.3048 13-Aug HAH36 Juvenile 36.08 0.5592 13-Aug HAH37 Juvenile 37.52 0.6825 13-Aug HAH38 Juvenile 36.89 0.5628 13-Aug HAH39 Juvenile 37.29 0.6453 13-Aug HAH40 Juvenile 33.89 0.4789 13-Aug HAH41 Juvenile 35.26 0.4905 13-Aug HAH42 Juvenile 32.81 0.3507 13-Aug HAH43 Juvenile 31.89 0.3355 13-Aug HAH44 Juvenile 30.52 0.2941 13-Aug HAH45 Juvenile 31.78 0.2658 13-Aug HAH46 Juvenile 34.48 0.5191 13-Aug HAH47 Juvenile 38.79 0.7013 13-Aug HAH48 Juvenile 37.13 0.6174 13-Aug HAH49 Juvenile 37.21 0.626 13-Aug HAH50 Juvenile 36.97 0.4998 13-Sep HAI01 Male 61.16 3.9626 0.0174 0.44 13-Sep HAI02 Female 64.38 4.1753 0.068 1.63 13-Sep HAI03 Female 56.61 2.92 0.0555 1.9 13-Sep HAI04 Male 60.23 3.5819 0.0068 0.19 13-Sep HAI05 Male 55.26 2.5289 0.0071 0.28 13-Sep HAI06 Female 55.71 2.3227 0.0101 0.43 13-Sep HAI07 Male 56.63 3.0544 0.0176 0.58 13-Sep HAI08 Female 53.94 2.2443 0.0377 1.68 13-Sep HAI09 Male 55.1 2.6195 0.0066 0.25 13-Sep HAI10 Male 58.76 3.3396 0.0064 0.19 13-Sep HAI11 Female 56.64 2.9072 0.035 1.2 13-Sep HAI12 Male 57.18 3.0103 0.0047 0.16 13-Sep HAI13 Male 51.72 2.1284 0.0209 0.98 13-Sep HAI14 Male 59.78 3.132 0.0098 0.31 13-Sep HAI15 Male 61.19 3.7279 0.0111 0.3 13-Sep HAI16 Juvenile 43.15 0.8489 13-Oct HAJ01 Male 67.75 3.2623 0.0165 0.51 13-Oct HAJ02 Male 59.09 1.94 0.0101 0.52 13-Oct HAJ03 Juvenile 49.39 1.1103 13-Oct HAJ05 Female 64.37 3.1771 0.0304 0.96

48

Body Mass Gonadal Month/Yr Fish ID Sex SL (mm) (g) Mass (g) GSI 13-Oct HAJ04 Male 64.86 3.4502 0.0168 0.49 13-Oct HAJ06 Female 64.05 2.5223 0.0164 0.65 13-Oct HAJ07 Juvenile 54.25 1.631 13-Oct HAJ08 Male 71.31 4.084 0.0155 0.38 13-Oct HAJ09 Male 67.63 3.2852 0.0043 0.13 13-Oct HAJ10 Female 62.18 3.0013 0.0384 1.28 13-Oct HAJ11 Female 64.48 3.5941 0.0732 2.04 13-Nov HAK01 Male 64.5 2.8608 0.0303 1.06 13-Nov HAK02 Male 72.33 4.3251 0.0298 0.69 13-Nov HAK03 Female 66.24 3.3682 0.0833 2.47 13-Nov HAK04 Female 68.98 3.6063 0.0521 1.44 13-Nov HAK05 Male 66.45 3.384 0.0187 0.55 13-Nov HAK06 Male 64.9 2.9321 0.0137 0.47 13-Nov HAK07 Male 65.75 2.8697 0.0216 0.75 13-Nov HAK08 Female 63.54 2.6055 0.0413 1.59 13-Nov HAK09 Male 65.12 2.9353 0.0263 0.9 13-Nov HAK10 Male 64.52 2.5409 0.0113 0.44 13-Nov HAK11 Male 63.19 2.4687 0.0115 0.47 13-Nov HAK12 Male 71.29 4.5033 0.0156 0.35 13-Nov HAK13 Female 66.07 3.1248 0.0967 3.09 13-Nov HAK14 Female 65.66 3.1414 0.1114 3.55 13-Nov HAK15 Male 65.53 2.7815 0.013 0.47 13-Nov HAK16 Male 65.48 3.0969 0.0123 0.4 13-Nov HAK17 Male 63.51 2.7232 0.0104 0.38 13-Nov HAK18 Male 65.45 3.0241 0.0139 0.46 13-Nov HAK19 Female 66.8 3.122 0.0603 1.93 13-Nov HAK20 Male 63.07 2.6377 0.0109 0.41 13-Nov HAK21 Male 63.11 2.7173 0.0176 0.65 13-Nov HAK22 Juvenile 57.05 2.61 13-Nov HAK23 Juvenile 56.21 2.1409 13-Nov HAK24 Juvenile 57.94 2.0494 13-Nov HAK25 Juvenile 55.97 1.8261 13-Nov HAK26 Juvenile 51.56 1.3199 13-Nov HAK27 Juvenile 48.92 1.0803 13-Dec HAL01 Female 61.89 2.2703 0.0873 3.85 13-Dec HAL02 Juvenile 56.93 1.5801 13-Dec HAL03 Juvenile 50.94 1.1603 13-Dec HAL04 Juvenile 50.95 1.0612 13-Dec HAL05 Juvenile 51.12 1.1505

49

Body Mass Gonadal Month/Yr Fish ID Sex SL (mm) (g) Mass (g) GSI 13-Dec HAL06 Juvenile 48.04 0.941 13-Dec HAL07 Male 74.35 5.0542 0.0314 0.62 13-Dec HAL08 Female 73.49 4.1091 0.1275 3.1 13-Dec HAL09 Female 68.93 3.5421 0.1033 2.92 13-Dec HAL10 Female 67.03 3.0778 0.1151 3.74 13-Dec HAL11 Female 65.42 2.9976 0.0756 2.52 13-Dec HAL12 Female 64.07 2.5472 0.067 2.63 13-Dec HAL13 Male 64.2 2.7837 0.028 1.01 13-Dec HAL14 Female 63.95 2.6644 0.0781 2.93 13-Dec HAL15 Female 68.59 3.4504 0.0873 2.53 13-Dec HAL16 Female 67.46 2.9524 0.073 2.47 13-Dec HAL17 Male 67.18 3.2422 0.024 0.74 13-Dec HAL18 Female 66.89 2.9921 0.0904 3.02 13-Dec HAL19 Female 68.2 3.3987 0.0205 0.6 13-Dec HAL20 Male 66.95 2.7866 0.0109 0.39 13-Dec HAL21 Male 67.04 3.1458 0.0114 0.36 13-Dec HAL22 Juvenile 57.03 1.985 13-Dec HAL23 Juvenile 57.04 1.866 13-Dec HAL24 Juvenile 56.57 1.6415 13-Dec HAL25 Juvenile 53.12 1.6497 13-Dec HAL26 Juvenile 54.81 1.4966 14-Jan HAA01 Male 71.46 4.1356 0.0242 0.59 14-Jan HAA02 Male 63.05 2.2089 0.0201 0.91 14-Feb HAB01 Male 54.86 1.8121 0.0211 1.16 14-Feb HAB02 Male 54.41 1.6733 0.0201 1.2 14-Feb HAB03 Juvenile 51.55 1.1303 14-Feb HAB04 Juvenile 42.21 0.6899 14-Feb HAB05 Female 63.44 2.6451 0.1879 7.1 14-Feb HAB06 Juvenile 53.18 1.333 14-Feb HAB07 Juvenile 53.14 1.5312 14-Feb HAB08 Juvenile 53.75 1.4112 14-Feb HAB09 Juvenile 53.08 1.4223 14-Feb HAB10 Juvenile 52.07 1.2642 14-Feb HAB11 Juvenile 51.5 1.1799 14-Feb HAB12 Juvenile 51.76 1.1388 14-Feb HAB13 Juvenile 49.46 0.9818 14-Feb HAB14 Juvenile 51.37 1.239 14-Feb HAB15 Juvenile 50.16 0.9337 14-Feb HAB16 Juvenile 49.73 0.9753

50

Body Mass Gonadal Month/Yr Fish ID Sex SL (mm) (g) Mass (g) GSI 14-Mar HAC01 Female 58.68 2.1011 0.0813 3.87 14-Mar HAC02 Female 59.31 2.273 0.0927 4.08 14-Mar HAC03 Male 55.08 1.6444 0.0206 1.25 14-Mar HAC04 Juvenile 52.01 1.2435 14-Mar HAC05 Juvenile 50.9 1.2474 14-Mar HAC06 Juvenile 52.68 1.2333 14-Mar HAC07 Juvenile 49.54 1.0875 14-Mar HAC08 Juvenile 52.7 1.2859 14-Mar HAC09 Female 66.56 3.9437 0.5101 12.93 14-Mar HAC10 Female 77.16 5.8041 0.3201 5.52 14-Mar HAC11 Female 77.02 5.6389 0.6766 12 14-Mar HAC12 Female 68.42 4.2303 0.4322 10.22 14-Mar HAC13 Male 61.61 2.7652 0.0373 1.35 14-Mar HAC14 Female 61.77 2.7679 0.1377 4.97 14-Apr HAD01 Juvenile 54.82 2.3006 14-Apr HAD02 Juvenile 48.92 1.089 14-Apr HAD03 Female 69.32 3.7802 0.4891 12.94 14-Apr HAD04 Male 65.04 3.0903 0.0665 2.15 14-Apr HAD05 Female 62.08 2.6306 0.3621 13.76 14-Apr HAD06 Juvenile 56.73 1.9401 14-Apr HAD07 Juvenile 56.79 2.0165 14-Apr HAD08 Juvenile 56.05 1.9901 14-Apr HAD09 Juvenile 52.32 1.4782 14-Apr HAD10 Female 70.82 4.4021 0.5677 12.9 14-Apr HAD11 Male 65.43 3.3812 0.0582 1.72 14-Apr HAD12 Male 64.7 2.843 0.0454 1.6 14-Apr HAD13 Male 59.87 2.2079 0.0341 1.54 14-Apr HAD14 Male 58.63 1.9728 0.0232 1.18 14-Apr HAD15 Female 61.89 2.0823 0.2763 13.27 14-Apr HAD16 Male 62.51 2.6212 0.0567 2.16 14-Apr HAD17 Juvenile 52.94 1.6685 14-Apr HAD18 Female 60.08 2.1003 0.1802 8.58 14-Apr HAD19 Juvenile 54.63 1.7165 14-Apr HAD20 Juvenile 52.75 1.5351 14-May HAE01 Female 76.17 5.877 0.7816 13.3 14-May HAE02 Female 71.5 4.2964 0.4553 10.6 14-May HAE03 Male 70.91 4.1501 0.0762 1.84 14-May HAE04 Male 68.86 3.5622 0.0588 1.65

51

Body Mass Gonadal Month/Yr Fish ID Sex SL (mm) (g) Mass (g) GSI 14-May HAE05 Female 70.83 4.1796 0.4372 10.46 14-May HAE06 Female 71.24 3.7314 0.446 11.95 14-May HAE07 Female 68.56 3.7022 0.4259 11.5 14-May HAE08 Male 65.69 3.0781 0.0398 1.29 14-May HAE09 Male 60.85 2.3282 0.0427 1.83 14-May HAE10 Male 66.68 3.378 0.0575 1.7 14-May HAE11 Male 71.35 3.8364 0.0598 1.56 14-May HAE12 Male 60.34 2.317 0.0449 1.94 14-May HAE13 Female 62.01 2.4351 0.4204 17.26 14-May HAE14 Male 58.56 1.9001 0.0285 1.5 14-May HAE15 Male 59.77 2.146 0.0311 1.45 14-May HAE16 Male 60.27 2.2682 0.0305 1.34 14-May HAE17 Female 58.01 2.2115 0.4631 20.94 14-May HAE18 Male 56.09 1.9073 0.0347 1.82 14-May HAE19 Female 57.51 2.1457 0.302 14.07 14-May HAE20 Female 57.76 1.9951 0.1671 8.38 14-May HAE21 Male 56.37 1.7702 0.0276 1.56 14-May HAE22 Juvenile 53.77 1.5803 14-May HAE23 Male 57.4 1.8441 0.0359 1.95 14-May HAE24 Male 54.56 1.4867 0.0304 2.04 14-May HAE25 Male 55.53 1.7093 0.0323 1.89 14-May HAE26 Male 55.46 1.5036 0.0288 1.92 14-May HAE27 Male 56.55 1.5302 0.0215 1.41 14-May HAE28 Female 58.04 1.7048 0.2704 15.86 14-May HAE29 Female 57.76 1.8002 0.1782 9.9 14-May HAE30 Juvenile 51.51 1.6114 14-May HAE31 Female 56.83 1.546 0.2169 14.03 14-May HAE32 Female 69.21 4.086 0.5647 13.82 14-May HAE33 Male 69.98 3.9882 0.0599 1.5 14-May HAE34 Male 65.5 3.597 0.0273 0.76 14-May HAE35 Male 69.07 3.685 0.0581 1.58 14-May HAE36 Male 65.65 3.2851 0.0473 1.44 14-May HAE37 Male 60.98 2.4266 0.0321 1.32 14-May HAE38 Female 60.15 2.101 0.2011 9.57 14-May HAE39 Female 57.39 2.1338 0.1893 8.87 14-May HAE40 Male 57.6 2.085 0.0344 1.65 14-May HAE41 Male 55.71 1.8049 0.0169 0.94 14-May HAE42 Female 55.74 1.6825 0.2193 13.03 14-May HAE43 Juvenile 52.09 1.3419

52

Body Mass Gonadal Month/Yr Fish ID Sex SL (mm) (g) Mass (g) GSI 14-Jun HAF01 Female 72.86 4.3179 0.2002 4.64 14-Jun HAF02 Male 67.51 3.659 0.028 0.77 14-Jun HAF03 Female 76.91 4.9836 0.2045 4.1 14-Jun HAF04 Male 72.15 4.482 0.0592 1.32 14-Jun HAF05 Male 69.6 3.8787 0.0452 1.17 14-Jun HAF06 Male 69.78 4.0669 0.0289 0.71 14-Jun HAF07 Male 67.68 3.5419 0.0286 0.81 14-Jun HAF08 Male 70.3 4.0379 0.0359 0.89 14-Jun HAF09 Female 65.58 2.9663 0.1025 3.46 14-Jun HAF10 Male 72.62 4.3213 0.0417 0.97 14-Jun HAF11 Female 68.5 3.5939 0.1498 4.17 14-Jun HAF12 Male 68.66 3.4044 0.0433 1.27 14-Jun HAF13 Female 66.05 2.9008 0.1239 4.27 14-Jun HAF14 Male 66.67 3.0561 0.0241 0.79 14-Jun HAF15 Male 61.54 2.5039 0.026 1.04 14-Jun HAF16 Female 58.59 2.1331 0.2006 9.4 14-Jun HAF17 Male 62.61 2.2777 0.0291 1.28 14-Jun HAF18 Male 59.32 1.9393 0.0225 1.16 14-Jun HAF19 Female 47.74 1.0471 0.1671 15.96 14-Jun HAF20 Male 58.16 2.0298 0.0283 1.39 14-Jun HAF21 Male 60.39 2.0978 0.0109 0.52 14-Jun HAF22 Female 64.37 2.1863 0.0773 3.54 14-Jun HAF23 Male 71.47 3.5236 0.0275 0.78 14-Jun HAF24 Female 62.74 2.1034 0.0314 1.49 14-Jul HAG01 Female 72.37 4.2534 0.1835 4.31 14-Jul HAG02 Female 73.45 3.9429 0.1713 4.34 14-Jul HAG03 Female 72.07 4.216 0.1029 2.44 14-Jul HAG04 Male 68.46 3.4728 0.0213 0.61 14-Jul HAG05 Male 67.07 3.1777 0.0188 0.59 14-Jul HAG06 Female 71.32 3.8351 0.0379 0.99 14-Jul HAG07 Male 68.27 3.166 0.0164 0.52 14-Jul HAG08 Female 66.12 3.0456 0.1567 5.15 14-Jul HAG09 Male 61.01 2.0906 0.0103 0.49 14-Jul HAG10 Male 61.26 2.2518 0.027 1.2 14-Jul HAG11 Female 61.52 2.1245 0.0224 1.05 14-Jul HAG12 Female 58.76 2.0203 0.0223 1.1 14-Jul HAG13 Female 59.2 1.9581 0.0259 1.32 14-Jul HAG14 Male 60.1 2.0586 0.0032 0.16 14-Jul HAG15 Male 58.11 1.8533 0.0055 0.3

53

Body Mass Gonadal Month/Yr Fish ID Sex SL (mm) (g) Mass (g) GSI 14-Jul HAG16 Female 59.87 1.8628 0.0129 0.69 14-Jul HAG17 Female 58.07 1.8311 0.0231 1.26 14-Jul HAG18 Juvenile 56.51 1.6017 14-Jul HAG19 Juvenile 56.26 1.6674 14-Jul HAG20 Juvenile 54.66 1.4308

54

Ovarian # Stage I # Stage II # Stage III # Stage IV Total # Month/Yr Fish ID Stage (I-V) Eggs Eggs Eggs Eggs Eggs 13-May HAEE01 13-May HAEE02 13-May HAEE03 13-May HAEE04 IV 61 440 282 12 795 13-May HAEE05 IV 26 335 562 4 927 13-May HAEE06 13-May HAEE07 13-May HAEE08 13-May HAEE09 13-Jun HAFF01 13-Jun HAFF02 13-Jun HAFF03 13-Jun HAFF04 V 56 382 109 3 550 13-Jun HAFF05 13-Jun HAFF06 13-Jun HAFF07 13-Jun HAFF08 13-Jun HAFF09 13-Jun HAFF10 13-Jun HAFF11 IV 345 817 548 30 1740 13-Jun HAFF12 IV 329 615 198 34 1176 13-Jun HAFF13 13-Jun HAFF14 13-Jun HAFF15 13-Jun HAFF16 13-Jun HAFF17 V 157 339 179 5 680 13-Jun HAFF18 13-Jun HAFF19 13-Jun HAFF20 13-Jun HAFF21 13-Jun HAFF22 13-Jun HAFF23 V 56 182 67 305 13-Jun HAFF24 13-Jun HAFF25 13-Jun HAFF26 13-Jun HAFF27 13-Jun HAFF28 13-Jun HAFF29

55

Ovarian # Stage I # Stage II # Stage III # Stage IV Total # Month/Yr Fish ID Stage (I-V) Eggs Eggs Eggs Eggs Eggs 13-Jun HAFF30 13-Jun HAFF31 13-Jun HAFF32 13-Jun HAFF33 13-Jun HAFF34 13-Jun HAFF35 V 34 141 326 39 540 13-Jul HAGG01 13-Jul HAGG02 V 232 317 7 556 13-Aug HAH01 V 394 129 15 538 13-Aug HAH02 13-Aug HAH03 13-Aug HAH04 13-Aug HAH05 13-Aug HAH06 13-Aug HAH07 13-Aug HAH08 13-Aug HAH09 13-Aug HAH10 13-Aug HAH11 13-Aug HAH12 13-Aug HAH13 13-Aug HAH14 13-Aug HAH15 13-Aug HAH16 13-Aug HAH17 13-Aug HAH18 13-Aug HAH19 V 55 108 33 10 206 13-Aug HAH20 13-Aug HAH21 13-Aug HAH22 13-Aug HAH23 13-Aug HAH24 13-Aug HAH25 V 284 72 35 391 13-Aug HAH26 13-Aug HAH27 13-Aug HAH28 13-Aug HAH29 13-Aug HAH30 13-Aug HAH31

56

Ovarian # Stage I # Stage II # Stage III # Stage IV Total # Month/Yr Fish ID Stage (I-V) Eggs Eggs Eggs Eggs Eggs 13-Oct HAJ04 13-Oct HAJ06 13-Oct HAJ07 13-Oct HAJ08 13-Oct HAJ09 13-Oct HAJ10 13-Oct HAJ11 13-Nov HAK01 13-Nov HAK02 13-Nov HAK03 I 97 17 79 193 13-Nov HAK04 13-Nov HAK05 13-Nov HAK06 13-Nov HAK07 13-Nov HAK08 13-Nov HAK09 13-Nov HAK10 13-Nov HAK11 13-Nov HAK12 13-Nov HAK13 I 117 179 296 13-Nov HAK14 I 181 77 258 13-Nov HAK15 13-Nov HAK16 13-Nov HAK17 13-Nov HAK18 13-Nov HAK19 13-Nov HAK20 13-Nov HAK21 13-Nov HAK22 13-Nov HAK23 13-Nov HAK24 13-Nov HAK25 13-Nov HAK26 13-Nov HAK27 13-Dec HAL01 I 547 36 583 13-Dec HAL02 13-Dec HAL03 13-Dec HAL04 13-Dec HAL05

57

Ovarian # Stage I # Stage II # Stage III # Stage IV Total # Month/Yr Fish ID Stage (I-V) Eggs Eggs Eggs Eggs Eggs 13-Dec HAL06 13-Dec HAL07 13-Dec HAL08 I 307 25 332 13-Dec HAL09 I 525 11 536 13-Dec HAL10 I 706 64 22 792 13-Dec HAL11 I 795 795 13-Dec HAL12 I 425 33 458 13-Dec HAL13 13-Dec HAL14 I 587 26 613 13-Dec HAL15 I 90 90 13-Dec HAL16 13-Dec HAL17 13-Dec HAL18 I 454 454 13-Dec HAL19 I 462 462 13-Dec HAL20 13-Dec HAL21 13-Dec HAL22 13-Dec HAL23 13-Dec HAL24 13-Dec HAL25 13-Dec HAL26 14-Jan HAA01 14-Jan HAA02 14-Feb HAB01 14-Feb HAB02 14-Feb HAB03 14-Feb HAB04 14-Feb HAB05 II 1083 164 1247 14-Feb HAB06 14-Feb HAB07 14-Feb HAB08 14-Feb HAB09 14-Feb HAB10 14-Feb HAB11 14-Feb HAB12 14-Feb HAB13 14-Feb HAB14 14-Feb HAB15 14-Feb HAB16

58

Ovarian # Stage I # Stage II # Stage III # Stage IV Total # Month/Yr Fish ID Stage (I-V) Eggs Eggs Eggs Eggs Eggs 14-Mar HAC01 III 285 261 188 734 14-Mar HAC02 III 470 396 10 876 14-Mar HAC03 14-Mar HAC04 14-Mar HAC05 14-Mar HAC06 14-Mar HAC07 14-Mar HAC08 14-Mar HAC09 IV 81 885 277 59 1302 14-Mar HAC10 III 531 699 73 17 1320 14-Mar HAC11 IV 545 1202 385 14 2146 14-Mar HAC12 III 401 557 387 38 1383 14-Mar HAC13 14-Mar HAC14 III 512 407 216 1135 14-Apr HAD01 14-Apr HAD02 14-Apr HAD03 IV 143 250 845 73 1311 14-Apr HAD04 14-Apr HAD05 IV 195 155 917 25 1292 14-Apr HAD06 14-Apr HAD07 14-Apr HAD08 14-Apr HAD09 14-Apr HAD10 IV 166 121 977 125 1389 14-Apr HAD11 14-Apr HAD12 14-Apr HAD13 14-Apr HAD14 14-Apr HAD15 IV 150 207 730 64 1151 14-Apr HAD16 14-Apr HAD17 14-Apr HAD18 III 612 88 303 2 1005 14-Apr HAD19 14-Apr HAD20 14-May HAE01 372 854 1282 58 2566 14-May HAE02 267 413 753 48 1481 14-May HAE03 14-May HAE04

59

Ovarian # Stage I # Stage II # Stage III # Stage IV Total # Month/Yr Fish ID Stage (I-V) Eggs Eggs Eggs Eggs Eggs 14-May HAE05 IV 113 362 577 189 1241 14-May HAE06 IV 219 264 525 33 1041 14-May HAE07 IV 88 426 596 137 1247 14-May HAE08 14-May HAE09 14-May HAE10 14-May HAE11 14-May HAE12 14-May HAE13 IV 155 426 372 50 1003 14-May HAE14 14-May HAE15 14-May HAE16 14-May HAE17 IV 250 399 313 53 1015 14-May HAE18 14-May HAE19 IV 471 281 388 21 1161 14-May HAE20 III 260 415 297 26 998 14-May HAE21 14-May HAE22 14-May HAE23 14-May HAE24 14-May HAE25 14-May HAE26 14-May HAE27 14-May HAE28 IV 508 416 268 8 1200 14-May HAE29 V 292 270 252 35 849 14-May HAE30 14-May HAE31 IV 208 324 259 72 863 14-May HAE32 IV 210 404 516 23 1153 14-May HAE33 14-May HAE34 14-May HAE35 14-May HAE36 14-May HAE37 14-May HAE38 V 197 187 9 2 395 14-May HAE39 V 109 222 227 19 577 14-May HAE40 14-May HAE41 14-May HAE42 IV 118 83 158 14 373 14-May HAE43

60

Ovarian # Stage I # Stage II # Stage III # Stage IV Total # Month/Yr Fish ID Stage (I-V) Eggs Eggs Eggs Eggs Eggs 14-Jun HAF01 V 401 106 8 515 14-Jun HAF02 14-Jun HAF03 V 257 578 21 4 860 14-Jun HAF04 14-Jun HAF05 14-Jun HAF06 14-Jun HAF07 14-Jun HAF08 14-Jun HAF09 V 262 390 179 8 839 14-Jun HAF10 14-Jun HAF11 V 11 73 335 83 502 14-Jun HAF12 14-Jun HAF13 V 231 245 104 68 648 14-Jun HAF14 14-Jun HAF15 14-Jun HAF16 V 251 258 140 649 14-Jun HAF17 14-Jun HAF18 14-Jun HAF19 IV 14-Jun HAF20 14-Jun HAF21 14-Jun HAF22 14-Jun HAF23 14-Jun HAF24 14-Jul HAG01 V 214 132 211 32 589 14-Jul HAG02 V 2 307 753 73 1135 14-Jul HAG03 69 746 23 838 14-Jul HAG04 14-Jul HAG05 14-Jul HAG06 14-Jul HAG07 14-Jul HAG08 V 1 9 587 26 623 14-Jul HAG09 14-Jul HAG10 14-Jul HAG11 14-Jul HAG12 14-Jul HAG13 14-Jul HAG14 14-Jul HAG15

61

River Stage I Stage II Stage III Stage IV Water Dischare Avg Diam Avg Diam Avg Diam Avg Diam Temp Monthly Month/Yr Fish ID (mm) (mm) (mm) (mm) (C) Mean in ft3/s 13-May HAEE01 21.9 1177 13-May HAEE02 21.9 1177 13-May HAEE03 21.9 1177 13-May HAEE04 0.485 0.5652 0.875 1.003 21.9 1177 13-May HAEE05 0.4053 0.4905 0.7128 1.02 21.9 1177 13-May HAEE06 21.9 1177 13-May HAEE07 21.9 1177 13-May HAEE08 21.9 1177 13-May HAEE09 21.9 1177 13-Jun HAFF01 25.7 417.5 13-Jun HAFF02 25.7 417.5 13-Jun HAFF03 25.7 417.5 13-Jun HAFF04 0.387 0.4183 0.5569 0.9505 25.7 417.5 13-Jun HAFF05 25.7 417.5 13-Jun HAFF06 25.7 417.5 13-Jun HAFF07 25.7 417.5 13-Jun HAFF08 25.7 417.5 13-Jun HAFF09 25.7 417.5 13-Jun HAFF10 25.7 417.5 13-Jun HAFF11 0.4716 0.6367 0.7707 0.9228 25.7 417.5 13-Jun HAFF12 0.4482 0.5379 0.7303 0.9097 25.7 417.5 13-Jun HAFF13 25.7 417.5 13-Jun HAFF14 25.7 417.5 13-Jun HAFF15 25.7 417.5 13-Jun HAFF16 25.7 417.5 13-Jun HAFF17 0.469 0.6361 0.9176 1.0406 25.7 417.5 13-Jun HAFF18 25.7 417.5 13-Jun HAFF19 25.7 417.5 13-Jun HAFF20 25.7 417.5 13-Jun HAFF21 25.7 417.5 13-Jun HAFF22 25.7 417.5 13-Jun HAFF23 0.5873 0.7571 0.8771 25.7 417.5 13-Jun HAFF24 25.7 417.5 13-Jun HAFF25 25.7 417.5 13-Jun HAFF26 25.7 417.5 13-Jun HAFF27 25.7 417.5 13-Jun HAFF28 25.7 417.5 13-Jun HAFF29 25.7 417.5

62

River Stage I Stage II Stage III Stage IV Water Dischare Avg Diam Avg Diam Avg Diam Avg Diam Temp Monthly Month/Yr Fish ID (mm) (mm) (mm) (mm) (C) Mean in ft3/s 13-Jun HAFF30 25.7 417.5 13-Jun HAFF31 25.7 417.5 13-Jun HAFF32 25.7 417.5 13-Jun HAFF33 25.7 417.5 13-Jun HAFF34 25.7 417.5 13-Jun HAFF35 0.3858 0.5087 0.6855 0.7853 25.7 417.5 13-Jul HAGG01 26.4 934.6 13-Jul HAGG02 0.317 0.472 0.6886 26.4 934.6 13-Aug HAH01 0.2661 0.5165 0.5932 25.1 818.8 13-Aug HAH02 25.1 818.8 13-Aug HAH03 25.1 818.8 13-Aug HAH04 25.1 818.8 13-Aug HAH05 25.1 818.8 13-Aug HAH06 25.1 818.8 13-Aug HAH07 25.1 818.8 13-Aug HAH08 25.1 818.8 13-Aug HAH09 25.1 818.8 13-Aug HAH10 25.1 818.8 13-Aug HAH11 25.1 818.8 13-Aug HAH12 25.1 818.8 13-Aug HAH13 25.1 818.8 13-Aug HAH14 25.1 818.8 13-Aug HAH15 25.1 818.8 13-Aug HAH16 25.1 818.8 13-Aug HAH17 25.1 818.8 13-Aug HAH18 25.1 818.8 13-Aug HAH19 0.333 0.5041 0.6758 0.871 25.1 818.8 13-Aug HAH20 25.1 818.8 13-Aug HAH21 25.1 818.8 13-Aug HAH22 25.1 818.8 13-Aug HAH23 25.1 818.8 13-Aug HAH24 25.1 818.8 13-Aug HAH25 0.3415 0.456 0.5964 25.1 818.8 13-Aug HAH26 25.1 818.8 13-Aug HAH27 25.1 818.8 13-Aug HAH28 25.1 818.8 13-Aug HAH29 25.1 818.8 13-Aug HAH30 25.1 818.8 13-Aug HAH31 25.1 818.8

63

River Stage I Stage II Stage III Stage IV Water Dischare Avg Diam Avg Diam Avg Diam Avg Diam Temp Monthly Month/Yr Fish ID (mm) (mm) (mm) (mm) (C) Mean in ft3/s 13-Aug HAH32 25.1 818.8 13-Aug HAH33 25.1 818.8 13-Aug HAH34 25.1 818.8 13-Aug HAH35 25.1 818.8 13-Aug HAH36 25.1 818.8 13-Aug HAH37 25.1 818.8 13-Aug HAH38 25.1 818.8 13-Aug HAH39 25.1 818.8 13-Aug HAH40 25.1 818.8 13-Aug HAH41 25.1 818.8 13-Aug HAH42 25.1 818.8 13-Aug HAH43 25.1 818.8 13-Aug HAH44 25.1 818.8 13-Aug HAH45 25.1 818.8 13-Aug HAH46 25.1 818.8 13-Aug HAH47 25.1 818.8 13-Aug HAH48 25.1 818.8 13-Aug HAH49 25.1 818.8 13-Aug HAH50 25.1 818.8 13-Sep HAI01 22 262.4 13-Sep HAI02 22 262.4 13-Sep HAI03 22 262.4 13-Sep HAI04 22 262.4 13-Sep HAI05 22 262.4 13-Sep HAI06 22 262.4 13-Sep HAI07 22 262.4 13-Sep HAI08 22 262.4 13-Sep HAI09 22 262.4 13-Sep HAI10 22 262.4 13-Sep HAI11 22 262.4 13-Sep HAI12 22 262.4 13-Sep HAI13 22 262.4 13-Sep HAI14 22 262.4 13-Sep HAI15 22 262.4 13-Sep HAI16 22 262.4 13-Oct HAJ01 14.4 159.6 13-Oct HAJ02 14.4 159.6 13-Oct HAJ03 14.4 159.6 13-Oct HAJ05 14.4 159.6

64

River Stage I Stage II Stage III Stage IV Water Dischare Avg Diam Avg Diam Avg Diam Avg Diam Temp Monthly Month/Yr Fish ID (mm) (mm) (mm) (mm) (C) Mean in ft3/s 13-Oct HAJ04 14.4 159.6 13-Oct HAJ06 14.4 159.6 13-Oct HAJ07 14.4 159.6 13-Oct HAJ08 14.4 159.6 13-Oct HAJ09 14.4 159.6 13-Oct HAJ10 14.4 159.6 13-Oct HAJ11 14.4 159.6 13-Nov HAK01 15.3 249.7 13-Nov HAK02 15.3 249.7 13-Nov HAK03 0.3225 0.5185 0.8034 15.3 249.7 13-Nov HAK04 15.3 249.7 13-Nov HAK05 15.3 249.7 13-Nov HAK06 15.3 249.7 13-Nov HAK07 15.3 249.7 13-Nov HAK08 15.3 249.7 13-Nov HAK09 15.3 249.7 13-Nov HAK10 15.3 249.7 13-Nov HAK11 15.3 249.7 13-Nov HAK12 15.3 249.7 13-Nov HAK13 0.3317 0.4287 15.3 249.7 13-Nov HAK14 0.3563 0.3825 15.3 249.7 13-Nov HAK15 15.3 249.7 13-Nov HAK16 15.3 249.7 13-Nov HAK17 15.3 249.7 13-Nov HAK18 15.3 249.7 13-Nov HAK19 15.3 249.7 13-Nov HAK20 15.3 249.7 13-Nov HAK21 15.3 249.7 13-Nov HAK22 15.3 249.7 13-Nov HAK23 15.3 249.7 13-Nov HAK24 15.3 249.7 13-Nov HAK25 15.3 249.7 13-Nov HAK26 15.3 249.7 13-Nov HAK27 15.3 249.7 13-Dec HAL01 0.362 0.401 13 1140 13-Dec HAL02 13 1140 13-Dec HAL03 13 1140 13-Dec HAL04 13 1140 13-Dec HAL05 13 1140

65

River Stage I Stage II Stage III Stage IV Water Dischare Avg Diam Avg Diam Avg Diam Avg Diam Temp Monthly Month/Yr Fish ID (mm) (mm) (mm) (mm) (C) Mean in ft3/s 13-Dec HAL06 13 1140 13-Dec HAL07 13 1140 13-Dec HAL08 0.4183 0.4034 13 1140 13-Dec HAL09 0.4092 0.413 13 1140 13-Dec HAL10 0.4703 0.4527 0.7018 13 1140 13-Dec HAL11 0.342 13 1140 13-Dec HAL12 0.3943 0.4047 13 1140 13-Dec HAL13 13 1140 13-Dec HAL14 0.355 0.429 13 1140 13-Dec HAL15 0.353 13 1140 13-Dec HAL16 13 1140 13-Dec HAL17 13 1140 13-Dec HAL18 0.3735 13 1140 13-Dec HAL19 0.4027 13 1140 13-Dec HAL20 13 1140 13-Dec HAL21 13 1140 13-Dec HAL22 13 1140 13-Dec HAL23 13 1140 13-Dec HAL24 13 1140 13-Dec HAL25 13 1140 13-Dec HAL26 13 1140 14-Jan HAA01 8 898.2 14-Jan HAA02 8 898.2 14-Feb HAB01 10.2 1248 14-Feb HAB02 10.2 1248 14-Feb HAB03 10.2 1248 14-Feb HAB04 10.2 1248 14-Feb HAB05 0.4411 0.495 10.2 1248 14-Feb HAB06 10.2 1248 14-Feb HAB07 10.2 1248 14-Feb HAB08 10.2 1248 14-Feb HAB09 10.2 1248 14-Feb HAB10 10.2 1248 14-Feb HAB11 10.2 1248 14-Feb HAB12 10.2 1248 14-Feb HAB13 10.2 1248 14-Feb HAB14 10.2 1248 14-Feb HAB15 10.2 1248 14-Feb HAB16 10.2 1248

66

River Stage I Stage II Stage III Stage IV Water Dischare Avg Diam Avg Diam Avg Diam Avg Diam Temp Monthly Month/Yr Fish ID (mm) (mm) (mm) (mm) (C) Mean in ft3/s 14-Mar HAC01 0.4294 0.4944 15.6 539 14-Mar HAC02 0.3533 0.4931 0.501 15.6 539 14-Mar HAC03 15.6 539 14-Mar HAC04 15.6 539 14-Mar HAC05 15.6 539 14-Mar HAC06 15.6 539 14-Mar HAC07 15.6 539 14-Mar HAC08 15.6 539 14-Mar HAC09 0.4014 0.6601 1.0303 1.1415 15.6 539 14-Mar HAC10 0.523 0.6595 0.6374 1.1228 15.6 539 14-Mar HAC11 0.5074 0.6387 0.8455 0.8628 15.6 539 14-Mar HAC12 0.5061 0.6114 0.8026 0.8627 15.6 539 14-Mar HAC13 15.6 539 14-Mar HAC14 0.461 0.522 0.788 15.6 539 14-Apr HAD01 17.2 1094 14-Apr HAD02 17.2 1094 14-Apr HAD03 0.4885 0.6205 1.0505 1.1922 17.2 1094 14-Apr HAD04 17.2 1094 14-Apr HAD05 0.4658 0.6049 1.0394 1.1116 17.2 1094 14-Apr HAD06 17.2 1094 14-Apr HAD07 17.2 1094 14-Apr HAD08 17.2 1094 14-Apr HAD09 17.2 1094 14-Apr HAD10 0.4612 0.6166 0.9104 1.0869 17.2 1094 14-Apr HAD11 17.2 1094 14-Apr HAD12 17.2 1094 14-Apr HAD13 17.2 1094 14-Apr HAD14 17.2 1094 14-Apr HAD15 0.4411 0.6263 0.7811 0.9006 17.2 1094 14-Apr HAD16 17.2 1094 14-Apr HAD17 17.2 1094 14-Apr HAD18 0.4924 0.6133 0.7733 0.9703 17.2 1094 14-Apr HAD19 17.2 1094 14-Apr HAD20 17.2 1094 14-May HAE01 0.4456 0.5574 0.9411 0.9939 21.1 542.2 14-May HAE02 0.4703 0.6049 0.9627 1.1167 21.1 542.2 14-May HAE03 21.1 542.2 14-May HAE04 21.1 542.2

67

River Stage I Stage II Stage III Stage IV Water Dischare Avg Diam Avg Diam Avg Diam Avg Diam Temp Monthly Month/Yr Fish ID (mm) (mm) (mm) (mm) (C) Mean in ft3/s 14-Mar HAC01 0.4294 0.4944 15.6 539 14-Mar HAC02 0.3533 0.4931 0.501 15.6 539 14-Mar HAC03 15.6 539 14-Mar HAC04 15.6 539 14-Mar HAC05 15.6 539 14-Mar HAC06 15.6 539 14-Mar HAC07 15.6 539 14-Mar HAC08 15.6 539 14-Mar HAC09 0.4014 0.6601 1.0303 1.1415 15.6 539 14-Mar HAC10 0.523 0.6595 0.6374 1.1228 15.6 539 14-Mar HAC11 0.5074 0.6387 0.8455 0.8628 15.6 539 14-Mar HAC12 0.5061 0.6114 0.8026 0.8627 15.6 539 14-Mar HAC13 15.6 539 14-Mar HAC14 0.461 0.522 0.788 15.6 539 14-Apr HAD01 17.2 1094 14-Apr HAD02 17.2 1094 14-Apr HAD03 0.4885 0.6205 1.0505 1.1922 17.2 1094 14-Apr HAD04 17.2 1094 14-Apr HAD05 0.4658 0.6049 1.0394 1.1116 17.2 1094 14-Apr HAD06 17.2 1094 14-Apr HAD07 17.2 1094 14-Apr HAD08 17.2 1094 14-Apr HAD09 17.2 1094 14-Apr HAD10 0.4612 0.6166 0.9104 1.0869 17.2 1094 14-Apr HAD11 17.2 1094 14-Apr HAD12 17.2 1094 14-Apr HAD13 17.2 1094 14-Apr HAD14 17.2 1094 14-Apr HAD15 0.4411 0.6263 0.7811 0.9006 17.2 1094 14-Apr HAD16 17.2 1094 14-Apr HAD17 17.2 1094 14-Apr HAD18 0.4924 0.6133 0.7733 0.9703 17.2 1094 14-Apr HAD19 17.2 1094 14-Apr HAD20 17.2 1094 14-May HAE01 0.4456 0.5574 0.9411 0.9939 21.1 542.2 14-May HAE02 0.4703 0.6049 0.9627 1.1167 21.1 542.2 14-May HAE03 21.1 542.2 14-May HAE04 21.1 542.2

68

River Stage I Stage II Stage III Stage IV Water Dischare Avg Diam Avg Diam Avg Diam Avg Diam Temp Monthly Month/Yr Fish ID (mm) (mm) (mm) (mm) (C) Mean in ft3/s 14-May HAE05 0.4287 0.5724 0.9593 1.0524 21.1 542.2 14-May HAE06 0.4729 0.6582 0.8666 1.0511 21.1 542.2 14-May HAE07 0.458 0.6211 0.9828 1.0615 21.1 542.2 14-May HAE08 21.1 542.2 14-May HAE09 21.1 542.2 14-May HAE10 21.1 542.2 14-May HAE11 21.1 542.2 14-May HAE12 21.1 542.2 14-May HAE13 0.4664 0.627 0.8764 0.9607 21.1 542.2 14-May HAE14 21.1 542.2 14-May HAE15 21.1 542.2 14-May HAE16 21.1 542.2 14-May HAE17 0.4482 0.6263 0.9749 1.0316 21.1 542.2 14-May HAE18 21.1 542.2 14-May HAE19 0.4346 0.5776 0.8601 0.9913 21.1 542.2 14-May HAE20 0.5249 0.6192 0.8457 0.9351 21.1 542.2 14-May HAE21 21.1 542.2 14-May HAE22 21.1 542.2 14-May HAE23 21.1 542.2 14-May HAE24 21.1 542.2 14-May HAE25 21.1 542.2 14-May HAE26 21.1 542.2 14-May HAE27 21.1 542.2 14-May HAE28 0.5152 0.6393 0.8889 0.9851 21.1 542.2 14-May HAE29 0.5035 0.6075 0.8993 1.0511 21.1 542.2 14-May HAE30 21.1 542.2 14-May HAE31 0.4554 0.6445 0.9697 1.0609 21.1 542.2 14-May HAE32 0.4996 0.6458 0.96 1.1428 21.1 542.2 14-May HAE33 21.1 542.2 14-May HAE34 21.1 542.2 14-May HAE35 21.1 542.2 14-May HAE36 21.1 542.2 14-May HAE37 21.1 542.2 14-May HAE38 0.4684 0.6296 0.9914 1.0388 21.1 542.2 14-May HAE39 0.4716 0.6549 0.9789 1.0765 21.1 542.2 14-May HAE40 21.1 542.2 14-May HAE41 21.1 542.2 14-May HAE42 0.4742 0.6257 0.8889 1.018 21.1 542.2 14-May HAE43 21.1 542.2

69

River Stage I Stage II Stage III Stage IV Water Dischare Avg Diam Avg Diam Avg Diam Avg Diam Temp Monthly Month/Yr Fish ID (mm) (mm) (mm) (mm) (C) Mean in ft3/s 14-Jun HAF01 0.344 0.3949 0.6608 24.1 765.6 14-Jun HAF02 24.1 765.6 14-Jun HAF03 0.471 0.6679 0.8321 1.0787 24.1 765.6 14-Jun HAF04 24.1 765.6 14-Jun HAF05 24.1 765.6 14-Jun HAF06 24.1 765.6 14-Jun HAF07 24.1 765.6 14-Jun HAF08 24.1 765.6 14-Jun HAF09 0.4469 0.6549 0.7635 0.9344 24.1 765.6 14-Jun HAF10 24.1 765.6 14-Jun HAF11 0.417 0.4677 0.6621 0.9665 24.1 765.6 14-Jun HAF12 24.1 765.6 14-Jun HAF13 0.4255 0.599 0.7193 0.8202 24.1 765.6 14-Jun HAF14 24.1 765.6 14-Jun HAF15 24.1 765.6 14-Jun HAF16 0.5113 0.6757 0.8273 24.1 765.6 14-Jun HAF17 24.1 765.6 14-Jun HAF18 24.1 765.6 14-Jun HAF19 24.1 765.6 14-Jun HAF20 24.1 765.6 14-Jun HAF21 24.1 765.6 14-Jun HAF22 24.1 765.6 14-Jun HAF23 24.1 765.6 14-Jun HAF24 24.1 765.6 14-Jul HAG01 0.4716 0.5639 0.9418 1.0901 24.5 219.8 14-Jul HAG02 0.3878 0.4918 0.6322 0.8431 24.5 219.8 14-Jul HAG03 0.3824 0.5587 0.8684 24.5 219.8 14-Jul HAG04 24.5 219.8 14-Jul HAG05 24.5 219.8 14-Jul HAG06 24.5 219.8 14-Jul HAG07 24.5 219.8 14-Jul HAG08 0.326 0.401 0.6933 0.8823 24.5 219.8 14-Jul HAG09 24.5 219.8 14-Jul HAG10 24.5 219.8 14-Jul HAG11 24.5 219.8 14-Jul HAG12 24.5 219.8 14-Jul HAG13 24.5 219.8 14-Jul HAG14 24.5 219.8 14-Jul HAG15 24.5 219.8

70

River Stage I Stage II Stage III Stage IV Water Dischare Avg Diam Avg Diam Avg Diam Avg Diam Temp Monthly Month/Yr Fish ID (mm) (mm) (mm) (mm) (C) Mean in ft3/s 14-Jul HAG16 24.5 219.8 14-Jul HAG17 24.5 219.8 14-Jul HAG18 24.5 219.8 14-Jul HAG19 24.5 219.8 14-Jul HAG20 24.5 219.8

71

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