INTERACTION BETWEEN DUCKWEEDS AND THEIR DIATOM EPIPHYTES

8/2/2019 INTERACTION BETWEEN DUCKWEEDS AND THEIR DIATOM EPIPHYTES

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UNIVERSITY OF OKLAHOMA

GRADUATE COLLEGE

INTERACTIONS BETWEEN DUCKWEEDS AND THEIR DIATOM EPIPHYTES

A THESIS

SUBMITTED TO THE GRADUATE FACULTY

in partial fulfillment of the requirements for the

Degree of

MASTER OF SCIENCE

By

NINA DESIANTINorman, Oklahoma

2012


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INTERACTIONS BETWEEN DUCKWEEDS AND THEIR DIATOM EPIPHYTES

A THESIS APPROVED FOR THEDEPARTMENT OF BOTANY AND MICROBIOLOGY

BY

______________________________Dr. Elizabeth A. Bergey, Chair

______________________________Dr. Wayne J. Elisens

______________________________Dr. Rebecca Sherry


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Copyright by NINA DESIANTI 2012All Rights Reserved.


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I dedicated my thesis to my advisor, Dr. Elizabeth A. Bergey and my beloved parents,

Djoko Pramono and Iswanti.


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Table of Contents

Acknowledgements .......................................................................................................

List of Tables ................................................................................................................. List of Figures ...............................................................................................................

Abstract .........................................................................................................................

Chapter 1: Introduction and Literature review ..............................................................

Periphyton communities .....................................................................................

Macrophytes and epiphyte interaction ................................................................

Duckweeds and epiphyte interactions .................................................................

Epiphytic Diatom Host Specificity ......................................................................

Research Questions .................................................................................................

Hypotheses ..............................................................................................................

Objectives ................................................................................................................

Thesis outline ........................................................................................................... Chapter 2: Epiphytic diatom composition on Duckweeds .............................................

Introduction .............................................................................................................

Materials and methods .............................................................................................

Study sites ...........................................................................................................

Macrophytes sampling and diatom processing ...................................................

Variation of Diatoms within Duckweeds ...........................................................

Sample preparation for SEM examination .........................................................

Data analysis .......................................................................................................

Results .....................................................................................................................


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Appendix F Test for differences in epiphytic diatom assemblage between roots and

leaves of Lemna minor . ...................................................................................... 65

Appendix G Test for differences in epiphytic diatom assemblages across root and leaf

from different ages of Spirodela polyrrhiza . ...................................................... 66

Appendix H Test for differences in epiphytic diatom density on leaf from different age

of Spirodela polyrrhiza . ...................................................................................... 66

Appendix I Epiphytic diatom taxa of duckweeds and artificial substrates ....................

Appendix J Test for differences in epiphytic diatom assemblages across different

nutrient and light treatments and substrate. ........................................................ Appendix K Test for differences in Lemnicola hungarica relative abundance on

duckweeds across different nutrients and light treatments. ................................

Appendix L. Test for differences in Achnanthidium minutissimum relative abundance on


Appendix M. Test for differences in Adlafia sp. relative abundance on duckweeds

across different nutrients and light treatments. ...................................................

Appendix N. Test for differences inGomphonema parvulum relative abundance on



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List of Tables

Table 1. Terminology of periphyton ..............................................................................

Table 2 Study sites, sampling dates and duckweed species ........................................... Table 3 Latitude and longitude of locations of the eleven survey sites ..........................


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List of Figures

Figure 1 PCA plot for epiphytic diatoms assemblage characteristic at eleven sites in

Oklahoma. ..................................................................................................................... Figure 2 MDS ordination of epiphytic diatom assemblages indicating different groups

from eleven sites in Oklahoma. .....................................................................................

Figure 3 Relationship between epiphytic diatoms species composition distances (Bray

Curtis distance) and the geographic distances of eleven sites in Oklahoma (Boot

n=1540)...........................................................................................................................

Figure 4 (a) Relation between maximum local abundance (n=115) (b) mean abundanc

(n=115) and regional occupancy of epiphytic diatoms taxa of eleven sites in Oklahom

.......................................................................................................................................

Figure 5 PCA plot for epiphytic diatoms assemblage characteristic at Glasses Creek,

Oklahoma. .....................................................................................................................

Figure 6 MDS ordination of epiphytic diatom assemblages at Glasses Creek, JohnstonCounty, Oklahoma .........................................................................................................

Figure 7 Physio-chemical parameters of algal mat, edge of stream and isolated pool

sites in Glasses Creek. ...................................................................................................

Figure 8 Epiphytic diatoms abundances, pH, PO4, NO3, SiO2 seasonal variation in

William Morgan Park, Oklahoma ..................................................................................

Figure 9 Diatom density on three sizes (ages) of Spirodela polyrrhiza leaves. ............. 29

Figure 10 Epiphytic diatoms .........................................................................................

Figure 11 (a) Racks; (b) shaded and unshaded treatments; (c)Spirodella, Lemna minor

andLemna valdiviana and agar nutrient inside . (d) Experimental pond ....................... 38


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Figure 12 MDS ordination of epiphytic diatom assemblages of different nutrient

treatments and shading treatments on artificial substrate and duckweeds. ....................

Figure 13 MDS ordination of epiphytic diatom assemblages of different light .............

Figure 14 MDS ordination of epiphytic diatom assemblages of different substrate ......

Figure 15 Diatoms relative abundance on artificial substrate and duckweeds on contro

nitrogen, nitrogen + phosphorus and phosphorus enrichment........................................ 46

Figure 16 Diatoms relative abundance on artificial substrate and duckweeds on contro

nitrogen, nitrogen + phosphorus and phosphorus enrichment........................................ 47

Figure 17 Relative abundance of Achnanthidium minutissimum, Adlafia sp., Lemnicolahungarica andGomphonema parvulum on three different species of duckweeds before

experiment and after experiment. ..................................................................................


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Abstract

The majority of epiphytic diatoms are not specific for particular plants. One exception

Lemnicola hungarica (Grunow) Round and Basson 1997 and the duckweeds, small

floating aquatic plants in the family Araceae . This research was conducted to examine

the habitat specificity of Lemnicola, which involved both a field survey of natural

habitats and an experiment in a University of Oklahoma Aquatic Research Facility

pond. From the field survey, Lemnicola occurred on three all encountered duckweeds

Lemna minor , Spirodela polyrhiza andWollfia sp., from eleven sites in Oklahoma.

Lemnicola was always present but not always the dominant species, and particularspecies (Cocconeis placentula, Diadesmis confervacea and Nitzschia perminuta ) were

also commonly present. The experiment used different nutrient enrichment and shadin

treatments in small floating chambers with duckweeds and plastic artificial substrates.

Nutrient treatments were phosphorus, nitrogen, a combination of phosphorus and

nitrogen and an un-enriched control, and the shading treatments were shaded and un-

shaded. Among nutrients, phosphorus (either alone or in combination with nitrogen)

altered diatom assemblage composition. Lemnicola was more abundant with

phosphorus enrichment and under shade. Lemnicola colonized floating artificial

substrates when phosphorus was enriched, indicating a nutrient interaction with

duckweeds involving phosphorus. Color of plastic (light transmission) of the artificial

substrates had no effect, indicating that refracted light is important to duckweedepiphytes. This research supported the occurrence of a characteristic diatom flora for

duckweeds and demonstrated that Lemnicola hungarica may colonize other substrates,

given appropriate conditions (i.e., high phosphorus and shade).


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Chapter 1: Introduction and Literature review

Diatoms are unicellular algae characterized by siliceous cell walls and storage o

oil reserves. Diatoms are ubiquitous in aquatic habitats, where they live in the water

column as plankton, attach to substrates, or occur in bottom sediments.

Substrate-dwelling diatoms can be ecologically classified based on the substrat

episammic diatoms live on sand grains, epipelic diatoms live in sediment, epilithic

diatoms live on rocks, epiphytic diatoms occur on plants, and epizoic diatoms are

associated with animals. Diatom assemblages in each of these specific habitats differ i

species composition although the habitats share many species (Stevenson, et al. 1996)Epiphytic algal assemblages may differ among plants species, although this

difference is more apparent in oligotrophic lakes than in lakes with higher nutrient

concentration (Eminson and Moss, 1980). Despite these assemblage differences, the

majority of diatom species are not specific for particular plants, and these differences

are differences in relative abundance within the species pool. One exception is the

monospecific Lemnicola hungarica (Grunow) Round and Basson 1997 and duckweeds

(Hustedt, 1930), small floating aquatic plants in the family Araceae formerly in the

family Lemnaceae . The genus name Lemnicola was erected by Round and Basson

(1997) and signifies this association.

Lemnicola hungarica subsequently referred as Lemnicola is commonly found on

duckweeds, although there is variation in the presence and density of this diatom,among species of duckweeds (Goldsborough, 1993; Buczk, 2007). Additional diatom

including the morphologically similarCocconeis placentula may also be present.


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The objective of my research was to explore the interaction of epiphytic

diatoms on duckweeds, with an emphasis on the diatom Lemnicola hungarica . Research

was conducted on diatom composition on three different species of duckweeds and

floating artificial substrates under different levels of nutrient concentration and light

intensity. In addition, duckweeds and associated diatoms from several sites were

surveyed and diatom microspatial distribution on duckweeds was observed.

Periphyton communities

A wide variety of aquatic organisms in freshwater can be found growing

attached to substrates; these organisms include bacteria, fungi, algae, protozoans andmulticellular plants and animals. These attaching organisms may be considered part of

the benthos, a term for organisms growing on the bottom of a water body, in contrast t

the plankton, which are floating or swimming free. Because of variations in habitat,

habitat requirements, mode of life, type of adaptation to the environment, and other

factors, these two primary groups of aquatic organisms are further divided into a

number of categories (Cooke, 1956).

According to Sldekov (1962), the term "benthos" and "benthic" are to be

used exclusively for organisms living freely in the upper layer of sediments, meanwhi

the organism growing attached to other kind of substrate are placed in the "Aufwuchs"

community, for which the term "periphyton" is often used.

rmek-Huek in Sldekov (1962) listed the types of periphyton according to

common substrate (Table 1.1).


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Table 1. Terminology of periphytonSubstratum Organism Community

Attached DependentVarious Epihola Nereidea

SessiliaHolobionta Epiholon

Plants Epiphyta Phytobionta EpiphytonAnimals Epizoa Zoobionta EpizoonWood Epidendron Dendrobionta Dendron

Epixylon Xylobionta EpixylonRock Epilitha Lithobionta Epilithon

Ecologically, periphyton contributes to aquatic food webs and increases

productivity. For example, phytoplankton, periphyton and macrophytes all contribute

the total primary productivity of shallow lakes; however, the relative contribution of

these autotrophs varies. In Borax Lake, annual productivity of phytoplankton and

periphyton were similar to each other and exceeded that of macrophytes (Wetzel 1964

Macrophytes and epiphyte interaction

Macrophytes, where present in the littoral zone, play major role as a substrate

for periphyton, including algae, bacteria and fungi (Hutchinson, 1975). For example,epiphytic algae were responsible for 31.3 % of the littoral production and for 21.4 % o

the total annual production, including phytoplankton production, for the whole lake

(Allen, 1971).

The specificity of epiphytic algae, including diatoms, for particular macrophyte

has been noted (Prowse, 1959; Sldekov, 1962; Pip & Robinson, 1985, Eminson and

Moss, 1980), yet the relationship between epiphytic algae and their host macrophytes

unclear. A number of macrophyte-epiphyte interactions have been proposed. Potential

benefits to epiphytes are that epiphytes are elevated in the water column, where there

greater access to light than at the sediment surface, and that epiphytes can access


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macrophyte-derived nutrients (Burkholder and Wetzel, 1990). Macrophytes may bene

from epiphytes protecting them from herbivore or pathogen damage (Hutchinson,

1975). Alternatively, macrophytes may serve as a neutral site for attachment or

contribute negligibly to epiphyte nutrient supplies (Cattaneo and Kalff, 1979).

Symbiosis between epiphytic algae and the host plant is considered to be more

beneficial to the algal colonizers than to the host plant. Epiphytes may cause harm; the

underlying macrophyte host may become mechanically stressed, light-limited, and/or

carbon-limited with increasing epiphyte colonization, as demonstrated in sea grass and

its epiphytes (Sand-Jensen, 1977). Macrophytes may compete with algal colonizers fonutrients and could release allelopathic substances that inhibit epiphyte growth

(Anthony, et al. 1980; Fitzgerald, 1969). Periphyton grazing by herbivorous snails can

increase production in some macrophytes (Martin et al. 1992), presumable by reducin

light limitation. The interactions between epiphytes and their host plant can vary

depending on the season, macrophyte condition and availability of water-column

nutrients (Stevenson et al., 1996).

Epiphytes may also affect nutrient cycling. In lakes, epiphytes can have a majo

role in the temporary storage of phosphorus derived from epilimnetic water and from

the sediment via macrophyte roots. Epiphyte can be important in phosphorus

transforming of un-reactive phosphorus into reactive phosphorus, some of which may

leach into the water (Riber, et al. 1983).


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Epiphytic Diatom Host Specificity

Although examples of interaction specificity are common in terrestrial systems

(e.g. insect-herbivore specificity and parasitoid-insect specificity), aquatic plant host

specificity is uncommon and has been much less studied. There are few examples of

specificity by diatoms for host macrophytes (Delbecque, 1983, Millie and Lowe, 1982

Buczk, 2007). One example of diatom host-macrophytes specificity is Navicula

endophytica Hasle, an endophyte found in receptacles of some common marine brown

algae, such as rockweed ( Ascophyllum nodosum ) (Hasle, 1968), bladderwrack (Fucus

vesiculosus ) and toothed wracks, (Fucus serratus ) (Tassen, 1972) andFucus evanescens (Main and McIntire, 1974).

A second example of a diatom species living in specific habitat is Lemnicola

hungarica . Hustedt (1930) commented on the occurrence of Achnanthidium hungarica

as apparently preferring Lemna. [Note: A.hungarica is a synonym of Lemnicola

hungarica .]

Research Questions

The main research questions are:

1. Does Lemnicola hungarica prefer duckweeds as a habitat?

2. Does microhabitat differences (between roots and leaves) affect diatom

composition on duckweeds?

3. Does growing condition, especially nutrients and light, affect diatom

composition on duckweeds?


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Hypotheses

This study was designed to test four hypotheses:

Hypothesis 1. Diatom composition differs among different species of duckweeds

(including the genera Lemna, Spirodela , andWolffia )

Hypothesis 2. Diatom composition differs between the leaf and root of duckweeds.

Hypothesis 3. Lemnicola will be present on different species of duckweeds but will be

relatively more abundant on duckweeds than on artificial substrates, whereas other

diatoms will not show this specificity.

Hypothesis 4. Growing conditions (nutrients and light) will affect the composition of diatoms on duckweeds.

Objectives

The specific objectives of the study are to:

1. Investigate the habitat, occurrence and distribution of Lemnicola

2. Investigate the microspatial distribution of diatoms on duckweeds

3. Provide information about factors, especially nutrients and light, associated with

diatom assemblage composition on duckweeds.

4. Describe the succession of diatom assemblages on plants and artificial substrates.

Thesis outline

Chapter 2 presents the field survey and microspatial distribution of epiphytic diatoms

duckweeds.

Chapter 3 covers an experiment on the effects of nutrients and light intensity on

epiphytic diatoms on duckweeds and artificial substrates.


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different substrates. Differential environmental shading can affect diatom assemblage

structure, resulting differences between shaded and un-shaded habitats (Millie and

Lowe, 1982).

The aims of the study were (1) to investigate the occurrence and distribution of

Lemnicola on duckweeds, (2) describe the succession of diatom assemblages on

duckweeds, and (3) to investigate the microspatial distribution of diatoms on

duckweeds. A survey of epiphytic diatoms on duckweeds was conducted to examine t

occurrence of Lemnicola on duckweeds from different locations. Different species of

duckweeds from several study sites were sampled for diatom species composition.Diatom assemblages on new (smaller) and older (larger) leaves were compared to

examine temporal effects. The microspatial distribution of diatoms between roots and

leaves of duckweeds were compared using both extraction of diatom assemblages and

examination using scanning electron microscopy.

Materials and methods

Study sites

Eleven study sites in Oklahoma were sampled for duckweeds (Table 2); between July

2010 and October 2011. All duckweed species present at each site were collected.

Table 2 Study sites, sampling dates and duckweed speciesNo. Study sites (County) Sampling Dates Duckweed species1. William Morgan Park

(Cleveland)21 Jul 2010, 22 Apr, 8May, 15 May, 20 June,

14 Aug , 5 Sep 2011

Lemna minor Phil.;Spirodela polyrrizha L.

Scheiden2. Sutton Urban Wilderness

Park (Cleveland)21 July 2010 L.minor

3. Glasses Creek (Johnston) 14, 20, 27 Aug, 4 Oct2011

L. minor

4. Canadian River(Cleveland) 21 Aug , 24 Sep 2011 L.minor , S. polyrrizha


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No. Study sites (County) Sampling Dates Duckweed species5. Roman Nose State Park

(Blaine)26 Sep, 8 Nov 2010 L.minor

6. Robbers Cave State Park(Latimer)

25 Sep 2011 S. polyrrizha

7. Big Flag Lake (Rogers) 9 Sept 2011 S. polyrrizha , Wollfia sp8. Oxley Park - north woods

(Tulsa)29 Sept 2011 S. polyrrizha

9. Oxley Park - marsh area(Tulsa)

29 Sept 2011 S. polyrrizha , Wollfia sp

10. Oxley Park - bottomlandforest (Tulsa)

29 Sept 2011 L.minor , S. polyrrizha

11. Chickasaw NationalRecreation Area (Murray)

16 Oct 2011 L.minor , S. polyrrizha

I collected samples from Cleveland County and Johnston County; other samples were

collected by Elizabeth A. Bergey, Bruce Hoagland and Amy Buthod. For my

collections, physical and chemical parameters were measured in situ and include

dissolved oxygen (DO) using a Hach HQ 10 DO meter (Loveland, Colorado);

conductivity, temperature, pH using an Ex Stick II EC 500 (Waltham, Massachusetts);

percent shade with a spherical densiometer (Forestry Suppliers, Jackson, MS), and

nutrients (nitrate, phosphate and silica) content of the water using water chemistry ass

kits (Hach DR/890 Colorimeter; Loveland, Colorado). Latitude and longitude of the

sampling sites were determined from the website maps.google.com (Table 3). Spatial

effects on diatom assemblages were assessed in terms of distance between sampling

sites. Distance between each site was calculated using the haversine formula, whichbased on latitude and longitude measurements (program by Movable Type Ltd.;

available online). William Morgan Park included two different sampling areas, a shad

area and an open (un-shaded) area. Glasses Creek site included three different samplin


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habitats (an isolated pool, the stream edge and the stream center within algal mat). All

other sites had a single sampling area.

Table 3 Latitude and longitude of locations of the eleven survey sites

No. Study sites (County) Longitude Latitude1. William Morgan Park (Cleveland) 35.23 -97.492. Sutton Urban Wilderness Park (Cleveland) 35.24 -97.423. Glasses Creek (Johnston) 35.18 -97.474. Canadian River (Cleveland) 34.08 -96.745. Roman Nose State Park (Blaine) 35.93 -97.476. Robbers Cave State Park (Latimer) 34.97 -98.437. Big Flag Lake (Rogers) 36.20 -95.628. Oxley Park - north woods (Tulsa) 36.22 -95.909. Oxley Park - marsh area (Tulsa) 36.45 -95.9010. Oxley Park - bottomland forest (Tulsa) 36.48 -95.9011. Chickasaw National Recreation Area (Murray) 34.43 -97.01

Macrophytes sampling and diatom processing

A minimum of 15 individual duckweeds of each species present were collected

haphazardly from each site. Samples were preserved with 2% formalin until processin

in the laboratory. Samples were processed to digest plants materials, separate diatoms

from their substrate and oxidize the organic part of diatoms using 30% hydrogen

peroxide and heating in a water bath at 80 C for one hour. Nitric acid was then added

the sample and heated for an additional one to two hours. Samples were repeatedly

rinsed with distilled water to remove all traces of acid by filling with water, settling, a

pouring off most of the water, and refilling. The cleaned diatom sample was used for

making permanent slides with Naphrax (Brunel Microscopes, Wiltshire, U.K.), a high

refractive index mounting medium specific for viewing diatoms. Identification and

enumeration of diatoms were done by scanning transects across the mounted coverslip


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under 1000x magnification on an Olympus CX41 microscope. A minimum of 400

valves (cell wall halves) were counted for each sample.

Variation of Diatoms within Duckweeds

Epiphytic diatom assemblages were compared between roots and leaves and

among different-aged leaves. Thirty Lemna minor from Roman Nose Park were selected

for the root-leaf comparison. Attached roots for each of the three leaf categories were

separated and placed in separate vials and processed for diatoms, as described above.

FifteenSpirodela polyrrhiza from Big Flag Lake were selected for the comparison

among different-aged leaves.Spirodela has a larger leaf with many roots compared to Lemna , which has smaller leaves, each with a single root. Every visible leaf was

separated and measured for length and width using a digital micrometer. Leaves

become more elongate with age and the ratio of length to width was used to assign sta

(age) categories. The youngest smallest leaves were nearly round and were assigned a

first stage leaves (length: width ratio about 1:1); moderate-aged leaves were the secon

stage, which had a ratio of about 1:1.3, and the older leaves were assigned the third

stage and had a higher ratio (2:1.3). Attached roots for each of the three leaf categorie

were separated. Each of the three categories of leaves and the three sets of roots was

placed in separate vials and processed for diatoms, as described above. Leaf surface

area was measured by the weighing cut outs of photographed leaves that were printed

on paper.


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Sample preparation for SEM examination

Duckweed specimens were air dried and mounted onto metal stages, coated with an

AuPd alloy and observed using a ZEISS DSM-960A scanning electron microscope.

Digital photographs were taken of roots and leaves with their epiphytes.

Data analysis

Relative abundance (percent of total numbers) was calculated from the diatom counts

each sample. Data were arc-sine square-root transformed to normalize the distribution

of the data prior to statistical analysis. A multivariate analysis, two-way crossed

ANOSIM (= Analysis of similarity) (PRIMER software ver.6.1.7, Plymouth MarineLaboratory, Plymouth, England) was used to compare epiphytic diatom assemblages

across duckweed species and across eleven sites in Oklahoma. Principal Component

Analysis (PCA); was used to determine which taxa were responsible for any differenc

and used PAST (Paleontological Statistic software) (version 2.08, available on-line)

(Hammer, et al., 2001). Ordination by Non-metric Multi-Dimensional Scaling (MDS i

PRIMER) used a similarity matrix created from Bray Curtis similarity analysis.

CLUSTER analysis (PRIMER) using group-average clustering of the similarity matrix

was used to group samples in the MDS ordination. Relative abundance of selected

diatom taxa were plotted as bubbles on MDS ordinations. The Mantel tests of matrix

correlations in PAST were used for testing the relationships between matrices of

distance in species composition (evaluated using BrayCurtis quantitative distance

measure) and the geographic distance of the localities (in kilometers). The relationship

between local maximum and mean abundances in terms of relative abundances, and

regional occupancy were examined using correlation analysis in PAST. One-Way


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percent (Figure 2a-c). The stress value of 0.17 indicates slight distortion of the plot, bu

overlay cluster analysis results strengthens the interpretation.

Cluster I was characterized by a high to moderately high abundance of

Lemnicola hungarica (= Lemnicola ). This cluster included samples from Big Flag Lake,

the three Oxley Park sites (north woods, bottomland forest, and marsh area), Robbers

Cave State Park and one sampling from Glasses Creek. Lemnicola was found in

moderately high to low abundance in other clusters. Samples from William Morgan

Park and Roman Nose State Park formed three overlapping clusters, consisting cluste

II, III and IV. Cluster V and VI was slightly overlapping and are characterized by a lowabundance of Lemnicola and consist of samples from Glasses Creek, the Canadian

River, Chickasaw Recreational Area and Sutton Urban Wilderness Park (Figure 2a).

Cocconeis placentula (=Cocconeis ) was abundant in the overlapping clusters II,

III and IV (Figure 2b). The William Morgan Park and Roman Nose Park sites had hig

abundance and moderately high abundance of Cocconeis , respectively.

Diadesmis confervaceae was found in a very high abundance at Sutton Urban

Wilderness Park, Canadian River, one sampling from Chickasaw Recreational Area an

Glasses Creek (cluster IV). Diadesmis was found in high to moderately high abundance

in cluster I that included samples from Big Flag Lake, Oxley Park bottomland forest

Oxley Park - marsh area and Robbers Cave State Park (Figure 2c).

Nitzschia perminuta was found in a moderately high abundance in samples

primarily from Glasses Creek and some samples from William Morgan Park, which

were included in clusters IV and V. Nitzschia also found in low abundance in samples

from Canadian River, Oxley Park bottomland forest, Oxley Park - marsh area, Roma


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Nose State Park, which were included in cluster I, and some samples from William

Morgan Park, which were included in clusters II and III (Figure 2d).

Mantel tests result of the correlation between matrices of diatom composition

and distance among sites showed threshold response with good correlation among

samples from nearby sites (e.g., within Oxley Park; distance=0), but no relationship

between distance and assemblage composition for all other combination of sites (Figu

3). Maximum local abundance were positively correlated with regional occupancy

(R2=0.55, p


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Figure 1 PCA plot for epiphytic diatoms assemblage characteristic at eleven sitesin Oklahoma.A=Big Flag Lake, B=Canadian River, C=Chickasaw National Recreation Area,

D=Glasses Creek, E=Oxley Park -marsh area, F=Oxley Park bottomland forest,G=Oxley Park-north wood, H=William Morgan Park, I=Roman Nose State Park,J=Sutton Urban Wilderness Park, K=Robbers Cave State Park.

Achnanthidium exigua

Psammothidium lauenburgianum

Achnanthidium minutissima

Achnanthidium rivulare

Adlafia sp.

Amphora copulataAmphora pediculusAnomoneoneis sphaerophoraAulacoseria granulataAulacoseria pusillaBacillaria paxiliferCaloneis amphisbaenaCaloneis bacillumCaloneis schumanniana

Cocconeis placentula

Cocconeis scutellumCraticula ambiguaCraticula cuspidataCraticula dissociataCraticula ripariaCtenophora pulchella

Cyclotella meneghiniana

Cymatopleura soleaCymbela tumidulaCymbella asperaDelicatula delicataDenticula subtilisDiploneis sp.Discotella stelligeraEncyonema silesiacumEolimna subminisculaEpithemia adnataEunotia arcus

Eunotia lunaris

Fallacia teneraFragilaria capucinaFragilaria construensFragilaria pinataFragilaria teneraFrustulia amphipleuroidesGeissleria decussis

Geissleria dolomiticaGomphonema acuminatumGomphonema augurGomphonema cleveiGomphonema gracileGomphonema grockei

Gomphonema parvulum

Gomphonema parvulum var parvulum

Gomphonema truncatumGyrosigma attenuatumHippodonta hungaricaHalamphora montanaHalamphora tumidaHalamphora venetaHantzschia amphioxysKaraveya laterostriata

Lemnicola hungarica

Luticola muticaMelosira variansMeridion circulare

Diadesmis confervacea

Navicula cryptocephalaNavicula cryptotenelloidesNavicula kotschyiNavicula trivialisFallacia pygmaeaNavicula radiosa

Navicula recensNavicula rostellataNavicula sp.Navicula streckeraeNavicula tripunctataNeidium ampliatumNitzschia acicularisNitzschia agnita

Nitzschia filiformisNitzschia bergiiNitzschia constricta

Nitzschia dissipataNitzschia microcephalaNitzschia gracilisNitzschia inconspicuaNitzschia liebetruthii

Nitzschia linearisNitzschia palea

Nitzschia sigma

Nitzshia perminuta

Orthoseira dendroteresPinnularia bertrandii var bertrandiiPinularia acrosphaeraPinularia borealisPinularia divergentissimaPinularia gibbaPinularia turbulentaPinularia viridiformis

Placoneis gastrum

Planothidium frequentissimumPlanothidium stewartiiPleurosira laevis

Psammothidium chlidanos

Rhoi cosphenia abbreviataRhopalodia gibbaRhopalodia operculata

Selaphora pupula

Stauroneis phoenicenteronSurirella brebisoniiSurirella ovalisSurirella peisonisSurirella splendidaTryblionella angustataTryblionella hungaricaTryblionella calidaUlnaria acusUlnaria ulna

A

A2

B1

B2B3

C1

C2

D1

D2

D3

D4D5

D6 D7D8

D9

D10D11

D12

D13D14

D15D16

D17D18D19D20

D21

F1

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H19

-50 -40 -30 -20 -10 10 20 30

-60

-50

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-10

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20

30


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Figure 2 MDS ordination of epiphytic diatom assemblages indicating differentgroups from eleven sites in Oklahoma.Bubbles on graphs display the relative abundance of selected taxa in each sample: a

Lemnicola hungarica , b =Cocconeis placentula . A=Big Flag Lake, B=CanadianRiver, C=Chickasaw National Recreation Area, D=Glasses Creek, E=Oxley Park -marsh area, F=Oxley Park bottomland forest, G=Oxley Park-north wood, H=WilliamMorgan Park, I=Roman Nose State Park, J=Sutton Urban Wilderness Park, K=RobbeCave State Park.

Lemnic

10

40

70

100

A

A2

B1

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B3C1

C2

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D4D5D6D7

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D15D16D17D18

D19D20

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G1G2

K

I1

I2

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H1

H2H3

H4

H5

H6H7

H8

H9

H10

H11

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H14H15H16

H17H18 H19

2D Stress: 0.17

Coccon

10

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A2

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B2

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C2

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D4D5D6D7

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H8

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H11

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H14H15H16

H17H18 H19

2D Stress: 0.17

a

Lemnicola hungarica

Cocconeis placentula

b

Relativeabundance

Relativeabundance

I

II

III

IV

V

VI

I

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Figure 2 MDS ordination of epiphytic diatom assemblages indicating differentgroups from eleven sites in Oklahoma.Bubbles on graphs display the relative abundance of selected taxa in each sample: c

Diadesmis confervaceae , d = Nitzschia perminuta . A=Big Flag Lake, B=CanadianRiver, C=Chickasaw National Recreation Area, D=Glasses Creek, E=Oxley Park -marsh area, F=Oxley Park bottomland forest, G=Oxley Park-north wood, H=WilliamMorgan Park, I=Roman Nose State Park, J=Sutton Urban Wilderness Park, K=RobbeCave State Park.

Diades 10

40

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100

A

A2

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Nitzshi

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2D Stress: 0.17


c

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Nitzschia perminutaI

II

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Relativeabundance

Relativeabundance

I

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Figure 4 (a) Relation between maximum local abundance (n=115) (b) meanabundance (n=115) and regional occupancy of epiphytic diatoms taxa of elevensites in Oklahoma

-0.8

-0.4

0

0.4

0.8

1.2

1.6

2

2.4

L o g

l o c a

l m a x a

b u n

d a n c e

12 24 36 48 60 72 84 96 108

Regional frequency (%)

-3

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-2

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0

0.5

1

1.5

L o g

l o c a

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b u n

d a n c e

R2=0.55, p =0.0001

R2=0.44, p =0.0001


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Variability among habitats was observed in the Glasses Creek site. Lemna grew

in three different habitats at this site: an isolated pool area; a filamentous algal mat in

middle of the stream and at the edge of stream (without apparent filamentous algae).

Epiphytic diatom assemblages were significantly different among these habitats

(ANOSIM; R = 0.909, p2.2 ppm and >2.75 ppm, respectively


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Figure 5 PCA plot for epiphytic diatoms assemblage characteristic at GlassesCreek, Oklahoma.A=Algal mats, P=isolated pool, S=streams edge

Achnanthidium exigua

Psammothidium lauenburgianumAchnanthidium minutissimaAchnanthidium rivulare

Adlafia sp.Amphora copulataAulacoseria granulataAulacoseria pusilla

Caloneis bacillumCocconeis placentulaCocconeis scutellumCraticula ambiguaCraticula cuspidataCyclotella meneghinianaDiploneis sp.Discotella stelligeraEncyonema silesiacumEolimna subminiscula

Epithemia adnataEunotia arcus

Eunotia lunarisFallacia tenera

Fragilaria capucina

Frustulia amphipleuroidesGeissleria decussisGeissleria dolomiticaGomphonema gracile

Gomphonema grockei

Gomphonema parvulumGomphonema parvulum var parvulum f saprophylum

Halamphora montanaHalamphora tumidaHalamphora venetaHantzschia amphioxys

Lemnicola hungarica

Luticola muticaMelosira varians


Navicula cryptocephalaNavicula cryptotenelloidesNavicula trivialisFallacia pygmaeaNavicula recensNavicula rostellataNeidium ampliatumNitzschia agnitaNitzschia filiformis

Nitzschia constrictaNitzschia microcephalaNitzschia inconspicuaNitzschia liebetruthiiNitzschia linearis

Nitzschia palea

Nitzschia sigma

Nitzshia perminuta

Pinularia borealisPinularia divergentissimaPinularia gibbaPlaconeis gastrumPlanothidium frequentissimumPlanothidium stewartiiPsammothidium chlidanosRhopalodia gibbaRhopalodia operculataSel aphora pupula

Surirella brebisoniiSurirella ovalisTryblionella calidaUlnaria acus

Ulnaria ulnaS5

S4

S3

S2S1

S6

A1

A2A3 A4A5A6

P1

P2

P3

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-20 -15 -10 -5 5 10 15

-25

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5

10

15


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Figure 7 Physio-chemical parameters of algal mat, edge of stream and isolatedpool sites in Glasses Creek.(a) pH (b) temperature (c) Dissolved Oxygen (DO) and (d) nitrate (n=3).

a b

dc


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A pattern of seasonal variation was observed across the seven William Morgan

Park samplings that spanned from 22 April to 5 September 2011 (Figure 8). There wa

significant differences in epiphytic diatom assemblages across sampling dates

(ANOSIM; R= 0.726, p0.05) (Appendix E).


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2 2 A p r

1 1

8 M a y

1 1

1 5 M a y

1 1

5 J u n

1 1

1 4 A u g

1 1

5 S e p

1 1

0

1 6 3 2 4 8 6 4 8 0

A d l_ s p

0 1 6 3 2 4 8 6 4 8 0

C o c

_ p

l a 0 1 6 3 2 4 8 6 4 8 0

L e m_

h u n

0 1 6 3 2 4 8 6 4 8 0

N i t

_ p e r

6

7

8

9

p H

0

1

2

3

P

O 4

0

1

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1

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S i O 2

- 2 0 0 2 0 0 6 0 0 1 0 0 0 1 4 0 0 1 8 0 0

C o n

d u c

t i v

i t y

F i g u r e

8 E p i p h y t

i c d i a t o m s a b u n

d a n c e s , p H , P

O 4 , N O 3 , S i O 2 s e a s o n a

l v a r

i a t i o n

i n W i l l i a m

M o r g a n

P a r

k ,

O k l a h o m a


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Microspatial distribution of diatoms on duckweeds

Diatoms were attached to the lower surface of the leaves and on the roots of

Lemna minor andSpirodela polyrrhiza , as shown on SEM photographs (Figure 10a-f).

Diatoms were not common on upper surface of leaves. Single-species patches were

commonly observed especially of Lemnicola (e.g. Figure 10 c,d) andCocconeis

(Figure 10 e,f). Lemnicola showed a more dispersed pattern whereasCocconeis

occurred in clusters (Figure 10-f).

Epiphytic diatom composition did not differ between leaves and roots of Lemna

minor (Wilcoxon Signed Ranks; Z= -0.553, p=0.580) (Appendix F) orSpirodela polyrhiza (ANOSIM; R= -0.258, p=0.91). There was also no difference in diatom

composition among the three sizes (ages) of Spirodela leaves and roots (ANOSIM; R=

-0.301, p=0.96) (Appendix G). Diatom density increased with leaf stage (increasing

age), however the density differences among leaf stage were not statistically significan

(Figure 9, Appendix H).

Figure 9 Diatom density on three sizes (ages) of Spirodela polyrrhiza leaves.(n=3).


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Figure 10 Epiphytic diatoms(a) on a root and (b) on the underside of a leaf of Lemna minor; (c) clump of Cocconeis on the root surface of Spirodela polyrrhiza and (d) on underside of a leaf of Lemna minor; Lemnicola on leaf underside of (e) Lemna minor; (f) Spirodela

polyrrhiza

e

c d

b

f

a 50 m1200X100 m600X


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31

Discussion

Duckweeds served as substrate for a variety of diatom taxa. Most of these taxa

were benthic diatoms with a prostrate growth form. The ability of these diatoms to

attach to a substrate is enabled by their ability to form mucilage pads (Round, et al,

2000). A prostrate growth form has lower susceptibility to grazing than upright or

filamentous growth forms (Steinman, et al., 1992) and is a common growth form on

smooth surfaces that lack protective crevices (Bergey, 2005).

Lemnicola hungarica occurred on all three surveyed species of duckweeds (Spirodela

polyrrhiza, Lemna minor, Wolffia sp.).Lemnicola was not always the dominant diatomon these duckweeds. Depending on the site and time of sampling, dominants included

not only Lemnicola , but alsoCocconeis placentula , Diadesmis confervaceae and

Nitzschia perminuta .

Lemnicola is classified as a hypereutrophic diatom, whileCocconeis placentula ,

Diadesmis confervaceae and Nitzschia perminuta are eutrophic diatoms (Van Dam et

al., 1994). Hypereutrophic and eutrophic diatoms occur in nutrient-rich waters.

Duckweeds sites are typically rich in nutrients (Landolt, 1986); thus, duckweeds grow

in nutrient conditions conducive to Lemnicola and other duckweed-characteristic

diatom taxa.

A strong spatial pattern of algal assemblage composition was not apparent. For

example, Lemnicola was very abundant in three areas: the close-by Big Flag Lake and

Oxley Park sites and one distant Glasses Creek site. Another example was Diadesmis

confervaceae , which had a limited distribution and was only abundant at two distant

sites. Most taxa with high abundance, such as Lemnicola hungarica, Diadesmis


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confervaceae andNitzschia perminuta , had wide spatial distribution, a pattern also

found by Soininen (2004). HoweverCoccineis placentula had high abundance, but was

found in about 60% of the sites.

Habitat variability within a site (i.e., at Glasses Creek) and seasonal sampling

(i.e., at William Morgan Park) demonstrated associated spatial and temporal variation

diatom assemblages. Diatoms are sensitive to many environmental variables, includin

light, moisture conditions, temperature, current velocity, salinity, pH, oxygen, nutrient

(carbon, phosphorus, nitrogen and silica) (Van Dam et al., 1994). Measured difference

or fluctuation of nutrients (nitrogen, phosphorus, and silicate concentration), pH andconductivity at my sites likely contributed to the observed variation in diatom

assemblages. Because this was a survey, the precise factors producing variation in

diatom assemblages cannot be determined.

Light level is likely an important factor for Lemnicola growth because

Lemnicola was recorded in significantly higher abundance in shaded than non-shaded

areas. Duckweeds ( Lemna minor andSpirodela polyrhiza ) were also more abundant

(and had darker green leaves) in shaded areas at William Morgan Park, where riparian

vegetation and an elevated walkway provided shade. This preference for low light is

consistent with a previous study of Lemnicola in Canada, where Lemnicola was most

abundant in dense duckweed mats where subsurface light was limited (Goldsborough,

1993).

Diatom assemblages were similar among different species of duckweeds at eac

site. I could find only one study comparing the diatoms of different species of

duckweeds (Buczk, 2007) and this study used dried herbarium specimens from many


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sites (comparisons were not made within a site). This study concluded that diatoms

varied among species of duckweeds but my results indicate that the confounding site

effect would have produced this pattern.

Diatom assemblage differences between roots and leaves were not observed in

this study. This result contrasts with previous studies, which revealed apparent

differences of diatom composition between roots and leaves of duckweed based on the

SEM observations. However, these studies (Buczk, 2007; Zuberer, 1994) were

conducted without quantification of diatom assemblages on the roots and leaves.

Similarity of diatom assemblages on root and leaf parts of Lemna minor andSpirodela polyrrhiza agreed with Simonsens hypothesis of random colonization of diatoms (in

Millie and Lowe, 1982). Unlike diatoms, N-fixing bacteria are more common on Lemna

roots than on leaves (Zuberer, 1994); but this pattern may result from nutrient

interactions involving nitrogen, a nutrient that was found unimportant in diatom

colonization of duckweeds (Chapter 3).

When viewed with SEM, the distributional pattern of Cocconeis and Lemnicola

differed.Cocconeis had a more clumped distribution than Lemnicola . Goldsborough

(1994) suggested thatCocconeis has weakly motile progeny after cell division, whereas

Lemnicola has relatively more motile progeny and, hence, is distributed more evenly. I

observed that Lemnicola was often located along the junction lines of adjacent

duckweed cells, a location that forms a low dip and results in less protrusion by the

Lemnicola cell. This lower protrusion may reduce grazing loss in this species.

Further study is needed to examine the interaction between epiphytic diatoms on

duckweeds and environmental factors, especially light and nutrients (see Chapter 3).


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Chapter 3: Diatom Colonization on Artificial Substrate and ThreeSpecies of Duckweeds on different nutrient and light treatments

Introduction

Although duckweeds often have a characteristic diatom flora dominated by Lemnicola hungarica andCocconeis sp., there is wide spatial and seasonal variation in

species composition (Chapter 2; Bowker & Denny, 1980; Goldsborough & Robinson,

1985; Goldsborough, 1993, Buczk, 2007). Reasons for these variations in taxonomic

composition of epiphytic diatoms remain unclear.

Comparison of epiphytes on plants and artificial substrates can indicate whethe

there is an interaction between epiphytes and plants. Plastic artificial substrates are

metabolically inert and serve as neutral substrates in the study of epiphytic algae.

Significant interaction between epiphytic algae and its host substrate would be reflecte

by differences in epiphytic algae assemblages on artificial substrate and plants. For

example, algal productivity was higher on plants than on artificial substrates in the

oligotrophic waters, indicating a nutrient interaction between epiphytes and theirvascular plant hosts (Burkholder and Wetzel, 1990; Cattaneo and Kalff, 1979).

In order to better understand factors affecting diatom epiphyte composition on

duckweeds, I conducted an experiment in which duckweeds and artificial substrates

were exposed to different nutrient and light levels. Other aims of this research were to

investigate whether Lemnicola would colonize artificial substrates and to compare the

composition of diatom assemblages on plants and artificial substrates.


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Materials and Methods

Experiment design

The overall objective of the experiment was to evaluate different factors that

might affect the composition of epiphytic diatoms on duckweeds. The experiment use

floating chambers with factorial combinations of nutrient enrichment (N, P, N=P and a

control) and light level (shade and open). Each chamber included duckweeds and

artificial substrates.

The experiment was conducted in a pond at the Aquatic Research Facility on th

University of Oklahoma campus for a period of two weeks, started in 13 October 2011The pond is plastic-lined and partially covered with soil. The water source is

combination of rain water and groundwater. Submerged plants includedChara spp. and

Potamogeton pectinatus L.

Three different species of duckweeds ( Lemna minor Phil. , Lemna valdiviana

Phil. andSpirodela polyrrhiza L. Scheiden) and four different colors of floating

artificial substrates were placed in each of 48 floating screen-walled plastic containers

with dimension of 10 cm x 10 cm x 10 cm (Figure 9a-e). Healthy duckweeds were

selected; counted and distributed; each plastic container had 20 Lemna minor (60 80

leaves); 10Spirodela polyrrhiza (20 30 leaves); 100 Lemna valdiviana (200 300

leaves). Lemna minor used in this experiment were collected from Canadian River,

Spirodela polyrrhiza from William Morgan Park and Lemna valdiviana from the

University of Oklahomas greenhouse.

Floating artificial substrates were constructed from 2.5 cm circles cut from

plastic sheets. Four different colors of plastic were used to either mimic the color of


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duckweeds (light and dark green) or to alter light quality (clear and black). The four

different colored circles were tacked together on the edges; and floated with small

pieces of Styrofoam.

The experiment simultaneously tested nutrients (4 treatments) and light level (2

treatments), using 6 replicates of each nutrient and light combination. (= 48 containers

Nutrient treatments used nutrient diffusing substrates, specifically nutrient-enriched

agar in open-topped glass scintillation vials (2.5 cm diameter and 4.5 cm tall). Nutrien

treatments are listed below:

1.

phosphorus treatment (P), agar solution enriched with 0.1 M KH2PO4 2. nitrogen treatment (N), agar solution enriched with 0.1 M NaNO3

3. nitrogen and phosphorus treatment (N+P), agar solution enriched with 0.1 M

NaNO3 and 0.1 M KH2PO4.

4. control (C), agar solution only

Before the experiment, the rate of nutrient leaching from the nutrient-enriched

agar was estimated by measuring the phosphorus leaching from agar under laboratory

conditions. Based on this data, nutrient vials were replaced after the first week of the

experiment to maintain high rates of nutrient release.

For light levels, the shaded treatments were covered by shade cloth on the top o

containers and unshaded treatment remained uncovered (Figure 11. a-c). Four racks

held containers, with each rack holding 12 containers. Racks were placed side by side

and were anchored in the middle of the pond (Figure 11. d-e). Experiment treatments

were randomly assigned using a program from Random.org.


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Diatoms sampling and processing

Samples were collected after two weeks. This time period was ample for colonization

Containers were removed from the pond, transferred into ice boxes and kept in a

greenhouse during sampling. Different duckweeds species and artificial substrates we

placed in separate vials and preserved with 2% formalin until processing in the

laboratory. Duckweed and plastic samples were treated with 30% hydrogen peroxide

and heat in a water bath at 80 C for one hour in order to separate diatoms from their

substrates and oxidize organic material in diatoms. Nitric acid was then added to the

sample and the sample was heated for an additional one to two hours. Samples wererepeatedly rinsed with distilled water by filling with water, settling, and pouring off

most of the water; to remove all traces of acid. The cleaned diatoms samples were

mounted into slides with Naphrax, a high refractive index mounting medium specific

for viewing diatoms. Identification and enumeration of diatoms were done by scannin

transects across the coverslips under magnification 1000x on an Olympus CX41

microscope. A minimum of 400 valves (cell wall halves) were counted for each sampl

Data analysis

Relative abundance was calculated from the diatom count of each sample. Only diatom

taxa with more than 5-% abundance were considered for the data analysis. Compariso

of epiphytic diatom assemblages across treatments (nutrients and shading) and substra

type (different plants species and different color of artificial substrates) were conducte

using distance-based permutational multivariate analysis of variance, PERMANOVA

(Anderson 2001; McArdle and Anderson 2001). The analysis is based on Bray-Curtis

distances of arc-sine square-root transformed data and was run using FORTRAN-


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written PERMANOVA.exe program (PERMANOVA v1.6) (Anderson 2005). Some

samples in this experiment were missing due to some loss of duckweeds or artificial

substrates from some chambers. Missing data were calculated as zero with the

assumption of zero presence of diatoms. Within the PERMANOVA program, pair-wis

a posteriori comparisons were conducted among different nutrient treatment (N, P, N=

and control) and among different substrates (the three duckweeds species and four

different colors of artificial substrates). The most common taxa were compared across

different nutrient and light level treatments and across different substrates using

PERMANOVA. Comparison between diatom assemblages on different duckweedspecies (Spirodela polyrrhiza, Lemna minor and Lemna valdiviana ) pre-experiment and

post-experiment was tested by two-way crossed ANOSIM (= Analysis of similarity)

(PRIMER software ver.6.1.7, Plymouth Marine Laboratory, Plymouth, England).

Figure 11 (a) Racks; (b) shaded and unshaded treatments; (c) Spirodella, Lemna minor and Lemna valdiviana and agar nutrient inside . (d) Experimental pond

a b c

d e


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Results

Eighteen diatom taxa were recorded in a high abundance (more than 5 percents

recorded in at least one sample) in this study. The most common taxa were Lemnicola

hungarica, Gomphonema parvulum, Adlafia sp. and Achnanthidium minutissima

(Appendix I).

The Non-metric Multi-Dimensional Scaling (MDS) ordination plot of all

samples showed overlapping-clouds of samples of diatom assemblages among differe

nutrient enrichment treatments and shading treatments (Figure 12 and Figure 13,

respectively). Coding the ordination by substrate type showed a distinction betweendiatom assemblages of artificial substrate and duckweeds (Figure 14.).

PERMANOVA results showed significant differences in diatom assemblages among

the nutrient enrichment treatments (F=9.12, p


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X Data

-0.20 -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15

Y D a

t a

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

Col 4 vs Col 5 - ControlCol 4 vs Col 5 - NitrogenCol 4 vs Col 5 - Nitrogen+Phosphorus

Col 4 vs Col 5 - Phosphorus

Cont rolNitrogenNitrogen+Phosphorus

Phosphorus

Different symbols showed different nutrient addition treatment.

Figure 12 MDS ordination of epiphytic diatom assemblages of different nutrienttreatments and shading treatments on artificial substrate and duckweeds.


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X Data

-0.20 -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15

Y D a

t a

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

Col 4 vs Col 5 - openCol 4 vs Col 5 - shadedopenshaded

Different symbols showed different shading treatments.Figure 13 MDS ordination of epiphytic diatom assemblages of different light


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X Data

-0.20 -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15

Y D a

t a

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

Col 4 vs Col 5 - ArtificialCol 4 vs Col 5 - DuckweedsArtificialDuckweeds

Different symbols showed different substrates.Figure 14 MDS ordination of epiphytic diatom assemblages of different substrate


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Comparison of diatom assemblages across different substrates showed two

significantly different groups, which were plant (duckweed) substrates and artificial

(plastic) substrates. These groups showed no significant differences within groups, bu

all pair-wise comparisons between the groups were significant (Appendix J).

Lemnicola hungarica showed significantly different patterns of relative

abundance on duckweeds and artificial substrates across different nutrient enrichment

and shading treatments. Lemnicola had higher abundance in the shaded than the

unshaded treatment (F=10.79, p


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p


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3.6). Adlafia abundance was relatively higher on duckweeds than on artificial substrate

(Appendix M).

Gomphonema parvulum showed differences in relative abundance across

nutrient treatments (F=10.51, p


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Figure 15 Diatoms relative abundance on artificial substrate and duckweeds oncontrol, nitrogen, nitrogen + phosphorus and phosphorus enrichment.(Top) Lemnicola hungarica (Bottom) Achnanthidium minutissimum.

a

b

c

ca

b

c

c

aa

b

c aa

b

c


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Figure 17 Relative abundance of Achnanthidium minutissimum, Adlafia sp., Lemnicola hungarica and Gomphonema parvulum on three different species of duckweeds before experiment and after experiment.


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Discussion

The most common taxa in this study were benthic diatoms with prostrate growt

form, such as Lemnicola hungarica, Achnanthidium minutissimum, Gomphonema

parvulum andAdlafia sp. These diatoms rapidly colonized artificial substrates and

duckweeds and dominated both types of substrate during the experiment. Each of thes

taxa has mucilage pads that allow firm attachment to the substrate (Round, et al, 2000

Diatom species composition responded to nutrient enrichment. In this experiment,

species composition was significantly different across control, N, N+P, and P

enrichment. Although the same species were found in all treatments, abundances varieamong treatments. Differences in diatom assemblages occur in response to changing

nutrient concentration (Pringle and Bowers, 1983; Fairchild and Lowe, 1984; Stevens

et al., 1991; Marks and Power, 2001), which makes diatoms excellent bioindicators of

environmental nutrients.

Comparison between shaded and unshaded treatment also showed significant

differences in diatom species composition. Some diatoms might be adapted to low

irradiation environment and better compete in this condition (Goldsborough, 1994).

Variability within nutrient and shading treatment in this experiment was very high. Th

might be influenced by nutrient leaching across treatments in the water experiment.

Lemnicola was phosphorus-limited. This diatom responded to phosphorus addition, but

addition of nitrogen alone had little effect on Lemnicola abundance. Lemnicola is

associated with eutrophic conditions and can grow vigorously under high nutrient

concentrations environment in its habitat (Van Dam, et al., 1994; Garcia and Foncesa

Souza, 2006).


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The nutrient experiment provided evidence of a Lemnicola -duckweed nutrient

interaction. In experimental treatments with added phosphorus, Lemnicola occurred in

high abundance on artificial substrates, but without added phosphorus, Lemnicola was

rare on artificial substrates. This dependence on phosphorus on artificial substrates bu

not on duckweeds implies a phosphorus interaction with duckweeds.

There was little evidence of Lemnicola s substrate specificity. Lemnicola was

found in high abundant on duckweeds as well as on artificial substrates, especially

where enriched with phosphorus.

Lemnicola also showed significantly higher in shade than in unshadedtreatments, an effect found on both duckweeds and plastic substrates. Lemnicola s

growth is apparently highly affected by light intensity (see also Chapter 2). In addition

differences in diatom assemblages on the underside of different colors of artificial

substrates were not detected. The lack of effect in variation of the quality of direct ligh

indicates that refracted light is an important light source for diatom assemblages on

duckweeds.

Goldsborough (1994) also looked at colonization of duckweed diatoms on

artificial substrates, using vertical plastic rods within a dense duckweed mat. Lemnicola

colonized the upper part of rods at the same depth as the duckweed; supporting the

concept that light is important. Although phosphorus was not measured, the site was a

drainage canal in a botanic garden, so likely had high nutrients. Thus, his results were

similar to ours in that Lemnicola can colonize other substrates, given the proper light

and nutrient conditions.


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Achnanthidium minutissimum rapidly colonized and became abundant on both

duckweeds and artificial substrates, although this high abundance was more pronounc

on artificial substrates. This finding is consistent with Achnanthidium s behavior as an

early colonizer with a fast immigration rate (Stevenson et al., 1991). Achnanthidium

was less numerically dominant on duckweeds, probably because duckweeds were

already colonized by diatoms.

Achnanthidium dominated the control and nitrogen treatments, whereas

Lemnicola dominated the phosphorus treatments. Although this pattern suggests

competition, an indirect nutrient effect is possible. Lemnicola is associated with highphosphorus, so reduced growth (and more space) in low phosphorus treatments might

promote colonization by Achnanthidium , an early-succession colonizer. Light

apparently is normally not a limiting factor to Achnanthidium growth and colonization

(this experiment and Stevenson et al., 1991).

Gomphonema parvulum andAdlafia sp. were less abundant than Lemnicola and

Achnanthidium on both duckweeds and artificial substrates.Gomphonema parvulum ,

like Achnanthidium , is an early colonizer and also indicates euthrophic conditions (Van

Dam, et al., 1994). Although not present at the start of the experiment, this species

colonized all substrates. Adlafia sp. was abundant on two species of duckweeds, Lemna

valdiviana andSpirodela polyrrhiza at the start of the experiment and spread to all

substrates during the experiment. NeitherGomphonema parvulum norAdlafia sp. were

affected by the shade treatments.

Diatoms on duckweeds vary in their response to nutrients and light levels.

Comparison of nutrient effects betweens artificial substrates and duckweeds


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demonstrated the role of phosphorus in promoting Lemnicola abundance and the ability

to colonize other substrates. Lemnicola was also limited by high light levels. In contrast,

early colonizing species of diatoms ( Achnanthidium andGomphonema parvulum ) were

unaffected by light level and showed an inverse pattern of abundance with Lemnicola .

This study highlighted the unique ecology of Lemnicola hungarica and indicated a

nutrient interaction between Lemnicola and duckweeds.


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Appendix A: Diatom taxa of duckweeds from 11 sites in Oklahoma K

* * * * *

* * * *

J* * *

*

I* * *

* * * * * * *

*

H L * * * * * * * *

* * * * *

*

S * * * * * *

* * * *

G W

* * * * *

* * *

S * * *

F L * *

S *

E S * * *

D L * *

* * * * * * * * * * * *

C L * * * * * * *

S * *

B L * * * * * * * * *

S * * * *

A W

S * * *

D i a t o m

t a x a

A c h n a n t h i d i u m e x i g u u m

( G r u n o w

) C z a r n e c

k i

A c h n a n t h i d i u m m i n u t i s s i m u m

( K t z i n g

) C z a .

A c h n a n t h i d i u m r i v u l a r e

P o t a p o v a

& P o n a

d e r

P s a m m o t h i d i u m l a u e n b u r g i a n u m

( H u s

t e d t ) O

. M o n n i e r

A d l a f i a s p .

A m p h o r a c o p u l a t a v a r . e p i p

h y t i c a

R o u n d

& L e e

A m p h o r a p e d i c u l u s

( K t z . )

G r u n .

A n o m o e o n e i s s p h a e r o p o r a P

f i t z e r

A u l a c o s e r i a g r a n u l a t a

( E h r e n b e r g )

S i m o n s e n

A u l a c o s e r i a p u s i l l a ( M e i s t e r )

A . T

u j i & A

. H o u

k i

B a c i l l a r i a p a x i l i f e r

( O . F . M

l l e r )

T . M a r s s o n

C a l o n e i s

b a c i

l l u m

( G r u n .

) C l e v e

C a l o n e i s a m p h i s b a e n a

( B o r y )

C l e v e

C a l o n e i s s c h u m a n n i a n a

G r u n .

C o c c o n e i s p l a c e n t u l a v a r . l i n e a t a

( H e r . )

V a n

H e u r c

k

C o c c o n e i s s c u t e l l u m

E h r .


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K S * * * *

**

J L * * * *

**

I

L * * * * * * *

H L * * * * * * * * * * *

S * * *

* * * *

G W

*

S

F L *

S * * *

E S * * *

*

D L * * * * * * * *

* * * * *

*

C L * * * * * * * * *

S * * * *

* * *

B L * * * * * *

S * * * *

A W

S * * *

*

D i a t o m

t a x a

C r a t i c u l a a m b i g u a

E h r .

C r a t i c u l a c u s p i d a t a

( K t z .

) M a n n

C r a t i c u l a r i p a r i a H u s

t .

C r a t i c u l a d i s s o c i a t a ( R e i c h a r

d t ) R e i c h .

C t e n o p h o r a p u l c h e l l a ( R a l

f s e x K u t z .

) W i l l i a m s

& R o u n

C y c

l o t e l l a m e n e g

h i n i a n a

K t z .

C y m a t o p l e u r a s o l e a

( B r e

b i s s o n )

W . S

m i t h

C y m b e l l a t u m i d u l a

G r u n .

i n S c h m

i d t e t a l .

C y m b e l l a a s p e r a

( E h r

. ) C l e v e

D e l i c a t a d e l i c a t u l a

( K t z .

) K . K r

a m m e r

D e n t i c u l a s u b t i l i s

G r u n .

D i p l o n e i s s p .

D i a d e s m i s c o n f e r v a c e a

K t z .

D i s c o t e l l a s t e l l i g e r a

( C l e

. e t G r u n .

) H o u

k & K l e e

E n c y o n e m a s i l e s i a c u m

( B l e i s c h i n

R a b e n

h o r s

t ) M a n n

E o l i m n a s u b m i n i s c u l a ( M a n g u

i n ) G

. M o s e r


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K S * * * * * *

J L *

Documents

INTERACTION BETWEEN DUCKWEEDS AND THEIR DIATOM EPIPHYTES