THE BACTERIAL COMMUNITY OF A FRESHWATER SPONGE, RADIOSPONGILLA CEREBELLATA: A COMPARISON OF TERMINAL

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (T-RFLP) AND 16S RRNA CLONE LIBRARY METHODS

by

Andrew Stephen Mobley

Robert W. Thacker, PhD Committee Chair Asim K. Bej, PhD

Julie B. Olson, PhD

A THESIS

Submitted to the graduate faculty of The University of Alabama at Birmingham,

in partial fulfillment of the requirements for the degree of Master of Science

BIRMINGHAM, ALABAMA

2010

ii

THE BACTERIAL COMMUNITY OF A FRESHWATER SPONGE, RADIOSPONGILLA CEREBELLATA: A COMPARISON OF TERMINAL

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (T-RFLP) AND 16S RRNA CLONE LIBRARY METHODS

Andrew Stephen Mobley

DEPARTMENT OF BIOLOGY

ABSTRACT

Although most sponges are found in marine environments, some are also

found in freshwaters. Sponges harbor diverse communities of sponge-associated

bacteria, and the study of the relationship between host sponge and sponge-

associated bacteria may demonstrate the existence of symbiotic relationships

which benefit both the host sponge and its bacterial symbionts. We examined the

bacterial community associated with the freshwater sponge, Radiospongilla

cerebellata using terminal restriction fragment length polymorphism (T-RFLP)

and Clone Library Construction (CLC) methods. Although T-RFLP allows us to

rapidly assess the diversity of the bacterial community associated with freshwater

sponges, it does not provide the highest level of taxonomic resolution. However,

when T-RFLP results are compared with traditional clone libraries, it is possible

to enhance the taxonomic specificity of the T-RFLP method. In this investigation,

T-RFLP and CLC analyses demonstrated that there are notable differences

between the bacterial community associated with hatched freshwater sponges

and that of their intact and crushed gemmules. Freshwater sponges and their

gemmules harbor a diverse community of bacteria which varies in its species

composition, diversity, and evenness at different stages of their lifecycles. A total

iii

of 14 major bacterial lineages were identified with T-RFLP and CLC analyses,

however each method identified some bacterial lineages not identified by the

other. The clone library method identified 7 of these bacterial lineages, while the

T-RFLP method identified 10 bacterial lineages. Forty-two unique bacterial

species were identified in the hatched gemmule clone library, with 31 of these

matching T-RFLP profiles from the hatched gemmules. Analysis of similarity

calculations were used to produce Bray-Curtis similarity dendrograms and Multi-

Dimensional Scaling plots to illustrate relationships among the three gemmule

treatments. Rarefaction curves estimated the T-RF diversity among the three

gemmule treatments. The traditional diversity indices of the Shannon Index (H’)

and Pielou's evenness index (J’) demonstrated that hatched gemmules harbor a

bacterial community that is less diverse and less even than those of the intact

and crushed gemmules These findings suggest that the bacterial community

associated with freshwater sponges may include host-specific bacterial species,

but further investigation is necessary to identify potential host-specific symbionts.

iv

DEDICATION

This thesis is dedicated to my precious wife, Elizabeth. Without her constant

support, encouragement, and occasional gentle prodding, it simply never could

have been.

v

ACKNOWLEDGEMENTS

It would have been impossible to complete this work without the support,

training, guidance, encouragement and efforts of many people. First, I would like

to sincerely thank my mentor, Dr. Robert Thacker for encouraging me to further

develop the values, abilities, and critical thinking skills necessary for one to

become a good scientist. By sharing his knowledge and experience, he helped

me to learn from both my mistakes as well as my successes. I would like to thank

my committee members, Dr. Asim K. Bej, and Dr. Julie Olson, both models

of good scientists, for sharing their time, expertise and knowledge to assist me

throughout this experience. I would also like to thank Dr. Christina Diaz and

Dr. Sally Leys for their help in teaching me the important foundations of sponge

biology and taxonomy. I am indebted to Dr. Stephen Watts, Dr. Ken Marion, and

Dr. Robert Fisher for their support in making it possible for me to attain an

education at UAB. I would also like to thank Maria Salazar for her assistance in

the CFAR sequencing lab, and all the graduate students at UAB who have

become my friends and colleagues. This work has been supported by the

Caroline P. Ireland research scholarship.

vi

TABLE OF CONTENTS

Page

ABSTRACT .......................................................................................................... iii DEDICATION ....................................................................................................... v ACKNOWLEDGMENTS ....................................................................................... vi LIST OF TABLES ................................................................................................ vii LIST OF FIGURES ............................................................................................. viii INTRODUCTION .................................................................................................. 1 MATERIALS AND METHODS .............................................................................. 6 RESULTS ........................................................................................................... 13 DISCUSSION ..................................................................................................... 19 LIST OF REFERENCES .................................................................................... 26 FIGURES .......................................................................................................... 29 TABLES .......................................................................................................... 49

APPENDICES

A T-RFLP Relative Abundance Matrices .............................................. 52

vii

LIST OF TABLES

Table 1 Number of bacterial species ubiquitous and unique to hatched gemmules ................................................................................................ 49 2 Major bacterial divisions identified in T-RFLP and clone library

analyses from hatched gemmules ........................................................... 49

3 Traditional diversity indices for each restriction enzyme treatment .......... 50

4 Comparison of Clone library and T-RFLP results .................................... 51

viii

LIST OF FIGURES

Figure Page

1 Adult specimen of Radiospongilla cerebellata from Shades Creek .......... 29 2 Gemmules of Radiospongilla cerebellata ................................................. 29 3 Hatched Radiospongilla cerebellata gemmules ....................................... 30

4 Megasclere and gemmosclere spicules of Radiospongilla cerebellata ............................................................................................... 30 5 Mean number of T-RF’s observed for each restriction enzyme Treatment……………………………………………………………………….31 6 Total number of unique T-RF’s identified for each gemmule and restriction enzyme treatment .................................................................... 31 7 Mean Shannon index of diversity for each gemmule treatment by restriction enzyme .................................................................................... 32 8 Mean Evenness for each gemmule treatment by restriction enzyme ....... 32

9 Bray-Curtis similarity dendrogram of relationships among gemmule treatments for the BstUI restriction enzyme ............................. 33 10 Bray-Curtis similarity dendrogram of relationships among gemmule treatments for the HaeIII restriction enzyme ............................ 33 11 Bray- Bray-Curtis similarity dendrogram of relationships among gemmule treatments for the MspI restriction enzyme .............................. 34 12 Multi-dimensional Scaling similarity plot of the BstUI restriction enzyme treatment ................................................................... 35 13 Multi-dimensional Scaling similarity plot of the HaeIII restriction enzyme treatment .................................................................... 35

ix

14 Multi-dimensional Scaling similarity plot of the MspI restriction enzyme treatment .................................................................... 36 14 Multi-dimensional Scaling similarity plot of combined data for all enzyme treatments .................................................................................. 36 16 Rarefaction curve for BstUI treated intact gemmules ............................... 37

17 Rarefaction curve for BstUI treated crushed gemmules .......................... 37

18 Rarefaction curve for BstUI treated hatched gemmules .......................... 38

19 Combined rarefaction curve for all BstUI treated gemmules .................... 38

20 Natural log transformation of rarefaction curves for the BstUI restriction enzyme .......................................................................... 39 21 Rarefaction curve for HaeIII treated intact gemmules .............................. 40

22 Rarefaction curve for HaeIII treated crushed gemmules .......................... 40

23 Rarefaction curve for HaeIII treated hatched gemmules .......................... 41

24 Combined rarefaction curves for all HaeIII treated gemmules ................. 41

25 Natural log transformation of rarefaction curves for the ........................... 42 HaeIII restriction enzyme 26 Rarefaction curve for MspI treated intact gemmules ................................ 43

27 Rarefaction curve for MspI treated crushed gemmules ........................... 43

28 Rarefaction curve for MspI treated hatched gemmules ........................... 44

29 Combined rarefaction curves for all MspI treated gemmules ................... 44

30 Natural log transformation of rarefaction curves for the MspI restriction enzyme ........................................................................... 45 31 Neighbor-Joining phylogenetic tree showing genetic distances among β-proteobacteria clones retrieved from the hatched gemmule clone library and nearest matching Genbank sequences ........................ 46 32 Neighbor-joining phylogenetic tree showing genetic distances among γ-proteobacteria clones retrieved from the hatched gemmule clone library and nearest matching Genbank sequences ........................ 47

x

33 Neighbor-joining phylogenetic tree showing genetic distances among α-proteobacteria clones retrieved from the hatched gemmule clone library and nearest matching Genbank Sequences ........ 47 34 Neighbor-joining phylogenetic tree showing genetic distances among Bacteriodetes clones from the hatched gemmule clone library and nearest matching Genbank sequences ........................ 48 35 Percent composition of major taxonomic groups of bacteria identified in the hatched sponge clone library ......................................... 48

1

INTRODUCTION

Although the vast majority of sponge species are known from marine

environments, some are known to inhabit freshwater lakes, streams, and rivers.

These freshwater sponges share many characteristics with their marine

counterparts, but have several important adaptations to the freshwater

environment. Marine sponges and freshwater sponges have distinctly different

lifecycles. Unlike freshwater sponges, marine sponges live in a relatively stable

environment and are not subjected to intense seasonal environmental changes

such as temperature extremes, anoxia, and desiccation (Loomis et al. 1996,

Pennak et al., 1989, Reiswig, Miller 1998). While marine sponges reproduce

sexually and may have life spans up to 2300 years (McMuray et al., 2008),

freshwater sponges are annuals which reproduce asexually by producing

gemmules. These gemmules are small packages of specialized thesocyte cells

protected by a thick protective layer of collagen fibers and specialized

gemmosclere spicules (Frost et al, 2001). During the winter, gemmules remain in

a dormant state known as diapause (Loomis et al. 1996; Reiswig, Miller 1988)

until spring and early summer when favorable environmental conditions such as

an increase in water temperature induce them to hatch and grow into adult

sponges (Loomis et al. 1996; Frost et al., 2001). Sponges grow throughout the

summer months and begin to produce gemmules in late summer and early fall in

response to environmental stimuli. As the water temperature decreases in the

2

fall, the adult sponge dies, and the dormant gemmules either disperse in the

water currents or remain attached to their substrate until the lifecycle repeats

again in the spring.

Both freshwater and marine sponges are known to harbor diverse

bacterial communities including photosynthetic algae and cyanobacteria as well

as a wide range of heterotrophic bacteria (Webster et al., 2001, Gernert et al.,

2005). Some of these bacterial symbionts may be generalists found both in the

sponge and its surrounding environment; however some have been shown to

exist as specialist inhabitants of marine sponges (Thacker, Starnes 2002; Taylor

et al. 2004). Mutualist relationships between sponges and sponge-associated

bacteria are thought to exist; however the mechanism of these relationships has

not yet been identified (Thacker et al., 2003). Previous studies have identified

unique species of sponge-associated bacteria and cyanobacteria in marine

sponges (Hill et al. 2005, Thacker et al. 2003). Cyanobacterial sponge-

associated bacteria have been demonstrated to benefit the host sponge by

increasing its growth rate compared to sponges whose cyanobacterial symbionts

were shaded from sunlight (Erwin et al. 2008).

Two mechanisms have been proposed for the transmission of bacterial

symbionts between individual sponges or succeeding generations of sponges. In

the horizontal transmission or environmental acquisition model, bacterial

symbionts are transferred from one individual to another indirectly through the

environment. The association between the light-producing bacterial symbiont

Vibrio fischeri and its host, the squid Euprymna scolopes, is an example of

horizontal transmission in which the squid acquires its symbionts from the

3

environment (Wei and Young, 1989). In the vertical transmission model, bacterial

symbionts are directly transferred from parent to offspring. Vertical transmission

of sponge associated symbionts has been confirmed for two marine sponges

Corticium sp. and Ircinia felix (Sharp, 2007, Schmitt et al., 2007). In both of these

studies, microscopy revealed the presence of bacteria packaged within the

larvae of these two species. It has been also been reported that some sponge

species may utilize a combination of both horizontal and vertical transmission

mechanisms. (Taylor et al., 2007).

To date, no studies have been conducted to assess transmission

mechanisms for sponge-associated microbes in freshwater sponges. Because

these sponges reproduce asexually by producing gemmules which can be

hatched and manipulated under controlled conditions (Funayama et al., 2005),

freshwater sponges may further elucidate the association between sponges and

their bacterial symbionts. Several studies have shown that the exterior surface of

gemmules can be sterilized with hydrogen peroxide and/or sodium hypochlorite

prior to hatching to eliminate bacterial contamination. (Funayama et al., 2005,

Strekal and McDiffett, 1974). Although these studies did not specifically

investigate sponge-associated micro-organisms, their methods suggest that

sterilization of the exterior surface of the gemmule could be utilized to isolate

vertically transmitted sponge-associated bacteria located within the gemmule

from microbes of environmental origin located on the surface of the gemmules.

Clone library construction (CLC) and DGGE analysis based on 16S

ribosomal RNA have previously been utilized to characterize the bacterial

community of both marine and freshwater sponges (Magnusson, 2005, Erwin,

4

2007, Olson et al. 2005, Gernert et al., 2005). Direct sequencing of clone libraries

generates the highest degree of taxonomic resolution; however it is very time and

resource intensive. Terminal Restriction Fragment Length Polymorphism analysis

(T-RFLP) is a method in which PCR amplified 16S rRNA gene segments are

labeled with a fluorescent primer and then digested with restriction enzymes (Liu

et al. 1997). The resulting products are then run through a sequencer to

determine the length of the terminal restriction fragments. Although T-RFLP can

be used to rapidly assess a diverse bacterial community, it sacrifices the high

degree of taxonomic resolution obtained by traditional clone library methods in

order to obtain results both rapidly and economically. However, it is possible to

utilize online applications to compare T-RFLP results with predicted T-RF’s of a

clone library to accurately identify known species of interest, thus increasing the

taxonomic resolution of the method (Kent et al. 2003, Shyu et al. 2007, Szubert

et al. 2007).

At least four species of freshwater sponges are found in the Cahaba River

and Shades Creek: Radiospongila cerebellata, Ephydatia muelleri,

Corvospongilla becki, and one additional unidentified species. These encrusting

sponges are found attached to the underside of rocks which are exposed to

flowing water. Radiospongilla cerebellata and Ephydatia muelleri appear very

similar and cannot be distinguished in the field, however their spicules and

gemmules are morphologically distinct. Microscopic observations of gemmule

sections,megascleres, and gemmoscleres, are necessary to properly identify

these species (Figures 1-4). Radiospongilla cerebellata forms flat encrusting

patches of soft tissue and produces many yellow/orange gemmules scattered

5

throughout the sponge tissue. Ephydatia muelleri forms similar flat encrusting

patches of tissue with a more abrasive texture and gemmules that are smaller

and lighter in color.

The objectives of this study are: (1) Collect freshwater sponges and their

gemmules from the Cahaba River and Shades Creek, (2) Confirm species

identification from spicule preparations, (3) Determine a suitable hatching

medium for germinating gemmules under controlled laboratory conditions, (4)

Characterize sponge-associated bacterial community of freshwater sponge

gemmules using both CLC and T-RFLP methods.

The hypotheses for this study are: (1) The exterior surface of freshwater

sponge gemmules can be sterilized without harming their ability to hatch, (2) The

bacterial community of gemmules is distinct from that of hatched gemmules, (3)

The bacterial community of hatched gemmules is less diverse than that of intact

and crushed gemmules, (4) Some bacterial symbionts of freshwater sponges are

packaged within dormant gemmules and may be transmitted vertically from adult

sponge to gemmules, (5) Sponge-associated bacteria identified in clone library

sequences will be present in T-RF profiles.

6

METHODS

Collection, Preparation, and Identification of Freshwater Sponges

Freshwater sponges (Radiospongilla cerebellata) were collected from the

Cahaba River and Shades Creek in September and October of 2006. Gemmules

were removed from the sponge tissue, washed with Milli-Q water autoclaved for

30 minutes at 121°C, and stored in bottled Evian spring water (autoclaved for 30

minutes at 121°C) for at least 3 months at 4°C to simulate conditions gemmules

would experience in the field (Loomis et al., 1996). Additional specimens for long-

term preservation were collected in the fall of 2007 and preserved in either

ethanol or RNA-Later™ (Ambion, Inc.). Sponge tissue was digested in bleach to

isolate spicules for microscopic identification with published dichotomous keys

(Penney and Racek, 1968, Pennak et al., 1989).

Hatching of Gemmules in EV-50 Media.

A mineral “M” medium was prepared according to the method described

by previous studies (Rasmont, 1961). This medium was determined to be

unsuitable as it was not possible to completely dissolve its ingredients. Water

quality analysis reports for Evian brand spring water (Evian) demonstrated that it

contained similar concentrations of most of the minerals dissolved in the “M”

medium, and an alternative medium, “ EV-50”, consisting of 50% bottled Evian

mineral water and 50% Milli-Q water was used. Gemmules were removed from

7

refrigerated storage at 4°C and hatched in the dark at ambient lab temperature

(~20°C). Gemmules were successfully hatched in sterile 24-well Falcon multi-well

plates, 1.5ml microcentrifuge tubes, or Petri dishes. For sterility testing and T-

RFLP analysis, gemmules were hatched in 1.5ml microcentrifuge tubes. For

photography purposes, gemmules were hatched in Petri dishes.

Gemmule Sterilization Study

The exterior surfaces of gemmules were treated for 5 minutes in 1% H2O2,

1% sodium hypochlorite for 1 minute, or 10ul DNase for one minute and then

rinsed with sterile water. As a control, gemmules were also treated with Milli-Q

water autoclaved at 121°C for 30 minutes. 16S Ribosomal RNA was amplified

from the treated gemmules according to the PCR amplification protocol, and

sterilization was determined by the presence or absence of bacterial 16S

ribosomal RNA. Gemmules were then hatched according to the hatching protocol

to determine if the sterilization methods affected their ability to hatch.

DNA extraction and PCR amplification

Genomic DNA consisting of DNA from freshwater sponge and associated

bacteria was extracted from intact gemmules, crushed gemmules, and hatched

gemmule sponge tissue using the Promega Wizard SV Genomic DNA

Purification system (Promega). Universal bacterial primers Eco8F forward primer

5'-AGAGTTTGATCTGGCTCAG-3’ (Reysenbach et al. 1994) and 1392R reverse

primer 5’-ACGGGCGGTGTGTAC-3’ (Ferris et al., 1996) were used to amplify a

partial sequence of the bacterial 16S rRNA gene. PCR Reactions intended for T-

8

RFLP analysis used the hexachlorofluorescein labeled forward primer 8F-Hex,

while reactions intended for clone library analysis used the unlabeled Eco8F

forward primer. Each 50ul PCR reaction contained 15pmol 8F-Hex, 10pmol

1392R, 0.2mM of each dNTP, 1X MasterTaq PC Buffer (Eppendorf), 1X

TaqMaster (Eppendorf), 3.75mM MgCl2, and 2 units MasterTaq DNA polymerase

(Eppendorf). Reactions were conducted in a thermocycler with the following

conditions: 5 min denaturing @ 85°C, then 35 cycles of 45 seconds at 94°C, 60

seconds at 55°C, and 90 seconds at 72°C. PCR products were purified in a 1.5%

agarose gel and then cleaned using the Promega Wizard SV Gel Clean-Up

System. For each sample, PCR products from 3 separate reactions were

combined, and DNA concentrations were measured using a ND-1000 NanoDrop

spectrophotometer.

Purified PCR products were digested with BstUI, MspI, and HaeIII

restriction enzymes. These enzymes were selected because previous studies of

sponge associated bacteria indicated that they would report a maximum number

of T-RFLP fingerprinting fragments (Erwin et al., 2008, Engebretson et al., 2003).

For HaeIII and MspI, each reaction contained 400ng PCR product, 0.5ul BSA,

5.0ul 10x buffer, 2ul (2 units) enzyme, and H2O to bring final volume to 50ul.

Reactions were incubated at 37°C for 8 hours. For BstUI, each reaction

contained 400ng PCR product, 5ul 10x buffer, 2ul (2 units) enzyme, and H2O to

bring final volume to 50ul. Reactions were incubated at 60°C for 8 hours.

9

T-RFLP Analysis

Samples were precipitated overnight at -20°C with 5ul 3M sodium acetate

and 110ul ethanol at -20°C. Samples were then spun in a microcentrifuge at full

speed for 15 minutes at 4°C, rinsed with 500ul 70% ethanol, and dried in a

SpeedVac centrifuge until completely void of water. 10ul formamide and 0.5ul

GeneScan 500 TAMRA standard or 0.5ul GeneScan 600-LIZ standard were

added to each dried sample. Samples were placed in a thermocycler at 94°C for

2 minutes, cooled on ice for 2 minutes, and then vortexed and spun in a

centrifuge. Samples were run on an AB1377 automated sequencer or an ABI

3130xl Genetic Analyzer. The size in base pairs of the T-RF’s was determined by

comparison against the standard using Peak Scanner software version 1.0

(Applied Biosystems).

Clone Library Construction

A clone library was constructed using the Promega pGEM-T Easy Vector

System II (Promega) following the manufacturer’s suggested protocol. To each

0.5ml PCR tube, 5ul 2X ligation buffer, 0.5ul plasmid vector, 3.5ul PCR product,

1ul T4 Ligase was added. The positive control substituted 2ul control DNA and

1.5ul Milli-Q water autoclaved for 30 minutes at 121°C, and the background

control substituted autoclaved Milli-Q water for the PCR product. Ligations were

incubated overnight at 4°C. For each transformation, 4ul ligation reaction and

50ul competent cells were mixed in a 1.5ul microcentrifuge tube and placed on

ice for 20 minutes. Each tube was heated at 42°C for 45 seconds and then

placed on ice for 2 minutes. 950ul SOC media was added to each tube prior to

10

incubation at 37°C for 1.5 hours. Forty ul 2% bromo-chloro-indolyl-

galactopyranoside, 25ul 100mM ITPG, and 100ul transformed cells were added

to 1.5ul microcentrifuge tubes. 100ul or 65ul of this mixture was plated on Luria

broth/ampicillin plates and incubated at 37’C for 16-24 hours. 100 clones were

selected and placed in 1.5ul microcentrifuge tubes containing 1000ul Luria broth

and 4ul 2.5% ampicillin and incubated at 37°C overnight. Plasmids were

harvested and purified for sequencing with the Qiagen QIAprep Miniprep

purification system (Qiagen) according to the manufacturer’s microcentrifuge

protocol. Each of the 100 clones were digested with the restriction enzymes

HaeIII and MspI. For each enzyme, the reaction contained 400ng PCR product,

0.5ul BSA, 5.0ul 10x buffer, 2ul (2 units) enzyme, and autoclaved Milli-Q water to

bring final volume to 50ul. Reactions were incubated at 37°C for 8 hours.

Restriction enzyme digest products were separated on a 1.5% agarose gel.

Restriction enzyme patterns which appeared to be unique were identified and 55

clones were selected for sequencing.

Phylogenetic Identification of T-RFs and Clone Library Sequences

Sequences obtained from the clone library were compared to highly

similar sequences obtained from a blast search in Genbank. A neighbor joining

phylogenetic tree consisting of 55 16S r-RNA sequences and 42 Genbank

matches was constructed using Mega 4 software build number 4026 (Tamura et

al., 2007).

T-RFs profiles from the hatched gemmules were analyzed with the

phylogenetic assignment tool (PAT) (Kent el al., 2003) to identify possible

11

species matches. The T-RF profiles for all three enzymes were compared to a T-

RF database of 42,452 high quality (>1200bp) 16-S gene sequences. This

database of T-RF profiles was created by an insilco enzyme digest of the MiCAIII

(Microbial Community Analysis III) (Shyu et al. 2007) RDP database of 322,864

high quality 16S rRNA gene sequences (>1200bp) using the primers 8f and

1392r.

Data Analysis

T-REX software (Culman et al., 2009) was used to process, align, and

standardize the raw Genescan Peak Scanner files to produce abundance and

presence-absence matrices for further analysis. Primer 6 (Clarke et al., 2006)

software was used to compute traditional diversity indices (S, H, J) and to

produce Bray-Curtis similarity matrices based upon a 4th root transformation of

the matrices. The mean for each group was compared by ANOVA. Analysis of

similarity (ANOSIM) compared the three similarity matrices. MDS plots and

dendrograms were produced to assess similarity among the three experimental

treatments. Rarefaction curves from presences/absence matrices and percent

coverage estimates were calculated using Estimate-S software version 8.2.0

(Collwell, R.K., 2006). Rarefaction curves were compared among gemmule

treatments using an analysis of covariance (ANCOVA) of log-transformed data.

Raw T-RF Genescan profiles for each restriction enzyme were analyzed

using the Phylogenetic Assignment Tool (PAT) which compares T-RF profiles

with a database of known bacterial sequences and reports possible species

matches for the T-RF profiles. A large general database of 42,452 sequences

12

using 8f and 1392r primers was generated from the provided RDP database of

high quality 16S sequences using the virtual digest function of the online

Microbial Community Analysis III application (MiCAIII) (Shyu et al., 2007). A

second database of 42 hatched gemmules clone library sequences was

generated with an in-silico restriction digest using the online Restriction Enzyme

Mapping Application (REMA) (Szubert et al. 2007). T-RF profiles were compared

with both the RDP database and the hatched gemmule clone library database.

13

RESULTS

Species Identification and Gemmule Sterilization/Hatching Study

Three species collected locally in the Cahaba River and Shades Creek

were identified: Radiospongilla cerebellata, Ephydatia muelleri, and

Corvospongilla becki. A fourth species could not be identified to the genus level,

as there were no gemmules or gemmoscleres present to confirm identification

(Figures 1-4).

Gemmules treated with both H2O2 and sodium hypochlorite did not

produce any PCR products and were therefore considered sterilized, but none of

the other treatments were effective for sterilization of the outer coating of the

gemmules; however, gemmules treated with all combinations of H2O2 and

sodium hypochlorite failed to hatch in EV-50 medium. Unsterilized gemmules

placed in EV-50 medium began hatching at day 4 with visible spicules observed

outside gemmules. Visible sponge tissue was observed at day 11 and the

sponges continued growth for a total of 15 days prior to harvest (figure 3).

Because it was not possible to sterilize the exterior surface of the gemmules

without negatively affecting their ability to hatch, it was not possible to determine

if sponge-associated bacteria were contained within the gemmule by hatching

them under sterile conditions.

14

T-RFLP Analysis

With the restriction enzyme BstUI, 77 T-RF’s were identified in total with

55 found in intact gemmules, 54 in crushed gemmules, and 38 in hatched

gemmules. With the restriction enzyme HaeIII, 130 T-RF’s were identified in total

with 91 of them found in intact gemmules, 94 in crushed gemmules, and 51 in

hatched gemmules. With the restriction enzyme MspI, 62 T-RF’s were identified

in total with 38 of them found in intact gemmules, 29 in crushed gemmules, and

32 in hatched gemmules. For each of the three enzyme treatments, there were

fewer T-RFs identified in the hatched gemmules than in either the intact or

crushed gemmules (Figure 5). There were no significant differences among the

mean number of T-RF’s per sample for each of the gemmule treatments and

restriction enzymes (Figure 4)

With the restriction enzyme BstUI, 23 bacterial species were ubiquitous

and 9 were unique to the hatched gemmules. With the restriction enzyme HaeIII,

19 bacterial species were ubiquitous and 12 were unique to the hatched

gemmules. With the restriction enzyme MspI, 16 bacterial species were

ubiquitous and 10 were unique to the hatched gemmules (Table 1).

Traditional Diversity indices: Shannon Index and Evenness

The Shannon diversity index (H’) (Figure 7) and evenness (J’) (Figure 8)

were calculated for each enzyme treatment. For each enzyme, the hatched

15

gemmules had lower diversity and lower evenness than either the intact or the

crushed gemmules (Table 3).

Analysis of Similarity, Bray Curtis similarity dendrograms and MDS Plots

For the BstUI treated samples, analyses of similarity showed a significant

difference between the bacterial communities of hatched gemmules and both the

intact gemmules (p=0.008) and crushed gemmules (p=0.008). For the MspI

treated samples, the only significant difference observed was between the

hatched and intact gemmules (p=0.016). No significant differences were

observed between any of the gemmule treatments in the HaeIII treated samples.

For all three enzymes, there were no significant differences observed between

the intact and crushed gemmules.

Bray Curtis similarity dendrograms provided visual support for the

ANOSIM statistical calculations. The BstUI dendrogram (Figure 9) clearly

distinguishes the hatched gemmule treatment from the intact gemmule and

crushed gemmule treatments, with the hatched gemmules occupying a distinct

separate branch of the dendrogram. Dendrograms for HaeIII (Figure 10) and

MspI (Figure 11) were less informative due to the lack of statistically significant

differences observed between the three gemmule treatments.

Two dimensional MDS (Multi-Dimensional Scaling) plots illustrated the

differences between the three gemmule treatment groups for each of the

enzymes with low stress values (0.05-0.13) indicating that these plots are

consistent with the ANOSIM calculations and Bray Curtis similarity dendrograms.

16

The BstUI plot (Figure 12) clearly distinguishes the hatched gemmule treatment

from the intact and crushed gemmule treatments. MDS plots for HaeIII

(Figure 13) and MspI (Figure 14) illustrate some distinction between treatment

groups, but lack of significance between groups is also clearly presented. Data

for all three enzymes was combined into a single matrix and an MDS plot created

from this data set clearly illustrates distinct differences among the bacterial

communities of intact, crushed and hatched gemmules (Figure 15).

Rarefaction Curves

Rarefaction curves for all three enzyme treatments illustrated the

differences in the rate of species accumulation for each of the gemmule

treatments. For the BstUI (Figures 16-19) and MspI (Figures 26-29) enzyme

treatments, there were no significant differences observed between the number

of species accumulated in the intact, crushed and hatched gemmules. For the

HaeIII enzyme treatment (Figures 21-24), hatched gemmules treated with HaeIII

accumulated significantly fewer species than both the intact and crushed

gemmules (p<0.05).

Natural log transformations of the rarefaction curves for each of the three

restriction enzymes allowed a comparison of the slope and y-intercepts of the

three gemmule treatments (Figures 20, 25, 30). Rarefaction curves were

compared among treatments using an analysis of covariance (ANCOVA) of log-

transformed data. ANCOVA analysis reported that the rates of species

accumulation among the three gemmule treatments were significantly different

for both the BstUI (p<0.001) and MspI (p<0.001) restriction enzymes. However

17

the rates of species accumulation among treatments were not significantly

different for the HaeIII restriction enzyme (P=0.120).

Percent Coverage of T-RFLP Profiles

Percent coverage of species in the community was estimated with the

Chao2 estimator for each enzyme and gemmule treatment to determine how

effectively the T-RFLP method estimates of the number of sponge associated

bacteria. Percent coverage ranged from 30.0% for the HaeIII intact gemmules to

91.5% for BstUI crushed gemmules (Table 3).

Clone Library and Taxonomic Identification of Bacteria

55 clones of 16S r-RNA genes were sequenced from the hatched

gemmule clone library. A blast search in Genbank identified 42 unique bacteria

species with highly similar matches to those from the clone library. Neighbor-

joining phylogenetic trees were constructed from the 55 clone library sequences

and 42 Genbank matching sequences (Figures 31-34). The most abundant major

bacterial taxa identified were alpha-proteobacteria, beta-proteobacteria, gamma-

proteobacteria, and bacteriodetes. Acidobacteria, Nitrospirae, and

Gemmatimonadetes were also present (Figure 35).

For the hatched gemmule T-RF profiles, the Phylogenetic Assignment

Tool (PAT) identified 724 unique potential species matches from the T-RF

database of bacterial species, with multiple possible bacterial species matches

for each of the individual T-RF profiles. For the enzyme BstUI, 55.5% of all T-

18

RF’s were matched with at least one bacterial species from the database. For the

enzyme HaeIII, 71.1% of all T-RF’s were matched with at least one bacterial

species from the database. For the enzyme MspI, 87.9% of all T-RF’s were

matched with at least one bacterial species from the database. When the

T-RFLP profiles were compared with the T-RF database of clone library

sequences, PAT identified 31 possible sequence matches representing 25

unique clones from the 42 unique clone library sequences. Nine of the clone

library sequences were matched by PAT using a database of all three restriction

enzymes, and 22 additional sequences were matched using databases of two

restriction enzymes (Table 4).

19

DISCUSSION

Although previous studies reported that it was possible to surface-sterilize

gemmules of different species than those used in this study without affecting their

ability to hatch (Funayama et al. 2005, Strekal and McDiffett 1974), all

combinations of sterilization treatments were unsuccessful in this study.

However, these previous studies relied on traditional bacterial culture techniques

which are not reliable for culturing most bacterial species (Hugenholtz et al.,

2008). Because it is not possible to culture most sponge-associated bacteria, the

PCR technique used in this study was able to identify the presence of bacteria on

treated gemmules which eluded previous sterilization methods. Although it was

possible to sterilize the outer surface of gemmules with a combined treatment of

hydrogen peroxide and sodium hypochlorite, none of the treatments sterilized the

gemmules without negatively affecting their ability to hatch. Because it was not

possible to hatch surface-sterilized gemmules, this study was could not

determine if bacterial symbionts were packaged inside gemmules, and therefore

transmitted vertically from adult sponges to their gemmules.

In this investigation, T-RFLP analysis demonstrated that there are notable

differences between the bacterial community associated with hatched freshwater

sponges and that of their intact and crushed gemmules. For the BstUI and HaeIII

enzyme treatments, the total number of species identified in the hatched

gemmules was lower than the total number of species identified in both the intact

20

and crushed gemmules. For the MspI enzyme treatment, the total number of

species indentified was similar among the hatched sponges and intact and

crushed gemmules. In addition, the MspI enzyme treatment identified fewer

species than both the BstUI and HaeIII enzyme treatments, indicating that this

enzyme was least effective for differentiating different species within the bacterial

community of freshwater sponges and their gemmules (Figure 6). This is

supported by a previous study in which the restriction enzyme MspI identified

fewer sponge-associated bacterial species than the restriction enzyme HaeIII

(Erwin, 2007 dissertation), and suggests that this enzyme is less informative than

the other enzymes used in this study for characterizing the bacterial community

of freshwater sponge gemmules.

The traditional diversity indices of the Shannon Index (H’) and

Pielou's evenness index (J’) demonstrated that hatched gemmules harbor a

bacterial community that is less diverse and less even than those of the intact

and crushed gemmules. This indicates that hatched sponges harbor fewer

bacterial species than their gemmules, and that some bacterial species

associated with hatched gemmules dominate the bacterial community, while

these same species are less prevalent in both the intact and crushed gemmules

prior to hatching. Previous feeding studies demonstrated that sponges were able

to distinguish between their bacterial symbionts and bacterial species found in

seawater, retaining and consuming the seawater bacteria while expelling their

symbionts. (Wilkinson et al., 1984, Wehrl et al. 2007). Our study supports these

previous studies, as the decrease in the diversity of the bacterial community

associated with hatched gemmules suggests that these freshwater sponges were

21

also selectively feeding upon the bacterial species which were not recognized as

symbionts.

These findings suggest that the bacterial community associated with

freshwater sponges may include host-specific bacterial species which are

favored by the sponges after hatching from their gemmules (Erwin et al. 2007,

Hill et al. 2005, Thacker et al. 2003, Taylor et al. 2004, Webster et al. 2004). This

is in contrast with previous studies which demonstrated that different species of

sponges harbor similar communities regardless of geography or taxonomic

relationship (Hentschel et al. 2002, Olson et al. 2005). In addition, the results of

this study are in contrast with those of another which reported that the bacterial

community of freshwater sponges resembled that of the lake from which they

were collected (Gernert et al., 2005). We found that the bacterial community

associated hatched sponges was less diverse and more even than that of the

gemmules from which they hatched. In both marine and freshwater sponges,

sponge-specific photosynthetic cyanobacteria have been identified (Erwin et al.

2007) and have been shown to benefit the sponge by improving the sponge’s

growth rate (Erwin et al. 2008). This type of mutualism may also be present

between freshwater sponges and their associated bacterial community, but

further investigation is necessary to identify potential host-specific symbionts.

Rarefaction curves created from T-RFLP profiles illustrated the differences

among the number of species accumulated for the three gemmule treatments.

For the BstUI and HaeIII enzyme treatments, the rarefaction curves for each

gemmule treatment demonstrated that the intact and crushed gemmules

accumulated more bacterial species than the hatched gemmules. For the MspI

22

enzyme treatment, the three rarefaction curves for each gemmule treatment were

similar and there was no clear distinction among the intact, crushed, or hatched

gemmules. ANCOVA analysis of natural log transformations of the rarefaction

curves for BstUI and MspI restriction enzymes indicated that the rate of species

accumulation was significantly different among the three gemmule treatments.

A clone library constructed from 55 clones identified 42 unique species

associated with the hatched freshwater sponges, but Chao2 diversity estimates

based upon T-RFLP data consistently reported that the number of sponge

associated bacterial species may be much higher than the clone library results

suggest. This may be because T-RFLP methods are capable of identifying rare

species that elude detection through traditional clone library methods. Also,

further sampling to increase the size of the clone library would likely identify more

rare species which were not identified in this study.

A total of 14 major bacterial lineages were identified with T-RFLP and CLC

analyses, however each method identified some bacterial lineages not identified

by the other. The clone library method identified 7 of these bacterial lineages,

while the T-RFLP method identified 10 bacterial lineages. This is likely due to T-

RFLP’s capability of identifying rare species which might otherwise elude

traditional clone library methods. Both methods identified alpha-proteobacteria,

beta-protobacteria, gamma-protetobacteria, nitrospira, and bacteriodetes

lineages.

When the T-RFLP profiles were compared to a large T-RF database of

known bacterial species profiles, the Phylogenetic Assignment tool (PAT)

identified over 700 potential species matches for bacteria associated with the

23

hatched sponges. In contrast, T-RFLP profiles recovered from the hatched

sponges identified a maximum of 51 T-RF’s in the HaeIII treated samples, and as

few as 32 in the MspI treated samples. This discrepancy is likely due to the

relatively low taxonomic resolution of T-RFLP analyses because T-RFLP only

reports the location of the site where the restriction enzyme cuts the sequence

but does not report any other information about the sequence. In contrast,

sequences derived from clone libraries produce the highest taxonomic resolution,

as they report the entire sequence which makes it possible to distinguish closely

related species that T-RFLP methods would identify as a single species. When

T-RFLP profiles of the hatched sponges were compared to a much smaller

internal databases of 42 bacterial species from the hatched sponge clone library,

PAT matched 31 T-RFLP profiles with sequences from the clone library,

demonstrating that it possible to rapidly identify sponge-associated bacterial

species of interest by comparing T-RF profiles to sponge-associated bacterial

species previously isolated from a clone library. Thus, it is possible to increase

the taxonomic resolution while maintaining the high throughput of the T-RFLP

method by directly matching T-RFLP profiles with known clone library sequences

using a combination of online analysis applications such as MicaIII, PAT, and

REMA.

There are several inherent biases which may affect the results of T-RFLP

analyses. Because T-RFLP is PCR based, it is possible that the efficiency of

amplification may vary among different species present in the bacterial

community. This amplification bias could cause some species to appear more or

less dominant in the community than they really are. There is also a discrepancy

24

between the predicted T-RF length determined by an in-silico digest of clone

library sequences and that which is reported by T-RFLP analysis, and it is

therefore necessary to compensate for this by defining a bin size of several base

pair for each predicted T-RF in order to match clone library sequences with T-RF

profiles (Kent et al., 2003). Additionally, T-RFLP makes the assumption that each

unique T-RF identified represents a single unique bacterial species (Marsh,

1999); however comparisons of T-RF profiles and an in-silico digest of clone

library sequences demonstrated that multiple bacterial species in the clone

library shared the same T-RF profile. Thus, the T-RFLP method tends to

oversimplify the true diversity of the bacterial community.

There are many possibilities for future studies of these freshwater

sponges. Analysis of the bacterial community in the field over time as well as

additional laboratory culturing of gemmules could provide much informative data

to further our understanding of the association between bacteria and their

freshwater sponge hosts. This study identified 2 sponge species not previously

reported in Shades Creek and the Cahaba River and correctly named three

species that had not previously been identified. The species identified were

Radiospongilla cerebellata, Ephydatia muelleri, and Corvospongilla becki. A

fourth species could not be identified to the genus level. Additional sampling of

Alabama watersheds may reveal additional species which have not previously

been reported. The use of fluorescent in situ hybridization methods to observe

the presence or absence of bacteria within the sponge gemmules could confirm

the vertical transmission hypothesis. Examination of gemmules using either

25

transmission or scanning electron microscopes could also identify the presence

of bacteria both inside the gemmules and on their exterior surfaces.

Freshwater sponges and their gemmules were found to harbor a diverse

community of bacteria which varies in its species composition, diversity, and

evenness at different stages of their lifecycles. Observation of these diverse

communities can be rapidly assessed with the use of T-RFLP methods which

provide significant information about the bacterial communities, and T-RFLP data

can be matched with that of traditional clone libraries to identify species of

interest more efficiently than previous methods allowed.

Freshwater sponges are particularly of interest for future research, as they

can be obtained locally and it is possible to store dormant gemmules for

extended periods of time and hatch them under controlled conditions when

desired. This is not possible with marine sponges, and thus freshwater sponges

are an ideal model organism for the study of sponges and their bacterial

communities under controlled conditions. Metazoan life often depends upon the

association between complex organisms and bacterial communities. Because

sponges are the earliest known metazoans, the elucidation of relationships

between sponges and their bacterial communities may help us to understand the

nature of symbiotic relationships at the most basal level, leading to a greater

understanding of symbiotic relationships between more complex species.

26

REFERENCES

Clarke, KR, Gorley, RN, (2006) PRIMER v6: User Manual/Tutorial. PRIMER-E, Plymouth. Colwell, R.K. (2006) EstimateS, Statistical Estimation of Species Richness and Shared Species from Samples Version 8. url <purl.gclc.org/estimates>. Culman, S.W.,et al. (2009) T-REX: Software for the Processing and Analysis of T-RFLP data. BMC Bioinformatics 10:171. Erwin, P. M., Thacker R.W. (2007) Incidence and identity of photosynthetic symbionts in Caribbean coral reef sponge communities. Journal of the Marine Biological Association of the United Kingdom 87: 1683-1692. Erwin, P. M., and Thacker R. W. (2008) Phototrophic nutrition and symbiont diversity of two Caribbean sponge-cyanobacteria symbioses. Marine Ecology Progress Series 362: 139-147. Frost, T.M., Reiswig, H.M., and Ricciardi, A. (2001) Chapter 4: Porifera. Ecology and Classification of North American Freshwater Invertebrates, Second Edition: p. 97-133. Academic Press. 2001. Funayama, N. et al. (2005) Isolation of the choanocyte in the freshwater sponge, Ephydatia fluvaitilis and its lineage marker, EF annexin. Develop. Growth Differ. 47: 243-253. Genert, C. et al. (2005) Micribial Diversity of the Freshwater Sponge Spongilla lacustris. Microbial Ecology 50: 206-212. Hentschel, U., et al. (2002) Molecular evidence for a uniform microbial community in sponges from different oceans. Appl Environ Microbiol 68: 4431-4440. Hill, M., Hill, A., Lopez, N., and Harriott, O. (2005) Sponge-specific bacterial symbionts in the Caribbean sponge, Chondrilla nucula (Demospongiae, Chondrosida). Marine Biology 148: 1221-1230.

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Hodges, T.W., Olson, J.B. (2008) Molecular Comparison of Bacterial Communities within Iron-Flocculent Mats Associated with Submarine Volcanoes along the Kermadec Arc. Applied Environmental Microbiology 75: 1650-1657. Loomis, S.H. et al. (1996) Metabolism of Gemmules from the Freshwater Sponge Eunapiusfragilis During Diapause and Post-Diapause States. Biological Bulletin 191: 385-392. Kent, A.D., Smith, D.J., Benson, B.J., and Triplett, E.W. (2003) Web-based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Polymorphism Profiles of Microbial Communities. Appl Environ Microbiol 69: 6768-6776. Liu, W., Marsh, T.L., Cheng, H., and Forney, L.J. (1997) Characterization of microbial diversity by determining terminal restriction fragment polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63: 4516-4522. Magnusson, T. (2005) Master’s Thesis: A Comparison of Microbial Communities Associated with the Freshwater Sponges Radiospongilla crateriformis and Eunapius fragilis. Loomis, S.H. et al. (1996) Metabolism of Gemmules from the Freshwater Sponge Eunapius fragilis During Diapause and Post-Diapause States. Biological Bulletin 191: 385-392. Marsh, T.L. (1999) Terminal restriction fragment polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. Curr Opin Microbiol 2: 323-327. Olson, J.B., and McCarthy, P.J. (2005) Associated bacterial communities of two deepwater sponges. Aquat Microb Ecol 39: 47-55. Osborne, C.A., et al. (2006) New threshold and confidence estimates for terminal restriction fragment length polymorphism analysis of complex bacterial communities. Appl Environ Microbiol 72: 1270-1278. Pennak, R.W. Fresh-Water Invertebrates of the United States: Protozoa to Mollusca 3rd Edition: p. 91-109. John Wiley & Sons, Inc., New York. 1989. Penney, J. T. and Racek A.A. (1968) Comprehensive revision of a worldwide collection of freshwater sponges (Porifera : Spongillidae). Bull. U. Nat. Mus. 272: 1-184. Reiswig, H.M., Miller, T.L. (1988) Freshwater Gemmules Survive Months of Anoxia. Invertebrate Biology 117: 1-8.

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Sharp, K.H. et al. (2007) Vertical Transmission of Diverse Microbes in the Tropical Sponge Corticum sp. Applied and Environmental Microbiology 73: 622-629. Schmitt, S. et al. (2007) Vertical Transmission of a Phylogenetically Complex Microbial Consortium in the Viviparous Sponge Ircinia felix. Applied Environmental Biology 73:7 2067-2078. Shyu, C., et al. (2007) MiCA: A Web-Based Tool for the Analysis of Microbial Communities Based on Terminal-Restriction Fragment Length Polymorphisms of 16S and 18S rRNA Genes. Journal of Microbial Ecology 53:562-570. Strekal, T.A., McDiffett W.F. (1974) Factors Affecting the Germination, Growth, and Distribution of the Freshwater Sponge Spongilla fragilis leidy (Porifera). Biological Bulletin 146: 267-278. Szubert, J., Et al. (2007) REMA: A computer-based mapping tool for analysis of restriction sites in multiple DNA sequences Journal of Microbiological Methods. 69: 411-413. Taylor, M.W., et al. (2004) Host specificity in marine sponge-associated bacteria, and potential implications for marine microbial diversity. Environmental Microbiology 6: 121-130. Thacker, R.W., and Sarnes, S. (2003) Host Specificity of the symbiotic cyanobacterium Oscillatoria spongeliae in marine sponges, Dysidea spp. Marine Biology 142: 643-648. Webster, N.S., Wilson, K., Blackall, L.L., and Hill, R.T. (2001) Phylogenetic diversity of the bacterial communities associated with the marine sponge, Rhopaloeides odorabile. Applied Environmental Microbiology 67: 434-444. Webster, N.S.,et al. (2004) Diverse Microbial Communities Inhabit Antarctic Sponges. Environ Microbiol 6: 288-300. Wehrl, M. et al. (2007) Bacterial Uptake by the Marine Sponge Aplysina aerophoba. Microbial Ecology 53: 355-365. Wilkinson, C.R. et al. (1984) Marine Sponges Discriminiate between Food Bacteria and Bacterial Symbionts: Electron Microscope Radioautography and in-situ Evidence. Proceedings of the Royal Society of London. Series B, Biological Sciences 220:1221 519-528.

29

FIGURES

Figure 1. Adult specimen of Radiospongilla cerebellata from Shades Creek.

Figure 2. Gemmules of Radiospongilla cerebellata. Gemmules are visible within disintegrating adult sponge tissue collected 10/15/2008.

30

Figure 3. Hatched Radiospongilla cerebellata gemmules. Hatched gemmules were grown under sterile laboratory conditions.

Figure 4. Megascleres and gemmosclere spicules of Radiospongilla cerebellata

31

Figure 5. Mean number of T-RF’s observed for each restriction enzyme treatment. For all restriction enzymes, there were no significant differences observed among the three gemmules treatments.

Figure 6. Total number of unique T-RF’s identified for each gemmule and restriction enzyme treatment.

0

5

10

15

20

25

30

35

40

45

BstUI HaeIII MspI

mean intact

mean crushed

mean hatched

Sp

ecie

s O

bse

rve

d

Restriction Enzyme

55

91

38

54

94

2938

51

32

77

130

62

BstUI HaeIII MspI

T-RFLP Profiles

Intact Gemmules Crushed Gemmules Hatched Gemmules Total T-RF's

32

Figure 7. Mean Shannon index of diversity for each gemmule treatment by restriction enzyme. Error bars indicate standard error of the means.

Figure 8. Mean Evenness for each gemmule treatment by restriction enzyme. Error bars indicate standard error of the means.

0

0.5

1

1.5

2

2.5

3

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4

BstUI HaeIII MspI

mean intact

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an

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BstUI HaeIII MspI

mean intact

mean crushed

mean hatched

Eve

nn

ne

ss

Restriction Enzyme

33

Figure 9. Bray-Curtis similarity dendrogram of relationships among gemmule treatments for the BstUIrestriction enzyme.

Figure 10. Bray-Curtis similarity dendrogram of relationships among gemmule treatments for HaeIII restriction enzyme.

34

Figure 11. Bray-Curtis similarity dendrogram of relationships among gemmule treatments for MspI restriction enzyme treatments.

35

Figure 12. Multi-dimensional Scaling similarity plot of the BstUI restriction enzyme treatment.

Figure 13. Multi-dimensional Scaling similarity plot of the HaeIII restriction enzyme treatment.

36

Figure 14. Multi-dimensional Scaling similarity plot of the MspI restriction enzyme treatment.

Figure 15. Multi-dimensional Scaling similarity plot of combined data for all enzyme treatments.

37

Figure 16. Rarefaction curve for BstUI treated intact gemmules. Dotted lines indicate 95% confidence values.

Figure 17. Rarefaction curve for BstUI treated crushed gemmules. Dotted lines indicate 95% confidence values.

0

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30

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50

60

70

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70

0 1 2 3 4 5

Spe

cie

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BstUI Crushed Gemmules

38

Figure 18. Rarefaction curve for BstUI treated hatched gemmules. Dotted lines indicate 95% confidence values.

Figure 19. Combined rarefaction curves for all BstUI gemmule treatments. Hatched gemmules accumulated fewer species than both intact and crushed gemmules.

0

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BstUI Rarefaction Curves

Intact

Crushed

Hatched

39

Figure 20. Natural log transformation of rarefaction curves for the BstUI restriction enzyme. The three gemmule treatments had similar slopes, but the Y-intercept of the hatched gemmules was lower than that of the intact and crushed gemmules. ANCOVA analysis reported that the rates of species accumulation among treatments were significantly different (p<0.001).

0

0.5

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0 0.5 1 1.5 2

ln s

pe

cie

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ln replicate

BstUI

Intact

Crushed

Hatched

40

Figure 21. Rarefaction curve for HaeIII treated intact gemmules. Dotted lines indicate 95% confidence values.

Figure 22. Rarefaction curve for HaeIII treated crushed gemmules. Dotted lines indicate 95% confidence values

0

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60

80

100

120

0 1 2 3 4 5

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120

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HaeIII Crushed Gemmules

41

Figure 23. Rarefaction curve for HaeIII treated hatched gemmules. Dotted lines indicate 95% confidence values.

Figure 24. Combined rarefaction curves for all HaeIII gemmule treatments. Hatched gemmules accumulated fewer species than both intact and crushed gemmules.

0

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HaeIII Rarefaction Curves

Intact

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42

Figure 25. Natural log transformation of rarefaction curves for the HaeIII restriction enzyme. The three gemmule treatments had similar slopes, but the Y-intercept of the hatched gemmules was lower than that of the intact and crushed gemmules. ANCOVA analysis reported that the rates of species accumulation among treatments were not significantly different. (p=0.120).

0

0.5

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ln s

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HaeIII

Intact

Crushed

Hatched

43

Figure 26. Rarefaction curve for MspI treated intact gemmules. Dotted lines indicate 95% confidence values.

Figure 27. Rarefaction curve for MspI treated crushed gemmules. Dotted lines indicate 95% confidence values.

0

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44

Figure 28. Rarefaction curve for MspI treated hatched gemmules. Dotted lines indicate 95% confidence values.

Figure 29. Combined rarefaction curves for all MspI gemmule treatments. No significant difference was observed between the three treatments.

0

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MspI Rarefaction Curves

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45

Figure 30. Natural log transformation of rarefaction curves for the MspI restriction enzyme. The three gemmule treatments had similar slopes, but the Y-intercept of the intact was greater than that of the hatched and crushed gemmules. ANCOVA analysis reported that the rates of species accumulation among treatments were significantly different (p<0.001).

0

0.5

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0 0.5 1 1.5 2

ln s

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Intact

Crushed

Hatched

46

Figure 31. Neighbor-Joining phylogenetic tree showing genetic distances among Beta-proteobacteria clones retrieved from the hatched gemmule clone library and nearest matching Genbank sequences.

47

Figure 32. Neighbor-Joining bootstrap consensus phylogenetic tree showing genetic distances among Gamma-proteobacteria clones retrieved from the hatched gemmule clone library and nearest matching Genbank sequences.

Figure 33. Neighbor-Joining bootstrap consensus phylogenetic tree showing genetic distances among clones of Alpha-proteobacteria, Acidobacteria, Nitrospirae, and Gemmatimonadales retrieved from the hatched gemmule clone library and nearest matching Genbank sequences.

48

Figure 34. Neighbor-Joining bootstrap consensus phylogenetic tree showing genetic distances among Bacteriodetes clones retrieved from the hatched gemmule clone library and nearest mat ching Genbank sequences

Figure 35. Percent composition of major taxonomic groups of bacteria identified in the hatched sponge clone library. The community was dominated by Beta-proteobacteria, followed by Alpha-proteobacteria, Bacteriodetes, and Gamma-proteobacteria.

43%

13%

20%

18%

2%

2% 2%

Beta-Proteobacteria

Gamma-Proteobacteria

Alpha-Proteobacteria

Bacteroidetes

Acidobacteria

Nitrospirae

Gemmatimonadetes

49

TABLES

Table 1. Number of bacterial species ubiquitous and unique to hatched gemmules identified with T-RFLP analyses.

Restriction Enzyme Ubiquitous Unique to Hatched

Gemmules

BstUI 23 9

HaeIII 19 12

MspI 16 10

Table 2. Major bacterial divisions identified in T-RFLP and clone library analyses from hatched gemmules. T-RF’s were analyzed with the phylogenetic assignment tool (PAT). Clone library was assembled from 16S rRNA clones. Clone library construction of 42 sequences identified 7 major taxonomic groups of bacteria. T-RFLP identified 10 major taxonomic groups of bacteria.

Bacterial Division CLC T-RFLP

Acidobacteria X

Bacteroidetes X X

Chlamydiae/Verrucomicrobia X

Chloroflexi X

Firmicutes X

Gemmatimonadetes X

Nitrospira X X

Planctomycetes X

Proteobacteria

Alpha-proteobacteria

Beta-proteobacteria

Gamma-proteobacteria

X

X

X

X

X

X

Tenericutes X

50

Table 3. Traditional diversity indices for each enzyme treatment. Percent coverage estimates are based upon total T-RF’s observed and Chao 2 calculations. The Significance level of pair-wise comparisons for mean T-RF’s, Pair-wise comparisons of Shannon Index and Evenness reported by ANOVA are represented by A,B,C, with different letters indicating p-values <0.05.

Total T-RF’s

Observed

Mean T-RF’s Observed

(± SE)

Mean Shannon Index (H’)

(± SE)

Mean Evenness (J’)

(± SE)

Chao 2 Estimate

Percent Coverage

BstUI

Intact Gemmules 55 31.0 ± 6.10

A 3.00 ± 0.17

A 0.903 ± 0.017

A 65.5 84.0%

Crushed Gemmules 54 32.8 ± 7.43

A 2.98 ± 0.400

AB 0.934 ± 0.016

B 59 91.5%

Hatched Gemmules 38 20.6 ± 1.81

A 2.30 ± 0.047

B 0.767 ± 0.012

C 70 54.3%

p=0.293 p=0.127 p<0.001

HaeIII

Intact Gemmules 91 33.8 ± 5.05

A 2.97 ± 0.141

A 0.855 ± 0.014

A 233.6 39.0%

Crushed Gemmules 94 31.0 ± 7.96

A 3.03 ± 0.221

A 0.927 ± 0.013

B 231.3 40.6%

Hatched Gemmules 51 19.4 ± 2.76

A 2.17 ± 0.105

B 0.748 ± 0.043

C 60.5 84.9 %

p=0.205 p=0.004 p<0.001

MspI

Intact Gemmules 38 17.0 ± 2.12

A 2.51 ± 0.197

A 0.89 ± 0.028

A 51 74.5%

Crushed Gemmules 29 10.6 ± 2.46

AB 2.07 ± 0.269

A 0.94 ± 0.011

B 39.5 73.4%

Hatched Gemmules 32 9.6 ± 2.38

B 1.21 ± 0.219

B 0.55 ± 0.058

C 50.7 63.1%

p=0.088 p=0.005 p<0.001

51

Table 4. Comparison of Clone library and T-RFLP results displaying the closest Genbank match to each clone matched with at T-RF profile, predicted T-RF length based upon in-silico restriction digestion of clone library sequences, and percent abundance for matching T-RF profiles and clone library sequences. Nine clones were matched with the database of T-RF profiles of all three restriction enzymes. Twenty-two additional clones were matched with databases of T-RF profiles of two of the three restriction enzymes.

Clone

ID

Closest matching

Genbank

Ascension #

Predicted T-RF’s

BstUI HaeIII MspI

T-RFLP P Mean

Abundance

BstUI HaeIII MspI

Abundance in

Clone Library

Clone 5 DQ917256 198 212 483 0.2% 10.9% 56.2% 1.8%

Clone 29 EF653576 198 212 483 0.2% 10.9% 56.2% 1.8%

Clone 30 DQ664252 198 193 483 0.2% 0.8% 56.2% 3.6%

Clone 34 DQ664252 198 193 483 0.2% 0.8% 56.2% 3.6%

Clone 33 AF336360 198 193 483 0.2% 0.8% 56.2% 1.8%

Clone 8 AF236008 203 217 488 0.7% 24.0% 4.7% 1.8%

Clone 24 FN428786 203 217 488 0.7% 24.0% 4.7% 1.8%

Clone 40 EU104140 204 218 489 0.7% 21.0% 4.7% 1.8%

Clone 10 AF005179 384 248 485 24.4% 0.1% 56.2% 1.8%

Clone 2 EU861860 381 218 0.3% 21.0% 1.8%

Clone 11 EF019693 396 211 0.1% 10.9% 3.6%

Clone 28 EF019693 396 211 0.1% 10.9% 3.6%

Clone 20 AB504926 394 258 0.2% 1.5% 1.8%

Clone 3 AB308368 380 475 14.6% 8.4% 5.5%

Clone 16 AB308368 380 475 14.6% 8.4% 5.5%

Clone 39 AB308368 380 475 14.6% 8.4% 5.5%

Clone 6 FJ593908 380 475 14.6% 8.4% 1.8%

Clone 18 AY250110 389 490 0.4% 4.7% 1.8%

Clone 27 EU193055 388 489 0.4% 4.7% 1.8%

Clone 38 AY247063 389 490 0.4% 4.7% 1.8%

Clone 41 AY512611 389 490 0.4% 4.7% 1.8%

Clone 43 CP000774 214 456 0.1% 0.4% 1.8%

Clone 1 AM397630 217 488 24.0% 4.7% 3.6%

Clone 13 AM397630 217 488 24.0% 4.7% 3.6%

Clone 7 FJ687971 225 437 15.1% 0.6% 3.6%

Clone 35 FJ687971 190 436 2.3% 0.6% 3.6%

Clone 15 FJ517716 219 490 2.8% 4.7% 1.8%

Clone 19 AB452984 219 490 2.8% 4.7% 1.8%

Clone 25 FJ517699 219 490 2.8% 4.7% 1.8%

Clone 32 AJ422163 219 490 2.8% 4.7% 1.8%

Clone 37 FM201185 219 490 2.8% 4.7% 1.8%

52

APPENDIX I

T-RFLP Relative Abundance Matrices

BstUI T-RFLP Relative Abundance Matrix

TRF I 1 I 2 I 3 I 4 I 5 C 1 C 2 C 3 C 4 5 H 1 H 2 H 3 H4 H5

101G 2.63 1.80 1.51 2.27 3.83 0.00 3.18 1.99 1.27 3.12 0.00 0.00 0.00 0.00 0.00

102G 1.83 0.46 0.00 0.00 0.00 0.00 1.16 1.02 1.23 0.69 0.00 0.00 0.00 0.00 0.00

103G 0.00 0.49 0.65 1.22 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

104G 7.51 4.96 1.82 0.85 6.29 0.00 1.14 0.76 1.57 4.21 0.00 0.00 0.00 0.00 0.25

107G 0.00 0.63 0.70 0.54 0.00 0.00 0.00 0.55 0.49 0.00 0.00 0.00 0.00 0.00 0.00

114G 0.00 0.55 0.34 0.38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.36 0.00 0.00 0.00

128G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.45 0.00 0.00 0.00 0.00 0.00 0.00

137G 0.00 0.00 0.00 0.42 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

144G 0.00 0.00 0.00 0.00 5.91 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

190G 5.88 4.06 3.86 5.89 9.74 0.00 2.28 5.55 4.10 5.27 2.00 0.94 1.33 1.44 1.31

191G 0.00 0.00 0.00 0.00 0.00 0.00 0.96 1.66 1.79 0.00 2.16 0.92 1.19 1.50 0.92

193G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.00 0.00 0.00

194G 2.04 1.49 2.06 2.20 0.00 0.00 5.68 1.17 2.13 1.90 0.00 0.00 0.00 0.00 0.00

195G 3.47 2.33 2.00 2.70 4.83 0.00 4.79 5.00 4.36 3.79 2.63 16.31 14.30 5.02 14.96

196G 0.00 0.90 1.35 1.24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.76 9.70

197G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.93 0.00 0.00 0.00

53

200G 0.00 0.00 0.34 0.00 0.00 0.00 0.00 0.00 0.00 0.00 11.23 4.87 7.92 5.30 3.12

201G 3.45 4.21 7.15 3.81 0.00 0.00 1.15 1.57 2.15 2.61 5.16 2.37 2.00 2.57 1.85

203G 3.50 4.22 5.86 3.84 3.55 0.00 1.38 2.44 2.59 2.49 2.16 0.89 0.00 1.27 1.48

205G 0.00 0.00 0.00 0.47 0.00 0.00 0.00 1.04 0.50 0.61 0.00 0.00 0.00 0.00 0.00

207G 0.00 0.55 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

207G 0.00 0.53 1.09 0.64 0.00 0.00 1.10 0.80 0.92 0.95 0.00 0.00 0.00 0.00 0.00

209G 0.00 0.00 0.32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

211G 0.00 0.46 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

214G 0.00 0.00 0.00 0.00 0.00 0.00 1.66 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

215G 0.00 0.00 0.00 0.00 0.00 27.57 0.75 0.00 0.00 0.00 0.00 0.30 0.00 0.00 0.00

226G 0.00 0.00 0.00 0.00 0.00 0.00 1.66 0.00 0.00 1.47 0.00 0.00 0.00 0.00 0.00

230G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.11 0.00 0.00 0.00 0.00

232G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.45 0.00 0.00 0.00 0.00 0.00 0.00

234G 0.00 0.00 0.00 0.54 0.00 0.00 0.00 1.00 1.02 0.00 0.00 0.00 0.00 0.00 0.00

235G 0.00 0.00 0.65 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

240G 0.00 0.88 1.43 1.42 0.00 0.00 1.28 1.32 0.99 1.04 0.00 0.00 0.00 0.00 0.00

243G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.46 0.00 0.00 0.00 0.00 0.00 0.00

263G 0.00 0.00 0.00 0.58 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

292G 0.00 1.03 0.00 0.80 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

324G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.29 0.00 0.00 0.00 0.00

357G 9.50 7.27 5.84 8.61 12.50 25.88 3.89 9.64 8.25 7.65 2.24 1.53 2.92 3.22 2.24

357G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.61 1.46 2.09 0.00 1.48

360G 0.00 0.92 0.70 1.05 0.00 0.00 4.34 1.55 2.86 2.49 0.00 0.55 0.00 0.00 0.29

361G 3.44 2.55 1.82 2.48 0.00 0.00 7.24 3.65 3.63 3.12 1.12 0.65 1.07 1.20 0.49

362G 0.00 0.86 0.50 0.76 0.00 0.00 1.32 1.90 1.54 1.37 0.00 0.00 0.00 0.86 0.00

367G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.28 0.00 0.00 0.00 0.00

370G 0.00 1.27 0.66 0.54 0.00 0.00 0.00 1.74 1.09 1.21 0.00 0.00 0.00 0.00 0.00

371G 0.00 0.00 0.00 0.46 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

54

372G 0.00 0.00 0.00 0.00 0.00 0.00 1.07 0.84 0.69 0.73 0.00 0.00 0.00 0.00 0.00

373G 0.00 0.75 0.88 0.00 0.00 0.00 0.00 0.52 0.50 0.00 0.00 0.00 0.00 0.00 0.00

374G 0.00 0.00 0.00 0.77 0.00 0.00 0.67 0.83 1.25 1.62 0.00 0.00 0.00 0.00 0.00

376G 3.11 1.45 0.96 1.37 0.00 0.00 2.52 0.88 0.68 0.00 0.00 3.90 1.12 3.07 0.37

378G 3.51 3.57 4.08 3.54 4.49 0.00 2.93 3.31 3.55 2.11 0.00 0.00 0.00 0.00 0.00

379G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 18.54 19.41 7.72 25.71

380G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.51 0.00 0.00 0.00 0.00

384G 3.19 0.00 0.00 0.00 3.85 21.12 9.07 4.22 0.00 9.44 36.47 19.33 24.62 29.68 11.94

384G 6.49 9.61 17.20 10.42 5.67 0.00 0.00 5.46 7.91 0.00 0.00 0.00 0.00 0.00 0.00

386G 0.00 3.38 3.04 1.64 0.00 0.00 4.11 1.28 3.88 1.80 15.29 13.95 11.89 18.92 14.62

386G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.72 0.00 0.00 0.00 0.00 0.00 0.00 0.00

388G 13.51 7.50 7.81 5.01 18.20 0.00 4.68 2.30 3.29 6.19 0.00 0.64 0.00 0.67 0.55

391G 6.70 8.67 6.46 6.62 6.52 0.00 5.93 5.89 4.45 6.43 0.00 1.20 2.28 1.74 1.87

393G 0.00 0.00 0.00 1.66 0.00 0.00 0.00 0.50 0.00 0.00 0.00 0.00 0.00 1.09 0.00

396G 0.00 0.86 1.00 1.23 0.00 0.00 0.69 1.31 2.16 1.25 0.00 0.00 0.00 0.68 0.00

398G 0.00 0.00 0.00 0.00 0.00 0.00 3.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

399G 2.52 2.32 1.44 3.00 0.00 0.00 0.00 1.66 1.62 1.19 2.06 0.00 0.00 0.00 0.00

405G 0.00 0.00 1.76 0.00 0.00 0.00 1.21 0.65 0.93 0.00 0.00 0.00 0.00 0.00 0.00

406G 0.00 1.24 0.00 1.60 0.00 0.00 0.00 0.75 0.00 1.26 0.00 0.00 0.00 0.00 0.00

408G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.99 0.00 0.00 0.00 0.00 0.00 0.00

456G 10.40 7.95 7.16 9.88 14.60 25.44 3.09 10.02 8.79 8.82 0.00 0.00 5.82 7.13 4.55

457G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 8.59 3.80 0.00 0.00 0.00

459G 0.00 0.79 0.64 0.98 0.00 0.00 3.13 1.09 2.49 2.39 0.00 0.45 0.00 0.00 0.00

461G 3.81 3.16 1.83 2.63 0.00 0.00 5.36 3.44 3.49 3.33 0.00 0.40 0.00 1.33 0.35

462G 0.00 0.00 0.62 0.77 0.00 0.00 1.11 1.17 1.09 1.14 0.00 0.00 0.00 0.00 0.00

468G 0.00 0.61 0.00 0.44 0.00 0.00 0.00 1.45 0.82 0.91 0.00 0.38 0.00 0.00 0.00

471G 0.00 0.64 0.00 0.61 0.00 0.00 0.00 0.59 0.00 0.00 0.00 0.00 0.00 0.00 0.00

479G 0.00 1.70 1.62 1.34 0.00 0.00 1.68 2.50 2.76 1.72 0.00 0.36 0.00 0.00 0.00

55

490G 0.00 1.53 0.00 1.36 0.00 0.00 2.01 3.16 3.48 3.84 0.00 0.00 0.00 0.00 0.00

491G 0.00 0.00 1.78 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.22

495G 3.52 1.87 1.07 1.47 0.00 0.00 1.72 1.12 0.95 0.00 1.08 4.97 2.04 4.13 0.70

496G 0.00 0.00 0.00 0.00 0.00 0.00 1.01 0.00 0.00 1.79 0.00 0.00 0.00 0.00 0.00

498G 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.68 1.04

56

HaIII T-RFLP Relative Abundance Matrix

TRF I 1 I 2 I 3 I 4 I 5 C 1 C 2 C 3 C 4 C 5 H 1 H 2 H 3 H4 H 5

126Y 0.50 0.00 0.00 0.00 0.00 0.46 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

167Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.98 0.00 0.00

174Y 0.00 0.00 0.00 0.00 0.00 0.00 0.42 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

180Y 1.38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

181Y 0.00 0.69 0.00 0.51 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

181Y 0.71 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

184Y 0.78 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

185Y 0.00 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

185Y 0.00 0.00 0.00 0.00 0.00 0.55 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

187Y 0.00 1.51 1.41 1.36 0.00 0.00 1.12 2.16 0.00 0.00 0.00 0.00 0.00 0.00 0.00

189Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.11 0.00 0.65 0.00

190Y 0.00 0.00 0.00 0.00 0.00 1.02 0.00 0.00 0.00 0.00 0.00 0.00 11.14 0.00 0.00

191Y 6.20 0.00 0.00 0.00 0.00 5.62 0.00 0.00 0.00 0.00 1.74 0.95 10.76 1.72 0.00

193Y 0.00 3.84 2.63 6.20 3.19 0.00 0.00 7.45 7.74 7.15 1.26 0.00 1.39 0.00 1.31

194Y 0.00 3.24 1.83 0.00 4.61 0.00 8.57 8.34 0.00 8.09 0.00 0.00 0.00 0.00 0.00

195Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.29 0.00 0.00 0.00 0.00 0.00 0.00

197Y 0.67 1.16 0.00 0.00 0.00 0.80 1.22 2.59 0.00 0.00 0.00 0.81 1.54 0.39 1.80

198Y 1.71 0.00 0.00 0.00 0.00 1.40 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

199Y 0.00 2.70 1.33 2.90 1.26 0.00 1.82 0.00 1.80 0.00 0.00 0.00 0.00 0.00 0.00

200Y 0.00 0.00 0.00 0.00 0.00 0.29 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

201Y 0.93 0.00 0.00 0.00 0.00 1.07 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

202Y 0.00 0.00 0.00 0.76 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

203Y 0.00 1.64 1.15 1.57 1.76 0.00 2.97 0.00 5.47 0.00 0.00 0.00 0.00 0.00 0.00

207Y 1.88 0.00 0.00 0.00 0.00 1.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.45 0.00

208Y 4.02 0.00 0.00 0.00 0.00 3.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.14 0.00

57

209Y 0.00 5.48 7.21 9.18 8.54 0.00 0.00 4.46 3.32 5.60 0.00 6.47 0.00 0.00 0.00

210Y 0.00 0.00 0.00 0.00 0.00 0.00 4.25 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

211Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 19.42 0.00 0.00

211Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 29.69

212Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 24.35 0.00 0.00 0.42 0.00

213Y 0.00 0.00 0.00 0.00 0.00 1.84 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

214Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 29.45 0.00 42.94 0.00

215Y 12.16 0.00 0.00 0.00 0.00 8.16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 23.21 0.00

216Y 4.75 0.00 29.01 0.00 0.00 3.95 0.00 12.68 13.00 16.60 0.00 15.10 0.00 0.00 0.00

217Y 0.00 16.65 0.00 16.81 16.40 0.00 9.89 0.00 0.00 0.00 18.61 9.73 26.39 0.00 36.49

218Y 0.00 0.00 4.59 4.73 6.39 0.00 7.34 9.62 7.84 14.15 13.36 0.00 0.00 0.43 0.00

220Y 0.00 14.26 3.80 2.21 0.00 0.00 1.27 3.89 3.78 0.00 0.00 0.00 0.00 0.00 0.00

220Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10.27 0.00 9.65 0.00 8.43

221Y 8.85 0.00 0.00 0.00 0.00 8.03 0.00 0.00 0.00 0.00 0.00 2.21 0.00 2.80 0.00

222Y 2.35 0.00 0.00 0.00 0.00 3.66 0.00 0.00 0.00 0.00 0.00 2.87 0.00 2.82 0.00

224Y 0.00 8.51 7.36 12.38 11.41 0.29 10.51 8.61 8.37 10.11 3.85 0.00 3.10 0.00 1.69

225Y 0.00 0.00 0.00 0.00 0.00 0.88 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

226Y 0.62 3.76 3.14 2.47 6.04 0.48 4.37 7.08 8.37 12.88 2.11 0.00 2.51 0.00 1.79

228Y 0.00 0.61 0.00 0.38 0.00 0.37 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

229Y 0.71 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

231Y 0.00 0.00 0.00 0.00 1.09 0.00 0.53 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

232Y 0.00 0.00 0.00 0.00 0.00 0.00 1.08 0.00 2.20 0.00 0.00 0.00 0.00 0.00 0.00

234Y 0.00 2.39 1.91 2.63 1.58 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

236Y 0.93 0.00 0.00 0.00 0.00 1.26 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

238Y 0.81 0.00 0.00 0.00 0.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

241Y 0.00 1.71 1.26 1.57 1.29 0.00 3.29 0.00 1.61 0.00 0.00 0.00 0.00 0.00 0.00

243Y 1.51 0.00 0.00 0.00 0.00 1.54 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

244Y 0.00 0.59 0.00 0.51 0.00 0.00 1.13 0.00 1.07 0.00 0.00 0.00 0.00 0.00 0.00

58

246Y 0.00 0.00 0.00 0.29 0.00 0.32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

249Y 0.77 0.00 0.00 0.55 0.00 1.54 1.41 0.00 0.97 0.00 0.00 0.00 0.00 0.00 0.00

249Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.42 0.00

250Y 0.59 0.00 0.00 0.00 0.00 0.00 0.76 2.54 0.00 0.00 0.00 0.79 0.00 0.73 0.00

252Y 2.19 0.00 0.00 0.00 0.00 0.91 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

253Y 5.00 0.00 0.00 0.00 0.00 2.68 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

254Y 0.00 1.39 2.59 1.75 1.54 0.00 4.13 4.23 2.50 0.00 0.00 0.00 0.00 0.00 0.00

255Y 4.71 0.00 0.00 0.00 0.00 2.76 0.00 0.00 0.00 0.00 0.00 1.19 0.00 0.40 0.00

256Y 0.00 5.26 8.46 7.99 17.95 0.00 1.06 0.00 1.69 0.00 0.00 0.00 0.00 0.00 0.00

257Y 0.49 0.00 0.00 0.00 0.00 0.68 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

258Y 0.00 0.00 0.00 0.00 0.00 0.00 1.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

259Y 0.00 6.37 3.19 5.54 2.93 0.00 2.31 2.88 3.78 0.00 2.75 0.00 3.59 0.00 1.14

261Y 0.00 1.61 3.05 0.78 0.00 0.00 2.91 0.00 1.76 0.00 0.00 0.00 0.00 0.00 0.00

262Y 0.59 0.00 0.00 0.00 0.00 0.86 0.00 3.43 0.00 0.00 0.00 0.00 0.00 0.00 0.00

263Y 0.00 0.00 0.00 0.41 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

264Y 0.85 0.00 0.00 0.00 0.00 1.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

265Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.33 0.00 0.94 0.00

266Y 0.00 1.50 0.88 0.53 0.00 0.00 3.25 2.51 2.13 0.00 0.00 0.00 0.00 0.00 0.00

268Y 0.00 1.48 0.00 1.06 0.00 0.00 1.06 0.00 1.24 0.00 0.00 0.00 0.00 0.00 0.00

270Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.96 0.00 1.90 0.00 1.00

278Y 0.00 0.00 0.86 0.53 1.28 0.61 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

279Y 1.62 0.00 0.00 0.00 0.00 0.89 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

281Y 0.00 0.70 0.00 0.44 0.00 0.27 1.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

287Y 5.42 0.00 0.00 0.00 0.00 7.58 0.00 0.00 0.00 0.00 0.00 1.53 0.00 1.21 0.00

288Y 1.72 3.60 3.52 2.21 4.90 2.50 3.64 4.80 4.34 8.45 0.00 1.26 0.00 0.85 0.00

290Y 0.00 0.00 0.00 0.78 0.00 0.00 1.57 2.16 1.84 5.26 0.00 0.00 0.00 0.00 0.00

291Y 0.00 0.00 0.00 0.58 0.00 0.00 1.22 0.00 1.17 0.00 0.00 0.00 0.00 0.00 0.00

292Y 2.75 0.00 0.00 0.00 0.00 3.69 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

59

293Y 2.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.85 0.00 1.15 0.00

294Y 0.00 1.12 1.67 0.90 1.28 0.00 2.53 2.91 2.31 6.11 0.00 0.00 0.00 0.00 0.00

294Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.29 0.00 0.00 0.00 1.03

297Y 1.09 0.00 0.00 0.00 0.00 0.60 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

300Y 0.00 0.00 0.00 0.00 0.00 0.47 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

302Y 0.00 0.00 0.00 0.00 0.00 0.31 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

304Y 0.00 0.00 0.00 0.00 0.00 0.35 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.47 0.00

306Y 0.00 0.00 0.00 0.51 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

308Y 1.46 0.00 0.00 0.00 0.00 1.88 0.00 0.00 0.00 0.00 0.00 1.32 0.00 0.00 0.00

309Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.66 0.00 0.00 0.00

310Y 0.60 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.64 1.66 1.04 0.00

311Y 0.00 0.00 0.00 0.44 0.00 0.00 0.95 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

312Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6.49 0.00 0.00 0.00 5.80

313Y 0.00 0.00 0.00 0.32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.72 0.00 0.00

314Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.08 0.00 4.59 0.00

315Y 1.79 0.00 0.00 0.00 0.00 1.83 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

316Y 0.74 0.00 0.00 0.45 0.00 1.04 0.00 0.00 0.00 0.00 0.00 0.95 0.00 0.36 0.00

317Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.08 0.00 0.00 0.00 1.44

318Y 0.63 0.00 0.00 0.00 0.00 0.75 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

318Y 0.00 0.00 0.00 0.00 0.00 0.00 1.24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

320Y 0.00 0.71 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

321Y 0.00 0.00 0.00 0.00 0.00 0.00 0.91 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

322Y 0.00 0.00 0.00 0.00 0.00 0.44 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

322Y 1.28 0.00 0.00 0.00 0.00 0.66 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

324Y 0.00 0.00 0.00 0.00 0.00 0.44 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

326Y 0.00 0.90 0.00 0.43 0.00 0.00 0.94 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

328Y 1.56 0.00 0.00 0.00 0.00 1.94 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.47 0.00

330Y 0.00 0.95 1.90 2.09 2.51 0.00 2.06 5.46 3.03 5.60 0.00 0.00 0.00 0.00 0.00

60

370Y 0.00 1.80 2.16 1.36 2.57 0.00 1.66 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

371Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.09 0.00 0.00 0.00 0.00 0.00 0.00

372Y 2.59 0.00 0.00 0.00 0.00 4.12 0.00 0.00 0.00 0.00 0.00 0.87 0.00 1.22 0.00

376Y 0.00 0.00 0.00 0.00 0.00 0.00 0.70 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

376Y 0.92 0.00 0.00 0.00 0.00 2.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.35 0.00

390Y 0.00 0.00 1.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

391Y 0.00 0.00 0.00 0.78 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

393Y 0.99 0.00 0.00 0.00 0.00 1.05 0.00 0.00 0.00 0.00 1.83 0.00 0.00 0.00 0.00

394Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.19

394Y 0.00 0.00 2.69 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.21 0.00 0.00 0.00

395Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.59 0.00

397Y 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

399Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 7.05 7.98 0.00 6.13 4.11

400Y 3.39 0.00 0.00 0.37 0.00 2.19 0.00 0.00 0.00 0.00 0.00 0.00 4.25 0.00 2.08

401Y 0.00 0.00 0.00 0.00 0.00 1.70 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

401Y 0.00 2.55 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.63 0.00 2.11 0.00

402Y 0.00 0.00 0.00 0.86 0.00 0.00 2.26 2.19 2.33 0.00 0.00 0.00 0.00 0.00 0.00

403Y 0.97 0.00 0.00 0.00 0.00 1.46 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

405Y 0.59 1.31 1.38 1.10 1.45 0.00 1.52 0.00 0.97 0.00 0.00 0.00 0.00 0.00 0.00

406Y 1.11 0.00 0.00 0.00 0.00 2.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

420Y 0.00 0.00 0.00 0.00 0.00 0.83 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

61

MspI T-RFLP Relative Abundance Matrix

TRF ID I 1 I 2 I 3 I 4 I 5 C 1 C 2 C 3 C 4 5 H 1 H 2 H 3 H4 H 5

122Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.61 0.00 0.00 0.00

123Y 3.46 3.91 4.61 2.51 7.03 9.51 4.47 5.90 8.58 0.00 1.30 1.46 1.48 0.00 1.34

128Y 2.97 2.22 0.00 1.75 4.22 0.00 3.63 4.82 0.00 0.00 0.00 0.54 0.00 0.00 0.00

135Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.50 0.50 0.00 0.00 0.00

136Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.28 0.00 0.00

137Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.92 0.97 0.00 0.00 0.00

138Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.63 0.00 0.00

146Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.61 0.00 0.00 0.00

147Y 0.00 0.00 0.00 0.00 0.00 7.50 0.00 0.00 0.00 0.00 0.00 0.60 0.00 0.00 0.00

149Y 4.36 3.43 0.00 3.12 4.32 0.00 4.59 5.28 9.89 0.00 0.00 0.00 0.00 0.00 0.00

155Y 0.00 0.00 0.00 0.00 0.00 0.00 2.11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

159Y 0.00 0.00 0.00 1.97 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

170Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.45 0.00 0.00 0.00

172Y 0.00 0.00 0.00 2.10 4.59 0.00 1.85 0.00 5.80 0.00 0.00 0.00 0.00 0.00 0.00

198Y 2.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

199Y 0.00 0.00 0.00 0.00 0.00 5.92 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

201Y 1.92 0.00 0.00 0.00 0.00 0.00 0.00 3.42 0.00 0.00 0.00 0.00 0.00 0.00 0.00

203Y 0.00 0.00 4.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

204Y 5.02 4.55 0.00 3.89 7.31 0.00 3.38 6.71 7.12 0.00 0.00 0.00 1.06 0.00 0.00

211Y 0.00 0.00 0.00 0.00 2.59 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

252Y 0.00 1.93 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

273Y 0.00 0.00 0.00 0.00 3.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

277Y 0.00 0.00 4.90 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

277Y 6.58 3.92 0.00 4.69 7.85 0.00 5.92 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

280Y 0.00 2.44 0.00 2.12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

62

289Y 0.00 2.61 0.00 2.63 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

302Y 0.00 2.63 0.00 0.00 0.00 0.00 3.15 4.07 0.00 0.00 0.00 0.00 0.00 0.00 0.00

385Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.83 0.00 0.00 0.00 0.00

395Y 5.24 3.80 0.00 7.58 2.79 0.00 3.48 3.89 0.00 0.00 0.00 0.00 0.00 0.00 1.41

396Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.11 2.19 0.00 0.00 0.00

399Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 8.11 0.00 0.00 0.00 0.00 0.00 0.00

400Y 0.00 0.00 0.00 0.00 0.00 6.71 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

417Y 0.00 0.00 0.00 2.10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

419Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.46 0.00 0.00 0.00

431Y 17.19 12.72 0.00 13.40 15.89 0.00 15.71 21.97 22.25 0.00 0.00 0.00 3.54 0.00 1.89

432Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 39.69 0.00 4.72 0.00 3.01 0.00

432Y 0.00 0.00 14.48 0.00 0.00 27.68 0.00 0.00 0.00 0.00 3.70 0.00 0.00 0.00 0.00

436Y 4.54 1.82 0.00 2.95 2.44 0.00 4.35 7.20 6.88 0.00 0.00 0.00 0.00 0.00 0.00

437Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.37 1.66 0.00 0.00 0.00

438Y 2.48 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

439Y 0.00 3.50 0.00 2.30 0.00 0.00 2.74 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

441Y 3.75 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

442Y 0.00 2.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

442Y 0.00 0.00 0.00 3.77 8.21 0.00 2.82 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

445Y 0.00 0.00 5.35 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

452Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.11 1.32 0.00 5.28 0.00

453Y 3.52 3.63 5.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

454Y 0.00 0.00 0.00 1.99 3.97 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.04

456Y 2.44 2.60 5.12 2.97 2.87 10.93 4.49 6.90 4.48 0.00 0.00 0.00 0.00 0.00 0.00

470Y 6.80 4.18 0.00 4.78 0.00 0.00 5.71 0.00 0.00 0.00 0.00 0.00 0.00 2.33 7.13

471Y 0.00 0.00 0.00 0.00 0.00 6.71 0.00 5.39 0.00 0.00 3.20 4.90 0.00 0.00 0.00

474Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.72 0.00 0.00 0.00

476Y 4.41 4.43 0.00 5.60 4.50 0.00 4.23 0.00 0.00 0.00 0.00 0.00 19.70 0.00 0.00

63

477Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 21.76 0.00 0.00

478Y 0.00 0.00 8.49 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 31.35

479Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 11.92 0.00 0.00 0.00

480Y 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 41.16 0.00 0.00 0.00 0.00

482Y 0.00 17.72 0.00 0.00 0.00 0.00 0.00 0.00 10.48 35.49 0.00 0.00 0.00 0.00 0.00

483Y 14.75 0.00 0.00 18.08 12.02 0.00 17.73 13.69 0.00 0.00 0.00 0.00 0.00 89.39 0.00

485Y 8.49 15.76 0.00 0.00 6.26 0.00 0.00 0.00 0.00 24.82 43.81 42.94 49.55 0.00 54.85

485Y 0.00 0.00 47.97 9.70 0.00 18.90 9.64 10.76 16.41 0.00 0.00 0.00 0.00 0.00 0.00

486Y 0.00 0.00 0.00 0.00 0.00 6.14 0.00 0.00 0.00 0.00 0.00 23.43 0.00 0.00 0.00