379j3

MORPHOLOGICAL VARIATION AND ECOLOGICAL STATUS

OF HYDRILLA VERTICILLATA (L.f.) ROYLE

IN GATUN LAKE, PANAMA

DISSERTATION

Presented to the Graduate Council of the

University of North Texas in Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

By

Jorge Bricefno M., B.S., M.S.

Denton, Texas

May, 1990 U-

Bricefio M., Jorge, Morphological variation and ecological status of Hydrilla verticillata (L.f.) Royle in

Gatun Lake, Panama. Doctor of Philosophy (Biology), May,

1990, 115 pp., 19 tables, 19 figures, bibliography, 114 titles.

Research provides biological and ecological information Panama on Hydrilla verticillate (L.f.) Royle in Gatun Lake,

for an ongoing management program of aquatic weeds in the

Panama Canal. Morphological and genetic variation,

standing crop and life cycle were determined. There were at

least three 'morphotypes' of Hydrilla in Gatun Lake.

Isoenzyme analysis of specimens from throughout the lake

showed no genetic variation, indicating that the Gatun

population is derived from a single clone. Standing crop

was seasonally dependent showing significant correlation

(P <0.05) with rain and temperature. Maximum biomass was

420 g dry mass/m2 during June and December (Bat Cove

station) and 390 g dry mass/m2 in June, August and December

(Salt and Pepper station). Chlorophyll concentration ranged

between 0.7-1.3 mg/g fresh mass in the lake and root:shoot

ratios averaged 0.57 in the Barro Colorado region. Density

of the stands was depth dependent (220 shoots/m2 at 5 m).

Extensive defoliation by the moth Parapoynx sp. occurred in

Hydrilla stands during the 1988 and 1989 dry seasons, suggesting that this species of Pyralidae may be a potential biological control of Hydrilla in the lake. Life cycle of

Hydrilla was affected by season of the year, turbulence and water transparency, and regulated partially by defoliation

(Parapoynx sp.) and epiphytic algae. Patterns of stand

decline were associated with micro-habitat variations in water flow, depth and substrate character. Flowering in

Hydrilla occurred from November through March. Only male

flowers were found in Gatun. Recommendations for integrated

management of aquatic weeds in the Gatun Lake are: (1)

modification in treatment timing in agreement with

seasonality of Hydrilla biomass in particular habitats; (2)

application of sublethal treatment concentration of

herbicides to weaken Hydrilla plants; and (3) use of

Parapoynx sp. as a biological control during periods of peak

biomass in combination with mechanical or chemical controls. ACKNOWLEDGEMENTS

Partial financial and logistical support were provided by the following institutions: Universidad de Panama,

Smithsonian Tropical Research Institute, Panama Canal

Commission and The Organization of American States. I greatly appreciate the collaboration and encouragement throughout the study of Yolanda Aguila, Jose A. Martinez,

Luis D'croz, Haris Lesios, Ricardo Gutierrez, Gerardo

Caceres, Alberto Taylor, Mireya Correa and Ligia and Axel

Calderon. Special thanks to Denis Serrano, Liliana Arauz,

Edgardo Munoz, Daniel Holness, Erick Viveros, Deisa Piedad

Lamela, Rosana Dosman, Marisol Murillo, Amarilis Gutierrez

and Martin Mitre, members of the 'Hydrilla team' for their

enthusiastic field work and technical assistance.

iii TABLE OF CONTENTS

Page

LIST OF TABLES ...... V

LIST OF FIGURES...... VII

CHAPTER

I. INTRODUCTION ...... --...... 1

II. STUDY AREA ...... --...... 6

III. MATERIAL AND METHODS...... 15

Morphology ...... --...... 15 Isoenzyme analysis ...... 18 Histology ...... -...... 19 Standing crop ...... 20 Shoot length and mass relations ...... 21 Chlorophyll ...... 22 Associated fauna ...... 23 Life cycle ...... 23

IV. RESULTS ...... -...... 24

Morphometric analysis ...... 24 Isoenzymes...... 43 Fresh mass:Dry mass relations ...... 44 Chlorophyll ...... 50 Length and mass relation ...... 53 Standing crop ...... 61 Associated fauna ...... 72 Life cycle ...... 76

V. DISCUSSION...... 84

Morphology ...... 84 Isoenzymes ...... 91 Mass:length relationship ...... 93 Standing crop ...... 96 Associated fauna ...... 97 Life cycle ...... 98 Summary...... 103

REFERENCES...... 106

iv LIST OF TABLES

TABLE Page

1. Stations sampled in Gatun Lake, Panama from September 1987 through February 1989 ...... --...... 8

2. Morphometric variables assessed in herbarium specimens of Hydrilla verticillata (L.f.) Royle from Gatun Lake, Panama ...... 16

3. Buffer description and characteristics of electrophoresis setting for isoenzymes tested ...... --...... 19

4. Descriptive statistics of leaf surface area index of Hydrilla verticillata in samples from Gatun Lake, Panama ...... 28

5. Descriptive statistics of number of teeth along the margin of leaves of Hydrilla verticillata in samples from Gatun Lake, Panama ...... -...... 29

6. Descriptive statistics of stem diameter measurements (SDM) in Hydrilla verticillata from samples collected in different sites of Gatun Lake, Panama ...... 30

7. Descriptive statistics of leaf form index of Hydrilla verticillata in samples from Gatun Lake, Panama ...... 31

8. Descriptive statistics of number of leaves in the main stem apex of Hydrilla verticillata in samples from Gatun Lake, Panama ...... 33

9. Eigenvalue, percent of discriminant power (%DP), and canonical correlation coefficient (CCORR) associated with each discriminant function...... 37

10. Correlation values for standardized discriminant coefficient and discriminant variables ...... 40

V 11. Isoenzyme phenotypes of SOD, MDH, PGI, TPI and PO in samples of Hydrilla verticillata from Gatun Lake, Panama ...... ----.----. 43

12. Fresh mass dry mass conversion factor estimated for Hydrilla verticillata in samples from Salt and Pepper station ...... ----..-. 44

13. Root to shoot ratio estimated in individual plants of Hydrilla verticillata from Bat Cove station in April 19880...... 49

14. Depth variation of shoot density, plant weight and biomass per unit of volume (B/V) of a stand in Arenosa station during April 1988...... --..-..-..-50

15. Concentration of chlorophyll pigments estimated in Hydrilla verticillata from samples of different stations in Gatun Lake, Panama0...... -.------54

16. Comparison of mean standing crop values (g dry mass/m2) of Panamanian rainy and dry season in Barro Colorado Island ...... 66

17. Pearson correlation coefficient of selected environmental parameters with standing crop (g dry mass/m2) data of Hydrilla verticillata ...... 71

18. Invertebrate fauna associate with Hydrilla verticillata in Gatun Lake ...... 75

19. Spearman correlation coefficients of water quality parameters and Hydrilla verticillata morphotypes in Gatun Lake, Panama ...... 88

vi LIST OF FIGURES

FIGURE Page

1. Map of Gatun Lake, Panama showing the main stations collected during September 1987 through February 1989 ...... 12

2. Shoot section of Hydrilla verticillata (L.f.) Royle, showing characters assessed in the morphometric analysis of Gatun Lake population ...... 26

:3. Dendrogram showing classification of 287 herbarium specimens of Hydrilla verticillata (L.f.) Royle, collected at 13 stations of Gatun Lake, Panama ...... 35

4. Plot of first and second canonical functions indicating group centroids (average group score) ...... 39

5. Transverse section of stems from (A) Dump 2 ,and (B) Chagres River stations showing central cylinder and lacunae ...... -...... 42

6. Electrophoretic phenotype of Triose Phosphate Isomerase in stem apex of Hydrilla verticillata (L.f.) Royle ...... 46

7. Electrophoretic patterns in Hydrilla verticillata (L.f.) Royle , for PO, TPI, SO, MDH, and PGI0...... 48

8. Relationship between fresh mass (mg) and size (cm) of Hydrilla verticillata (L.f.) Royle...... 52

9. Biomass allocation in individual shoot (shoot weighed each 10 cm) of Hydrilla verticillata (L.f.) Royle. Total shoot length 1.24 m, total shoot mass 3.6 g...... 56

10. Biomass allocation in individual shoot (shoot weighed each 10 cm) of Hydrilla verticillata (L.f.) Royle. Total shoot length 2.19 m; total shoot mass 5.5 g ...... 58

vii 11. Biomass allocation in individual shoot (shoot weighed each 10 cm) of Hydrilla verticillata (L.f.) Royle. Total shoot length 7.35 m; total shoot mass 14.9 g...... 60

1:2. Schematic drawing of branching patterns observed at (A) Dump 2, and (B) Cuipo stations in Gatun Lake, Panama in May 1988...... ------0...... 63

13. Schematic drawing of branching patterns observed at (A) Bat Cove and (B) Salt and Pepper stations in Gatun Lake, Panama in May 1988...... ------65

14. Seasonal variation of standing crop (g dry mass/m2) of Hydrilla verticillata (L.f.) Royle, at Salt and Pepper station in Gatun Lake, Panama...... ------..--...68

15. Seasonal variation of standing crop (g dry mass/m2) of Hydrilla verticillata (L.f.) Royle, at Bat Cove station in Gatun Lake, Panama...... 70

16. Adult (A) female, and (B) male of Parapoynx sp. (Lepidoptera) collected above the stand of Hydrilla verticillata (L.f.) Royle ...... ------.-.-.-.-.-.-. 74

17. Relative percentage of Hydrilla stands decline in (A) Salt and Pepper and (B) Lacruces stations during 1988-1989...... ------.--.78

18. Schematic representation of the life cycle of Hydrilla verticillata (L.f.) Royle, in Gatun Lake, Panama...... 80

19. Carpellate flowers of Hydrilla verticillata (L.f.) Royle, collected at Bat Cove station during November 1988...... 83

viii CHAPTER I

INTRODUCTION

Hydrilla verticillata (L.f.) Royle is a serious aquatic nuisance in many regions of the world, including the United

States, Southeast Asia, Australia, New Zealand, Africa and

Central America, specially in the Panama Canal. It resists effective control measures through the ability to grow profusely and produce large standing crops under a variety of environmental conditions. Hydrilla has been the subject of many research efforts, demonstrating a continuing worldwide interest in its biology (e.g., Bowes et al., 1977;

Cook and Luond, 1982; Kulshrestha and Gopal, 1983; Barko et

al., 1988). As a member of the family Hydrocharitaceae,

this submersed perennial has been reported as both dioecious

and monoecious, the latter able to produce viable seeds

(Haller et al., 1976; Bowes et al., 1979; Environmental

Laboratory, 1985; Steward and Van, 1987).

Since introduction into the United States Hydrilla has

spread from Florida north into Virginia and Washington D.C.,

and westward through the southern states into California,

demonstrating an extraordinary colonizing capability (Gore,

1976; Maceina and Shireman, 1980). Globally, it ranges from 55* N to 400 S latitude, showing a clear adaptation for

warmer regions (Environmental Laboratory, 1985). 1 2

Major reasons for the success of Hydrilla in colonizing different environments involve special physiological, reproductive, morphological and genetic characteristics. of Hydrilla is one of the few aquatic plants capable has been photosynthesis at low light levels. This ability

studied in detail since the early 1970's (Bowes et al., and 1977; Van et al., 1977; Holaday et al., 1983; Ascencio

Bowes, 1983). Bowes (1987) indicated that, in general, and aquatic macrophytes have low rates of photosynthesis

productivity resulting from low diffusion rates of CO2 and has a biochemical 02 in water. As a C-4 plant Hydrilla mechanism, involving cytosolic phosphoenol pyruvate

carboxylase, that facilitates concentration of CO2 .

Aquatic weeds invade and dominate the flora of lakes and

reservoirs because of their vegetative reproductive

potential (Haynes, 1988). Sexual reproduction in many

aquatic plants appears to be a secondary reproductive tactic

(Philbrick, 1988). Hydrilla, in general, fits the

reproductive model of other aquatic weeds (e.g.,

Myriophyllum spicatum, Elodea canadensis, Potamogeton

nodosus). Its reproductive mechanisms and environmental

correlates have been reported by Cook and Luond (1982), and

Haynes (1988). Pollination occurs on the water surface.

Male flowers burst when they reach the surface and pollen is 3

scattered in the air. The female flower has to be close by grains that since dispersion of pollen is limited. Pollen land on the water are lost for reproductive purpose (Cook, Bowes et al. 1988; Haynes, 1988). Haller et al. (1976) and conditions (1979) indicated that under adverse growing

Hydrilla produces tubers and turions (i.e., specialized a asexual structures) in sufficient density to reestablish

complete population vegetatively.

Plant variation has been recognized as environmentally

related, particularly by taxonomists trying to identify

different samples taken from a single 'species' (Briggs and

Walters, 1969; Crawley, 1986). In weeds and clonal of the organisms phenotypic plasticity (i.e., the proportion

phenotype affected by the environment) is a major adaptation

for dispersal and colonization of different environments

(Harper, 1965; Cook, 1983). The external morphology of

Hydrilla, described in detail by Yeo et al., (1984), is

highly plastic and readily modified by changes in habitat

conditions (Cook and Lound, 1982). Furthermore, Cook and

Luond, in their review of Hydrilla, emphasized its wide

geographical distribution, and its special morphological and

reproductive adaptations that contribute to success in

colonizing new lakes or reservoirs.

Genetic variability in Hydrilla has been documented

recently (e.g., Verkleij et al., 1983; Pieterse et al., 4

1984; Pieterse et al., 1985 and Ryan, 1988), and has been

suggested as another principal reason for its highly

successful dispersion and colonization throughout Southeast

Asia, Australia, Central Africa and the Americas. The above

studies focused on among population comparisons; none

addressed genetic or morphological variation within a single

population. Silander (1985) indicated that the relation

between genetic variation and phenotypic plasticity in

clonal organisms (e.g., weeds) is highly dependent on

environmental conditions, and to understand the behavior in

a particular species requires both between and within

population studies.

Since there appears to be no published information on

within population variation of Hydrilla I began the study of

the population in Gatun Lake, the main reservoir for the

Panama Canal and the largest artificial lake in the Republic

of Panama. Hydrilla has been present in Gatun for

approximately 30 years (Zaret, 1984) where it is now the

dominant species, and the object of an aggressive and costly

management program.

Gatun Lake provides an excellent environment for

studying within variation in a single Hydrilla population.

Physical characteristics such as an intricate shoreline with

numerous coves having shallow waters, complex wave and wind

effects, different types of sediments, and rapid renewal or

I - IN 5

turnover rate of water provide a heterogenous habitat throughout the lake which allows studying relations between

Hydrilla and local environmental conditions. Since these among habitat variations within Gatun Lake are nearly as diverse as those encountered geographically by Hydrilla, this study should enable comparison of within-population variability with among-population variability reported in

the literature.

The specific objectives of my research were to: (1)

determine morphological variation of Hydrilla within the

lake; (2) estimate genetic variation by a preliminary survey of isoenzyme patterns; (3) examine the histology of selected

tissues; (4) examine seasonal variation of standing crop;

(5) assess shoot length-mass relations; (6) survey

organisms having potential for biological control of

Hydrilla; and (7) relate Hydrilla's life cycle to key biotic and abiotic factors within Gatun Lake. The overall goal was

to correlate biological variation within Hydrilla to

environmental variation throughout Gatun Lake, compare that

to published among-population variation data and evaluate

the patterns in terms of adaptive responses. CHAPTER II

STUDY AREA

Gatun Lake, located in the Republic of Panama, was formed in 1914 as part of the construction of the Panama

Canal. Its main purpose has been to provide the elevated water level necessary for passing ships through the Canal

(PCC Brochure, 1986). Gatun Lake, has a surface area of 425

Km2 with a total drainage area of 2,313 Km2 that extends from Gatun locks in the Atlantic to Miraflores locks in the

Pacific. At present it has an average storage capacity of

642 X 106 m3 of water with a total estimated watershed of

4.81 X 109 m3 (Zaret, 1984). Until 1936 when Lake Mead was formed as part of the Hoover Dam constructions, Gatun Lake was considered the largest man-made lake in the world.

The Panama Canal uses freshwater to raise ships entering into the Canal in three steps (using a system of locks) from sea level to Gatun Lake level (ca. 23 m). Since the locks operate by gravity, 1.96 X 108 L of water is lost for every ship passing through the Canal. Conservation of freshwater becomes a continuous priority for Canal operation and water levels in Gatun Lake are kept as high as possible throughout the year. Undoubtedly, Panama's special climate has been a key factor contributing to the success of the

Panama Canal.

6 7

In Panama, two well defined seasons occur during the

year. The dry season which lasts from January through

April, is characterized by low precipitation (<30 mm),

strong northern winds of about 30 km/hr and intense solar

radiation (ca. 405 langleys/day). The wet season occurs

during May through December when precipitation rises to ca.

2400 mm. The total annual rainfall during 1987 and 1988

(period of the study) at Barro Colorado Meteorological

Station were 2,953 and 2,602 mm, respectively. Radiation

decreases to a daily average of 230 langleys whereas

prevailing winds are typically from the east, southeast and

northwest and are less intense (<15 km/hr) than in the dry

season.

Air temperature over the lake was highest during April

(320C) and lowest during November (250C) . Water temperature

during the study ranged from 28 to 300 C during the dry

season and 25 to 26.50C during the wet season. The mean

annual relative humidity in the Canal area was 83 percent

with a range of 75 to 90% during the year.

Based on phosphorus concentrations, Gatun Lake is

considered to be in a mesotrophic state of eutrophication

(Gonzalez et al., 1975; Margalef, 1983; Canfield et al.,

1983). The main source of freshwater for the lake is the

Chagres River, which originates in the central mountains of

rwilmw, 41 pion 8

TABLE 1

Stations sampled in Gatun Lake, Panama from September 1987 through February 1989, numbers of the stations correspond to map numbers in Figure 1. Coordinates provided by Panama Canal Commission Cartographic Services and expressed in standard order (i.e., degrees, minutes and seconds).

GEOGRAPHIC COORDINATES STATION REGION LOCALITY LATITUDE LONGITUDE

1 CHAGRES AGUARDIENTE 09 09 00 79 40 06

2 CHAGRES LACRUCE 09 07 37 79 57 39

3 GAMBOA DUMP 2 09 06 41 79 42 16

4 GAMBOA DUMP 4* 09 07 36 80 19 53

5 GAMBOA PUENTE* 09 06 53 79 42 04

6 GATUN GATUN 1 09 15 44 79 54 56

7 GATUN GATUN 2 09 10 17 79 59 04

8 GATUN GATUN 3 09 07 48 79 48 18

9 GATUN LIRIOIS 09 14 55 79 51 17

10 BARRO COL. FARO* 09 07 36 79 48 11

11 BARRO COL. BAT COVE 09 05 00 79 45 00

12 BARRO COL. SALT&PEPPER 09 10 00 79 45 00

13 MIRAFLORES MIRAFLORES* 08 59 37 79 35 22

14 ESCOBAL ESCOBAL 09 08 35 79 57 39

15 CUIPO CUIPO 09 06 02 79 59 25

16 ARENOSA ARENOSA 1 09 02 22 79 57 20

17 ARENOSA ARENOSA 2 09 03 57 79 57 50

18 ARENOSA ARENOSA 3 09 02 22 79 57 30

* Stations only sampled for the genetic survey. 9

the Continental Divide (Pacific side of Panama) and runs to

outfall in the Atlantic side. Its waters are considered to

be of superior drinking quality, soft (<50 ppm hardness),

clear and almost free of major pollutants (Gonzalez et al.,

1975).

Specific locations of the sampling stations in this

study are summarized in Table 1. Characteristics of the

lake such as transparency, dissolved oxygen, pH, depth and

physical appearence of the stations are described according

with major regions in a subsequent section.

Chagres-Gamboa region is characterized by waters that

originate from the hypolimnion of Alajuela Dam in the upper

sections of Chagres River. This dam was built to control

water levels at Gatun Lake and as a reservoir for dry

periods when water levels become critical. Waters below the

dam are turbid (Sechii readings 0.5 - 1 m), low in dissolved

oxygen (<4 ppm) and high in organic matter that provide

appropiate conditions for aquatic plants. Besides the

Chagres River, another important source of sediments and

nutrients is the Chilibre River which contributes an

important amount of water specially during the wet season.

Three stations were sampled in this region: Aguardiente,

Lacruce and Puente.

The town of Gamboa hosts the facilities of the Dredging

Division of the Panama Canal. The Dredging Division's main 10

task is to deepen the channels and clean any debris that hamper free navigation in the Canal. This section (Dredging

Division facilities) of the Canal is called the Gaillard Cut and several dump areas are allocated to collect waste products of dredging operations. These areas are shallow

(ca. 1 - 5 m deep)., with abundant aquatic vegetation. Dump

2 and Dump 4 were included as stations for the study.

North of Gamboa (see Fig.1) is Barro Colorado Island which was considered a region to be examined in this study.

This region comprises the Canal channel between Barbacoa

Island and Bohio Peninsula including Gigante and Frijoles bays. Barro Colorado is the largest island in the lake and was recently declared a national monument for nature protection and wildlife preservation under the management of the Smithsonian Institution. In this region the Canal depth varies between 25 and 35 m in the main channel, but also it is common to find shallow areas from 1 - 5 m at both sides of the Canal. Transparency is variable depending on the season and the traffic of large vessels since turbulance originated by waves is common in the area (range of transparency during this study 2.5 - 7.0 m). During the dry season, winds enhance mixing of water with waves large enough (up to 1.5 m) to interfere with navigation of small boats.

______11

Fig. 1. Map of Gatun Lake, Panama showing in numbers the main localities collected during this study from September 1987 through February 1989.

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The Escobal region is located in the west section of the lake, providing the access channel to the towns of

Escobal and Cuipo. These small towns are farm and cattle oriented with noticeable signs of deforestation. Abundant aquatic vegetation covers the shorelines of this region indicating high productivity (eutrophication). Transparency measured as Sechii disk reading ranged between 3 and 4 m, pH and dissolved oxygen presented regular values for the lake

(7.3 and 7.2 ppm, respectively). Samples were collected in shallow bays (<4 m) of both towns and the stations were named accordingly.

Arenosa region is located at the southwest side of the lake and includes the town (Arenosa) and the access channel connecting with Trinidad Bay (Fig.1). The main channel is the deepest area (ca. 10 m) in this region. In general the region is quite shallow with abundant stumps and logs that make navigation difficult. Aquatic vegetation is abundant especially during the end of the dry season (June and July).

Transparency ranged between 3 - 5 m during the study. Three stations were sampled in this region: Arenosa dock, Buoy 1

(Arenosa 1) and Buoy 2 (Arenosa 2) on the access channel.

The north region of Gatun lake comprises the locks, recreation dock, and surrounding islands around buoys 6, 7 and 8 of the Canal. In this region water transparency reached its maximum readings (up to 9 m) during the dry 14

season. The main channel of the Canal is approximately 30 m deep. Three stations were used for collection in this region: Gatun 1 (dock), Gatun 2 (Buoy 7), and Gatun 3 (Buoy 8). East of the bascule bridge at the northeast section of the lake is located Monte Lirio region. Depth in the area ranges widely, from 0.5 to 20 m. Emergent stumps and logs are common in this section of the lake. Water quality parameters were typical of other regions of the lake

(dissolved oxygen 6.8 ppm, pH 7.6 and transparency of 4 m Sechii reading). Only Lirio island (Liriois) station was used for collection in this region. CHAPTER III

MATERIAL AND METHODS

Morphology

Morphometric analysis

Healthy shoots of Hydrilla verticillata (L.f.) Royle were collected from 17 different stations in Gatun Lake from

November 1987 through December 1988 (Table 1). Collections were made by hand (skin diving) at depths ranging between

0.5 to 3 m (Raschke and Rusanowski, 1984). Samples were kept in plastic bags and taken the same day to the Centro de

Ciencias del Mar y Limnologia, Universidad de Panama.

After rinsing with tap and distilled water to eliminate periphyton, plant material was gently blotted with paper towel, and prepared for herbarium specimens. Plants were stacked in a plant press, bound as tightly as possible and dried at 320 C for approximately a week (Wood, 1975). For each station 20 - 35 specimens were prepared keeping appropriate record of station and catalog number. A total of 285 herbarium specimens were prepared during the study

(available in the Centro de Ciencias del Mar y Limnologia,

Universidad de Panama).

For each herbarium specimen, the state of eight non reproductive morphological characters (Table 2) was

15 16

TABLE 2

Morphometric variables* assessed in herbarium specimens of Hydrilla verticillata (L.f.) Royle from Gatun Lake, Panama.

CHARACTER DESCRIPTION OF MEASUREMENT

NLA Number of leaves in the whorl (apex)

NTL Number of teeth around the leaf margin

SDM Stem diameter measure at the base of the shoot (widest point) in mm.

LL Total length of leaf (mm)

LW Width of the leaf (mm)

IND Internode distance (mm)

LFI Leaf shape expressed as the ratio between length/width.

SAI Surface area index expressed as length X width.

* Values of morphometric variables were determined in five replicates and the means were used for further calculations.

determined. These characters were selected based on

previous descriptions of Hydrilla in the literature (Den

Hartog, 1973; Cook and Luond, 1982; Yeo et al., 1984). Five

readings of each character were taken per specimen to

estimate the arithmetic mean. Descriptive statistics (mean,

standard deviation, range, coefficient of variation) of the

complete data set (N=1,425 observations) were estimated for

each morphological variable (Zar, 1984). 17

Numerical analyses of the data were performed on mainframe equipment of the Computer Science facilities,

University of North Texas, Denton, Texas. Clustering of collected stations was performed to determine similarities between sites indicated by general morphology of Hydrilla plants. The cluster analysis was performed using the average linkage between group method (UPGMA), available in the SPSS-X procedure (SPSS-X, 1986). The data base used in the classification and ordination of stations was tested for departure from normality using the UNIVARIATE procedure (SAS

Institute Inc., 1985). Each morphometric variable was transformed by log10 (continuous data) or a square root transformation (discrete data) to fulfill the assumption of a multivariate normal distribution (Pimentel, 1979; Reyment, et al., 1984). Euclidean distances of standardized morphometric values were used as the clustering criterion

(Sneath and Sokal, 1973).

A stepwise discriminant analysis (Sanathanan, 1975;

Tabachnick and Fidell, 1983; Norusis, 1986) was performed to select morphological variables that contain most of the classification information (discriminant labels) to differentiate among the sampled stations (variables to be classified). Only discriminant functions which were significant (P <0.05) when tested against Wilk's lambda 18

(SPSS-X, 1986) were used as discriminant criteria to identify potential 'morphotypes' in Gatun Lake.

Isoenzyme analysis

Fresh stems from the apex of Hydrilla were collected in selected areas of Gatun Lake (Table 1). Samples were placed in plastic bags with water, kept in a cooler and transported to the laboratory during the next 24 h. After rinsing with tap and distilled water the samples were blotted and frozen

0 at -850C. Frozen stem apices were ground at ca. 4 C in a phosphate buffer, pH 7.2 (Verkleij et al., 1983) with sand that was previously sieved, rinsed in distilled water and dried. The homogenates prepared were kept in the freezer

(-850C) until electrophoresis was conducted.

Electrophoresis was performed using horizontal starch gel techniques (e.g., Brewbaker et al., 1968; Brewer, 1970;

Shaw and Prasad, 1970; Vallejos, 1983). Gels were prepared with Electrostarch lot #86 (Otto Hiller Co.) at 11% concentration. Filter paper wicks (Whatman 3) were dipped into stem apex extracts, blotted to remove excess of liquid and inserted into starch gel recently prepared (i.e.,

<24 h). Table 3 summarizes technical details of each experiment including buffers, time and current set for the different enzymes analyzed. After electrophoresis, the gels were sliced horizontally and the enzymes were identified following staining procedures of Vallejos (1983) and Shaw and Prasad (1970). Eight enzyme systems were investigated: 19

TABLE 3

Buffer description and characteristics of electrophoresis settings for isoenzymes tested in shoots apex of Hydrilla verticillata (L.f.) Royle from Gatun Lake, Panama. Volts (constant voltage), mA (miliampers) and time in hours.

BUFFER ENZYME VOLTS m A TIME

TEB1 PO, MDH, PGI 200 80 4:00

LiOH2 AAT, SOD 250 80 5:00

TRIS-GLY3 PGM, SkDH 200 40 4:30

4 5:30 PC7 TPI 50 150

1. Continuous buffer, pH 8.7, reference Vallejos (1983). 2. Discontinuous buffer, pH 8.1 (gel), pH 8.4 (electrodes),reference Vallejos (1983). 3. Discontinuous buffer, pH 8.9 (gel), pH 8.3 (electrodes),reference Verkleij et al. (1983). 4. Continuous buffer, pH 7.0, reference Vallejos (1983).

peroxidase (PO), shikimate dehydrogenase (Shk), superoxidase dismutase (SOD), malate dehydrogenase (MDH) phosphoglucomutase (PGM), aspartate aminotransferase (AAT), phosphoglucose isomerase (PGI) and triosephosphate isomerase

(TPI). Stained gels were preserved in 70% glycerine and photographed (Polaroid equipment) for future analysis. Histology

To detect possible histological differences in Hydrilla plant sections (leaves, roots, stem apex) were collected from different sites in the lake (Table 1), and preserved in 20

the field in a mixture of formalin, ethanol and acetic acid

(Yeo et al., 1984). In the laboratory, samples were dehydrated in absolute ethanol series, cleared in xylene and embedded in paraffin. Microtome sections were prepared, mounted and stained with safranin-fast green (Sass, 1958;

Drury and Wallington, 1980; Kiernan, 1980).

Ecology and Life Cycle

Standing Crop

To explore how the environmental conditions in Gatun influence the growth and distribution of Hydrilla, two stands located in Barro Colorado Island (Salt and Pepper and

Bat Cove, Tables 1, 2) were sampled periodically (i.e., monthly) from September 1987 through February 1989.

Standing crop samples were taken randomly from each stand using the harvesting method (Westlake, 1965; Lind, 1979;

Wetzel and Likens, 1979). A bottomless metal cage (35 cm x

35 cm), defining a sample area of 0.12 m2 was dropped from a boat at depth between 1 - 3 m, divers then removed shoots and roots enclosed within the cage and lifted the sample to the boat where they were washed and placed in plastic bags and returned to the laboratory during the same day.

Five replicates were collected from each station according to sample criteria that consider stand density, sample unit and standard error established by Downing and

Anderson (1985). This number of replicates represent a 21

compromise between sample effort, cost efficiency and a standard error of approximately 20% from the mean. With the number of replicates and sample size used, the standard error of Bat Cove station was about 24% whereas in Salt and

Pepper station the standard error was approximately 35% .

In the laboratory the samples were re-washed and dried with blotting paper, fresh mass (precision, 0.10 g) was determined immediately after a sub-sample (15 - 20% of the total biomass of each sample) was dried to constant mass in an oven at 700 C. Standing crop values are reported as g dry mass/m2 1 standard deviation from the mean. Ash content was estimated for samples (September 1987 - April 1988) by combustion at 5000 C to determine seasonal variation of ash free biomass between stands (APHA, 1976).

Shoot length and mass relations

Complete plants were also removed by divers to determine root:shoot ratio, length, biomass allocation in single shoot. All mass measurements were taken as fresh mass (precision 0.01 g) and reported as mean 1 standard deviation from the mean. Weights for biomass allocation were taken by selecting complete Hydrilla plants (root and shoot) defining the main shoot (the longest one) and weighing each 10 cm from the top to the bottom of the plant.

Lateral shoots were included within their respective section

(i.e., for each 10 cm). Branching of plants was recorded in 22

similar fashion measuring the main and lateral shoots to scale in fresh specimens.

Seasonal variation and relationship of standing crop data with environmental parameters as well as length mass relations were analyzed using UNIVARIATE, STEPWISE, RSQUARE and GLM procedures available within the SAS package (SAS

Institute, 1985). Statistical tests used, to further interpret the data included t-test, ANOVA, regression and correlation, all the tests were conducted at P = 0.05 (Zar,

1984).

Chlorophyll

Extraction of photosynthetic pigments (Chlorophyll a + b) from the stem apex of Hydrilla was accomplished according to a modified method for vascular plants described by

Vollenweider (1974). Fresh shoot apices (three replicates, approximately 150 mg) collected from the field were extracted with 10 ml of acetone (90%) and buffered with 2 ml of MgCO3 . Samples were stored in a freezer in the dark, after 24h the samples were centrifuged at 3,000 rpm and readings were taken at 665 nm and 645 nm. Values of chlorophyll a + b were determined using equations described by Jeffry and Humprey (1975). Final chlorophyll values arereported as mg Chlorophyll per g fresh mass (Van et al.,

1976; Bowes et al., 1977; Pokorny et al., 1984). 23

Associated fauna

Samples of Hydrilla were taken by hand (divers) and weighed in the field ( 0.1 g), placed in plastic bags and transported to the laboratory. A sample unit of 250 g fresh mass was selected to assess diversity of macroinvertebrates in visited stands from November 1987 through July 1988

(Table 1). Three to six replicates from each stand were taken and inspected for associated organisms. According to taxa, different preservative solutions were used (ethanol

70%, AGA solution, formalin 5%; Needhan and Needhan, 1966).

Identification of organisms was accomplished with different taxonomic keys such as Pennak (1978), Merrit and Cummins

(1984), and Stehr (1987).

Life Cycle

Field observations in sporadic and permanent stations within the lake provided data to describe production and decline patterns of Hydrilla in Gatun Lake. Evaluation of zonation, condition (i.e., healthy or declining) and senescence in the stands were made from boat transects parallel and perpendicular to shoreline and from direct underwater observation (Wood, 1975; Lind, 1979). The growth pattern observed in Gatun is compared with a general growth model for aquatic plants available in the literature

(Westlake, 1965; Wetzel, 1983). CHAPTER IV

RESULTS

Morphological and Genetic variation

Morphometric analysis

The principal morphological characters used to assess phenotypic variation within the Hydrilla verticillata (L.f.)

Royle population in Gatun Lake are illustrated in Figure 2.

These are used as key nonreproductive characters in the identification of Hydrilla specimens. Superficially, the basic shoot morphology was similar within the Gatun Lake population. However, detailed measurements of herbarium specimens revealed significant differences in specific characters. The mean leaf surface area index (SAI) expressed as length (mm) X width (mm) ranged from 18.9 to

37.7 (Table 4) reflecting different environmental conditions in the lake (turbidity, radiation, and substrate). Plants with largest SAI occurred in Chagres

River region (Lacruce and Aguardiente stations) which is mostly characterized by frequent runoff and low transparency

(<3 m) whereas the smallest SAI values were found in plants from Barro Colorado Island (Salt and Pepper, Bat Cove, and

Frijoles stations) where transparency throughout the year had a wider range (3-7 m).

24 25

Fig . 2. Shoot section of Hydrilla verticillata (L,.f.) Royle, showing characters assessed in the morphometric analysis of Gatun Lake population. Indicated are number of leaves in a whorl (a), internode distance in mm (b), number of teeth along the leaf margin, and leaf length (c), and width of the stem in mm (d) . 26

ba

C

d 27

The number of teeth or leaf margin serration showed slight variation about a mean of 26 for the Gatun population

(Table 5). There was a significant difference between mean serrations in plants with large and small leaves

(independent t-test, t=2.10, df=174, P <0.05). Plants with large leaves (>SAI) occurred in Cuipo, Aguardiente, and Salt and Pepper areas; small leaves (

Means for stem diameter (SDM) measurements varied from

0.8 to 2.0 mm in the Gatun population (Table 6). The coefficient of variation (CV) for SDM in almost all areas was about 17%. Stems of plants from Chagres River (Lacruce and Aguardiente sites) and Dump 2 station were consistently thicker than other sites (Escobal, Frijoles, Miraflores).

The leaf form index (LFI) expressed as the ratio of length (mm)/width (mm) indicates broadness or slenderness of a leaf. Values of LFI measured in Hydrilla from Gatun Lake ranged between 4-9 with a maximum CV of 16% in the Salt and

Pepper area. There was a considerable plasticity in leaf form in the Gatun population (Table 7). The mean number of leaves in the stem apex (whorls) was a good indicator of stand productivity. The highest number of leaves were found in areas of Cuipo (9), Escobal (8) and Arenosa (8.5) during the period of maximum standing crop (July and August), whereas lower numbers (ca. 5; Table 8) occurred when 28

TABLE 4

Descriptive statistics of leaf surface area index* of Hydrilla verticillata in samples from Gatun Lake, Panama. For each station N=number of samples, SD=Standard deviation, and CV=Coefficient of variation. Grand mean (GM) and Grand coefficient of variation (GCV) are provided.

STATION N X SD RANGE C.V.%

AGUARDI 120 37.7 4.6 29.3 - 45.8 12.2

AREBOYA 60 24.1 2.9 19.2 - 30.0 12. 1

ARENOS1 100 19.4 2.5 15.4 - 24.4 12.7

ARENOS2 40 21.1 2.7 17.5 - 26.2 17.7

BATCOVE 92 32.6 4.0 22.4 - 39.1 12.4

ESCOBAL, 100 26.4 2.5 22.0 - 32.1 9.5

GACUIPO 100 22.8 3.3 18.0 - 30.0 14.6

GADUMP2 104 20.4 1.1 18.2 - 22.0 5.4

GATUNP1 100 23.3 1.8 19.9 - 28.6 7.8

GATUNC2 100 20.9 1.4 17.8 - 23.9 6.5

LACRUCE 76 29.8 2.5 26.5 - 34.3 8.3

LIRIOIS 72 24.0 2.3 17.6 - 28.6 9.7

SALTYPI 81 18.9 4.4 8.2 - 26.4 23.3

GM=22 .4 GCV=11. 7

* SAI=Length (mm) Width (mm) 29

TABLE 5

Descriptive statistics of number of teeth along the margin of leaves of Hydrilla verticillata in samples from Gatun Lake, Panama. For each station N=number of samples, SD=Standard deviation, and CV=Coefficient of variation. Grand mean (GM) and Grand coefficient of variation (GCV) are provided.

STATION N X SD RANGE C.V.%

AGUARDI 120 26.4 2.8 20.0 - 32.0 10.5

AREBOYA 60 23.3 2.7 20.2 - 28.0 11.7

ARENOS1 100 26.7 1.9 23.0 - 32.0 7.2

ARENOS2 40 24.2 2.4 21.0 - 27.6 9.7

BATCOVE 92 25.1 2.6 22.0 - 30.6 10.5

ESCOBAL 100 29.6 1.9 26.5 - 33.8 6.4

GACUIPO 100 28.2 3.1 24.0 - 35.5 11.1

GADUMP2 104 26.9 2.0 22. 0 - 30.0 7.5

GATUNP1 100 23.8 2.2 18.5 - 28.6 9.3

GATUNC2 100 21.8 2.1 17.5 - 27.0 9.6

LACRUCE 76 26.1 3.1 19.3 - 33.6 11.9

LIRIOIS 72 27.5 2.5 22.0 - 32.0 9.0

SALTYPI 81 25.1 3.0 20.0 - 32.0 12.0

GM=2 5. 7 GCV=9 .7 30

TABLE 6

Descriptive statistics of stem diameter measurements (SDM) in different Hydrilla verticillata from samples collected in sites of Gatun Lake, Panama. For each station N=number of of samples, X=Mean, SD=Standard deviation, and CV=Coefficient variation. Grand mean (GM) and Grand coefficient of variation (GCV) are provided.

STATION N X SD RANGE C.V.%

AGUARDI 120 1.8 0.3 1.0 - 2.0 18.4

AREBOYA 60 1.0 0.1 1.0 - 1.2 5.5

ARENOS1 100 1.0 0.3 0.6 - 2.0 24.8

ARENOS2 40 1.0 0.2 0.8 - 1.5 17.1

BATCOVE 92 1.1 0.2 0.7 - 1.5 16.3

ESCOBAL 100 1.0 0.2 0.5 - 1.2 16.1

GACUIPO 100 0.9 0.2 0.5 - 1.2 20.8

GADUMP2 104 2.0 0.3 1.0 - 2.6 13.3

GATUNP1 100 0.9 0.2 0.5 - 1.1 21.8

GATUNC2 100 0.9 0.1 0.5 - 1.1 14.4

LACRUCE 76 2.0 0.3 1.0 - 2.4 15.0

LIRIOIS 72 1.0 0.1 0.5 - 1.1 14.6

SALTYPI 81 0.8 0.2 0.6 - 1.0 17.8

GM=1.2 GCV=16.6 31

TABLE 7

Descriptive statistics of leaf form index* of Hydrilla verticillata in samples from Gatun Lake, Panama. For each station N=number of samples, SD=Standard deviation, and CV=Coefficient of variation. Grand mean (GM) and Grand coefficient of variation (GCV) are provided.

STATION N X SD RANGE C.v.%

9.8 AGUARDI 120 6.4 0.6 5.1 - 7.4

AREBOYA 60 6.1 0.9 4.8 - 8.0 14.1

ARENOS1 100 5.8 0.7 4.6 - 7.2 11.4

ARENOS2 40 5.9 0.5 5.3 - 6.9 9.2

BATCOVE 92 6.5 0.6 5.3 - 8.3 11.7

ESCOBAL 100 7.1 1.0 5.5 - 9.5 14.5

GACUIPO 100 6.7 1.0 4.8 - 9.8 14.7

GADUMP2 104 5.1 0.3 4.5 - 5.5 5.4

GATUNP1 100 6.2 0.4 5.5 - 7.1 7.2

GATUNC2 100 5.6 0.5 4.0 6.4 8.3

LACRUCE 76 6.9 0.6 6.0 - 8.1 8.2

LIRIOIS 72 6.2 0.8 4.4 - 7.6 13.1

SALTYPI 81 5.2 0.8 3.6 - 6.9 16.3

GM=6.1 GCV=11.1

* LFI=length (mm) /width (mm) 32

Hydrilla stands in the lake showed signs of low productivity

(e.g., thinning leaves, loss of photosynthetic pigment,

defoliated stems).

A dendrogram showing classification of the 287 her

barium specimens prepared for morphological analysis is

presented in Figure 3. Cluster analysis (Clark and Afifi,

1984) of Gatun stations clearly separated Dump 2 station

from the others. Plants from this area are morphologically

unique. They are small with short leaves and numerous

branches. The largest cluster is divided in two sub-groups,

the first one was formed by stations located in the open

areas of the lake (e.g., Liriois, Escobal, Arenosa, Cuipo).

The second sub-group contained three stations, Bat Cove,

Aguardiente, and Lacruce of which all are located in

enclosed areas with poor water circulation, relatively

shallow waters (<4 m depth) and low transparency (<3 m

sechii reading) during the year.

To test the hypothesis of no difference in Hydrilla

morphology among localities (i.e., stations) a stepwise

discriminant analysis (Tabachnick and Fidell, 1983) was

performed using the morphometric variables as classification

'labels' for the herbarium specimens prepared from Gatun

Lake samples. Therefore, plants from each locality (13

stations) were used as a 'group' (objects to be classified)

by the analysis.

lfilmwm 33

TABLE 8 in the main stem Descriptive statistics of number of leaves Lake, verticillata in samples from Gatun apex of Hydrilla SD=Standard Panama. For each station N=number of samples, (GM) deviation, and CV=Coefficient of variation. Grand mean are provided. and Grand coefficient of variation (GCV)

CV.% STATION N X SD RANGE

8.0 7.2 AGUARDI 120 7.3 0.5 6.0 - 7.6 AREBOYA 60 7.3 0.6 6.0 - 8.0 5.3 ARENOS1 100 7.6 0.4 7.0 - 8.5 6.2 ARENOS2 40 7.2 0.4 6.6 - 8.0 8.4 BATCOVE 92 7.2 0.6 6.3 - 8.0 4.6 ESCOBAL 100 7.5 0.3 6.8 - 8.0

GACUIPO 100 7.6 0.8 5.6 - 9.8 10.2

GADUMP2 104 6.2 0.3 5.4 - 6.8 5.1

GATUNP1 100 7.6 0.5 6.8 - 8.3 6.0

GATUNC2 100 7.0 0.6 6.0 - 8.0 9.0

LACRUCE 76 7.1 0.7 5.0 - 7.8 9.4

LIRIOIS 72 7.7 0.4 7.0 - 8.0 5.0

SALTYPI 81 6.6 0.8 5.0 - 8.3 12.1

GM=7. 2 GCV=7. 4 34

Fig. 3. Dendrogram showing classification of 287 herbarium specimens of Hydrilla verticillata (L.f.) Royle, collected at 13 stations of Gatun Lake, Panama. Method used is average linkage group (UPGMA), morphometric variables were standardized (z-scores) before comparing stations. Similarity indicated as rescaled euclidean distance from 0 to 25. 35

LC) *CN

.0 ~1 1

CL E 0)

CL) 36

On the basis of morphometric variables (Fig.2), five of values discriminant functions (i.e, weighted combinations that maximizes the ratio of the between group variance estimated relative to the pooled within group variance) were removal with a total Chi-square (96)=1,388 P <0.0001. After of the fifth function by the stepwise procedure (Wilkis' lambda test) there was still significant discriminant power,

Chi-squared (21)=54.4 P <0.001. The first two discriminant the functions accounted, respectively, for 59 and 22% of (between morphological variability of plants from Gatun the unexplained 'group' variation). A small percentage of variability was accounted for by the other functions (<10%)

and therefore they will not be considered further (Table 9).

The eigenvalue (variance matrix) and canonical correlation and coefficient (relation between the discriminant function

the classification labels) associated with the first two

functions indicate that morphometric variables and plants

from different regions (groups) were highly associated with

the discriminant functions (canonical correlation of 0.94

and 0.85, respectively; Table 9). The classification results

of the discriminant analysis indicated that 65% of the cases

were correctly classified.

A plot of mean discriminant scores (i.e., mean values

of discriminant function on the herbarium samples) of each 37

TABLE 9 and canoni percent of discriminant power (%DP), Eigenvalue, with each cal correlation coefficient (CCORR) associated discriminant function.

CCORR FUNCTION EIGENVALUE % DP

1 7.159 59.6 0.94

2 2.658 22.1 0.85

3 1.138 9.4 0.73

4 0.604 5.0 0.61

5 0.237 2.0 0.44

1 and 2 indicated 'group' with the discriminant functions in the how these functions discriminate between stations and lake. On the first discriminant function, Aguardiente

Lacruce stations were separated, but other areas (e.g., to Gatun, Salt and Pepper, Escobal, Bat Cove) were close

each other. It is the second function that clearly

distinguished Dump 2 station from all the other localities between (Fig. 4). The loading matrix of correlations

standardized discriminant functions with the predictor

variables (i.e., morphometric characters) indicated that

stem diameter (SDM), leaf surface area index (SAI), and

leaf length (LL) contribute significantly (P <0.0001) to

discriminant function 1 and 2 respectively (Table 10). 38

functions 4. Plot of first and second canonical Fig. after indicating group centroids (average group score) classification of the stations (groups) by discrimant analysis. 39

12

z '8 0

*4Dump2

.. . Las Cruces Z Gatun Aguardiente z Gatun0 0 0 Bat Cove

-8 -81-.

-12 -8 -4 0 4 8 12 CANONICAL FUNCTION 1 40

TABLE 10 discriminant coefficient Correlation values for standardized and discriminant variables. SDM=stem diameter in mm, index expressed as length (mm) X width SAI=leaf surface area the in mm, NTL=number of teeth around (mm), LL=leaf length of leaves in IND=internode distance in mm, NLA=number leaf, form index expressed the apex, LW=leaf width in mm, LFI=leaf as length (mm) /width (mm) .

FUNCTION 2 VARIABLE FUNCTION 1

0.556 SDM 0.646 * -0.712 * SAI 0.590 -0.664 * LL 0.401 0.032 NTL 0.051 0.124 IND -0.023 -0.355 NLA -0.089 -0.296 LW 0.419 -0.357 LFI 0.060

*P <0.0001

These morphometric variables properly define both

functions since they were the most distinguishing

characteristics under field conditions.

Histological examination indicated little micro of anatomical variation in Hydrilla. A comparison Dump 2 transverse sections of stems from Lacruce and and dis stations revealed minor differences in lacunae size (Fig. tribution and quantities of collenchyma and aerenchyma roots, and the 5). Other structures such as the leaves, among the areas. apex did not show observable differences 41

(A) Dump 2 Fig. 5. Transverse section of stems (100x) from central cylinder and and (B) Chagres River stations showing lacunae. Note the difference in collenchyma, aerenchyma, and lacunae distribution. Symbols represent: (ae) aerenchyma tissue; (e) epidermal cells; (cc) central cylinder; and (1) lacunae. 42

- Or

a e -

/AlA ae CC

aOt

B 43

Isoenzymes from different Electrophoresis of stem apex samples isoenzyme sites in the lake did not indicate a variable tested, showed enough resolution pattern. Five of 8 enzymes

TABLE 11 of bands detected in Isoenzyme phenotypes showing number of Hydrilla SOD, MDH, PGI, TPI and PO from samples Barro verticillata from Gatun Lake, Panama. Gamboa, where Colorado and Miraflores represent regions of the Lake to April shoot samples were collected from December 1988 1989.

MDH PGI REGIONS SOD TPI PO

3 GAMBOA 1 2 1 1 1 3 BARROC 1 2 1 1 3 MIRAFL 1 2 1

analyzed stations to suggest genetic homogeneity among the

of the lake. Table 11 summarizes results of typical isomerase monomorphic patterns obtained in Phosphoglucose isomerase (TPI), (PGI), Peroxidase (PO), Triosephosphate

Malate dehydrogenase (MDH), and Superoxidase dismutase PGM and Shk were (SOD). Isoenzyme phenotypes of AAT, repeated inconsistent and with poor resolution, but upon no indication of comparisons in the samples, there was isoenzyme variation.

WAAWWWWO - -WWAVANOWOOMM 44

The pattern of the isoenzymic bands of Triosephosphate the first time in isomerase (TPI) is being reported for in samples from stem Hydrilla. Two loci were identified under 3 min whereas the apex, the first band developed 370 C (Fig. second appeared after 3 - 5 min of incubation at consistently different in 6). Activity of isoenzyme was than MDH, dicating that PO and TPI were easier to detect

SOD, and PGI (Fig. 7). Ecology and Phenology

Fresh mass:Dry mass relations Hydrilla Fresh to dry mass ratio was estimated in

TABLE 12 for Hydrilla Fresh mass dry mass conversion factor estimated station in verticillata in samples from Salt and Pepper Standard Gatun Lake, Panama. Values represent Mean deviation, all values based on N=10.

RATIO DATE FRESH MASS DRY MASS FM:DM (G) (G)

0.066 Sept.87 418.54 85.41 27.62 4.05

Oct.87 102.74 17.34 8.01 1.36 0.078 0.082 Nov.87 77.48 11.62 6.37 0.97

Dec.87 67.19 10.07 5.64 0.67 0.087 0.075 Jan.88 60.80 10.34 5.28 0.74 0.076 Feb.88 52.51 10.50 3.94 0.59 0.078 Mar.88 42.71 5.12 3.25 0.33 X= 0.078 SD= 0.006 45

phenotype of Triose Phosphate Fig. 6. Electrophoretic (L.f.) Isomerase in stem apex of Hydrilla verticillata of Gatun Lake, Panama. Royle, from different stations 46

4

? 4 s p y in: 1. thank ef's,.....es. - 47

Fig. 7. Electrophoretic patterns in Hydrilla verticillata (L.f.) Royle, for PO, TPI, SO, MDH, and PGI. Origen and direction of anode is indicated. Position of isoenzymes are indicated according with their relative mobilities. Solid bands indicate strong activity, clear bands moderate activity, dotted band inconsistent and weak activity. 48

Po TPI SOD MDH PGI

amnooIZ]e

+ 49

plants from Barro Colorado (Salt and Pepper station) and summarized in Table 12. Conversion factors for the rainy

(September - December) and dry season (January - March) allowed the calculation of a grand mean (X SD) value of

0.078 0.006 which was used as the conversion factor for

Barro Colorado area. Water content in Hydrilla samples at this station range from 82 - 92% depending on the season of the year and locality of samples. Ash content was approximately 15% of the dry mass after combustion at 500C.

These values were typical of other regions of the lake such

TABLE 13

Root to shoot ratio estimated in individual plants of Hydrilla verticillata from Bat Cove station in April 1988, Gatun Lake. Ratio estimated on plant dry mass. Indicated total SD.

MASS

PLANT SIZE (CM) SHOOT (MG) ROOT (MG) ROOT/SHOOT RATIO

45 67.5 35.8 0.53

50 72.9 36.6 0.50

75 96.2 55.7 0.56

87 120.2 59.8 0.49

95 148.7 50.2 0.34

98 90.0 88.6 0.47

240 260.7 158.5 0.61

X=0.50 0.08 50

as Gamboa, Gatun, Miraflores and Arenosa. Below and above ground biomass ratios (R:S) calculated in plants from the

same station (Barro Colorado area) presented variation of biomass distribution between roots and shoots within the

stand. This variation suggests that allocation of biomass

between R:S was not related to shoot size. In general 50%

of the biomass was allocated to shoot production (Table 13).

Chlorophyll

Total chlorophyll concentration at different stations

of Gatun Lake ranged from 0.7 - 1.3 mg/g fresh mass (Table

14).

TABLE 14

Concentration of chlorophyll pigments (in mg/g fresh mass) estimated in Hydrilla verticillata from samples of different stations in Gatun Lake, Panama. Each value is based on three replicates. X SD, N=10 for all stations.

CHLOROPHYLL CONC. (MG/G F.M.)

STATION a b TOTAL CHL.a/b

Bat Cove 0.68 0.06 0.66 0.06 1.34 1.04

Salt Pepper 0.69 0.07 0.60 0.07 1.28 1.15

Las Cruces 0.47 0.07 0.38 0.07 0.85 1.24

Dump 2 0.56 0.04 0.43 0.04 0.99 1.39

BCI dock* 0.58 0.02 0.43 0.03 1.01 1.31

Frijoles* 0.42 0.02 0.34 0.03 0.76 1.24

* Sporadic stations in Barro Colorado region. 51

Fig. 8. Relationship between fresh mass (mg) and size (cm) of Hydrilla verticillata (L.f.) Royle, shoots were collected at Barro Colorado Island in Gatun Lake, Panama. Predicted line and 95% confidence belts around the line are given. 52

x .4 (0 .4 .4 0 .4 .4 x .4 C\I .4 X .4.4.4 .4 x LO .4 .4 .4 + .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 (0 0 Cl, .4 .4 .4 .4 .4 .4 .4 .4 couI'~, .4 .4 .4 .4 .4 .4 .4 .4 .4 .4.4 .4.4 II II It II 0 .4.4.4 .4.4.4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 N .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 .4 LO .4 .4 .4 .4 .4 .4 .4 .4 .4.4 .4.4 0 .4.4 .4.4 cl, .4.4 .4.4.4 .4 .4 .4 .4 .4 I I ~ A I 0 0 0 0 0qO2 0 0 LO LO LO 01 LO Cl cl N CN T- (6w) SSVIJ HS31Hd 53

Samples were taken between 1 and 2 m depth in the morning

(8:00 - 12:00) with an average illuminance of 3,200 foot candles. The amount of photosynthetic pigments (i.e.,

Chlorophyll a + b) available in stem shoot apex was relatively higher in stations located in open areas of the lake (Salt and Pepper, Bat Cove and Barro Colorado dock), whereas enclosed sites (Frijoles and Lacruce) presented lower values. These estimations were made between November

1987 through January 1988. The Chl.a/Chl.b ratio showed slight variation among stations, the maximum value (1.39) was found in Dump 2 station and the minimum (1.04) at Bat

Cove station. These values suggest the important role that

Chlorophyll 'b' concentration has in Hydrilla photosynthesis

(Table 14).

Length and mass relation

Measurements of length and mass of Hydrilla shoots for

Barro Colorado area (Bat Cove station) followed a simple linear model with a high correlation coefficient (r2=0.97,

P <0.001, Fig. 8). Regression analysis of these data indicated that fresh mass of shoots in milligrams for this station could be predicted by the following equation:

Fm(mg)= 388.64 + 21.6 (length in cm) where Fm refers to total fresh mass of each shoot after blotting, and length corresponds to a shoot range from 10 to

150 cm which was the plant size found in January 1989 in Bat 54

Cove station. The slope of the regression model had a standard error of 0.28.

Biomass distribution along shoots for different size plants follows a regular pattern of biomass allocation regardless of length of the shoot. Figures 9 - 11 show that the first 20 cm from the apex accounts for 5 - 10% of total biomass. Fifty percent of biomass allocation occurs in the mid section of the total shoot length. Less than 10% of the biomass is allocated in the last third of the stem. Shoot density per area (i.e., packing) in Arenosa station changed with depth (Table 15). Maximum density occurred at

TABLE 15

Depth variation of shoot density, plant weight and biomass per unit of volume (B/V) of a stand in Arenosa station during April 1988; Gatun Lake, Panama. Depth and length range in meters, N represent the number of shoots measured per area, mass in g fresh mass/plant (X SD), and B/V ratio expressed in g fresh mass/m3 .

DEPTH N SHOOT/MV LENGTH MASS B/V

3 11 170 0.80 - 2.20 3.59 1.26 393.74

5 10 220 0.85 - 4.40 3.97 2.53 450.21

6 31 85 0.90 - 2.90 3.82 2.21 153.89

9 32 60 0.25 - 1.15 1.65 0.82 162.29

5m with 220 shoots per m 2. The biomass per unit volume ratio estimated using these values gave a ratio of 450.21 g 55

Fig. 9. Biomass allocation in individual shoot (shoot weighed each 10 cm) of Hydrilla verticillata (L.f.) Royle, from Barro Colorado Island in Gatun Lake, Panama. Total shoot length 1.24 m; total shoot mass 3.6 g. 56

0

0

*- E0

co * z w 0 ...j

- 0 0

0

'TrJ

(Bw)ssVpj HS3HUI .LOOHS 57

Fig. 10. Biomass allocation in individual shoot (shoot weighed each 10 cm) of Hydrilla verticillata (L.f.) Royle, from Barro Colorado Island in Gatun Lake, Panama. Total shoot length 2.19 m; total shoot mass 5.5 g. 58

.

C0

. 0

0

0 1

14 N

c o - J 0 o 0 co O 0 (0

0

0

2 a III I 0 0 0 (0 0 0 0 LO Cf 0\

(Bw)SSVIj HS3HId .LOOHS

S SS 5 S 59

Fig. 11. Biomass allocation in individual shoot (shoot weighed each 10 cm) of Hydrilla verticillata (L.f.) Royle, from Barro Colorado Island in Gatun Lake, Panama. Total shoot length 7.35 m; total shoot mass 14.9 g. 60

0 0

* 0 00

0 Nw

COE

0

*co0

(6w) SSVIN HS3Hi.A OOHS

F.41 61

fresh mass m-3. Although quantification of shoot density was not estimated in other regions the observed density distribution appeared similar to Arenosa station. Biomass per unit of volume of Hydrilla in Gatun Lake gave an indication of the relationship between stand density, mass, and volume.

The branching pattern of Hydrilla was distinctive in the different areas of the lake. Plants from Dump 2 station were short and heavily branched, whereas plants from Gatun

and Salt and Pepper areas were slightly branched and slender

(Figs. 12, 13). The differences in plant geometry were in

agreement with morphological variation (Table 1 - 5) found

in the lake.

Standing crop

Seasonal change in standing crop of Hydrilla was

estimated periodically at two stations of Barro Colorado

Island at 3 m depth (Figs. 14, 15). The total mean standing

crops in Bat Cove and Salt and Pepper stations were

significantly different [Independent t-test, t=2.06, DF=136,

P <0.05)]. Although both stations are located on the island

(see Chapter II description of the study areas, and Fig. 1),

each Hydrilla stand experienced different environmental

conditions. The Bat Cove stand was more homogenous in shoot

size and depth, little wind and current action were

responsible for relatively calm waters throughout the year. 62

Fig. 12. Schematic drawing of branching patterns observed at (A) Dump 2 and (B) Cuipo stations in Gatun Lake, Panama in May 1988. Stem buds are indicated at the end of each branch. 63

0 0

0~ (0 (0

t1 0 0N 0

I a I I 0 %fwo 0Y 0 0 N

z 0

C* 0 (NI

0 0 (0 0

(LUO) HON3~1

0AW, 64

Fig. 13. Schematic drawing of branching patterns observed at (A) Bat Cove and (B) Salt and Pepper stations in Gatun Lake, Panama in May 1988. Stem buds are indicated at the end of each branch. 65

0

I w. z cc

oO

LO)

CLC) (mo) HLON~L~ HIOL() 66

The Salt and Pepper stand was more variable in depth, shoot

composition, and frequently affected by waves of passing

ships. Sechii disc transparency in both areas had a range

of 1 - 7 m with the minimum visibility in November and the maximum in April.

In general both biomass curves were similar. The Bat

Cove Hydrilla stands reached seasonal maximum of 395.97 g dry mass/m2 in June and August,1988; while Salt and Pepper attained two peaks of maximum standing crop (ca. 420 g dry

2 mass/m ), one in June and a second during December 1988.

Infrequent standing crop measurements at Gamboa and

Chagres River regions indicated that a maximum of 349 and

272 g dry mass/m2 respectively, occurred during the local dry season. Their minimum biomass was attained during

November and December (98 g dry mass/m). TABLE 16

Comparison of mean standing crop values (g dry mass/M2) Of Panamanian rainy and dry season in Barro Colorado Island during 1987-1988.

STATION N SEASON X SD T-STUDENT PROBABILITY*

SALT & 18 RAINY 320 132.47 PEPPER 0.75 NS 20 DRY 270 110.33

BATCOVE 18 RAINY 350.94 57.78 3.72 ** 20 DRY 212.23 51.58

*NS = P >0.2 ** = P <0.005 67

Fig. 14. Seasonal variation of standing crop (g dry mass/im) of Hydrilla verticillata (L.f.) Royle, at Salt and Pepper station in Gatun Lake, Panama. Values represent means of five replicates, bars indicated 1 Standard deviation. 68

2 U.

~c COo

b

0 z

-I

4000 (1)co -400

0

-co

z -.00000

(1)co .1i1t1111111I 0 o000 0 0)"'00 L''' 1"0 C1) C1 C') C'4 (Zw/ssew A.p 6) SSVI/OI8 69

Seasonal variation of standing crop (g dry Fig. 15. at Bat Cove of Hydrilla verticillata (L.f.) Royle, mass/m2) means of station in Gatun Lake, Panama. Values represent deviation. five replicates, bars indicated 1 Standard 70

LL.

- Zco

z

0 -I)

0

c o

z

t I I I I 1 I I I I

CO LOC) ) 0) LO - Of 0) (,'s sseVcJ ) 4O 4i (Zw/ssew Aip 6) ssvyqole 71

Comparison of mean standing crop for the rainy and dry season in each of the stations indicated different behavior.

Hydrilla stands at Bat cove site showed a statistical significant rainfall effect (independent t-test P <0.005), whereas Salt and Pepper station did not indicate any difference in its standing crop during the year (Table 16).

TABLE 17

Pearson correlation coefficient of selected environmental parameters with standing crop (g dry mass/m2 ) data of Hydrilla verticillata collected at stations in Barro Colorado Island in 1987-1988. N=18, probabilities of RHO=0 indicated in parenthesis.

PARAMETER* BAT COVE SALT AND PEPPER

RAIN (M) 0.56 0.49 (0.030) (0.063)

RADIATION (LANGLEY) -0.55 -0.44 (0.032) (0.100)

TEMPERATURE (*C) -0.50 -0.45 (0.060) (0.064)

LAKE LEVEL (M) -0.38 0.14 (0.206) (0.560)

TRANSPARENCY (M) -0.18 -0.30 (0.500) (0.318)

* Provided by Panama Canal Commission Meterological Services.

Correlation analysis of environmental parameters in the lake with biomass from both stations confirmed a positive association with precipitation and a negative one with radiation (Table 17). Further multiple regression analysis 72

indicated that 55.8% of the (Stepwise, forward selection) was explained by variation in biomass of Bat Cove station 3.04, P <0.05). Only the regression model (F from ANOVA = Pepper biomass was 40% of the variation of Salt and model that was not accounted for by the regression 1.63, P >0.22). The best significant (F from ANOVA = in both stations combination of independent variables 2 (R 2=0.56 ,and R =0.36 included temperature and rain SAS 1985). respectively ; RSQUARE Procedure, Associated fauna of the lake A survey conducted in different sites controls of Hydrilla in searching for potential biological revealed a diverse the lake (October 1987 to April 1988) were the most invertebrate fauna (Table 18). Mollusks five species of abundant taxa (six genera and at least in the lake. This group gastropods) associated with Hydrilla Hydrilla where numbers was abundant in roots and shoots of (sample unit reach up to 200 per 250 g of fresh mass

selected as standard mass for the survey). important taxon was the Among the Arthropoda, the most of the genus Lepidoptera because of the interaction 16 shows the adult of this Parapoynx with Hydrilla. Figure vegetation at the moth which is abundant around trees and aquatic stage shoreline of the lake. Parapoynx sp. has an leaves. This during which it depends solely on Hydrilla of the selectivity is responsible for severe defoliation 73

Fig. 16. Adult (A) female, and (B) male of Parapoynx sp. (Lepidoptera) collected above the stand of Hydrilla verticillata (L.f.) Royle, at Bat Cove station in March, 1988 in Gatun Lake, Panama. 74

IE

E

ipAll-m V*

B 75

TABLE 18

Invertebrate fauna associated with Hydrilla verticillata in Gatun Lake. Each taxon is followed by life stage (A=Adult, I=Immature) , number of organisms and percentage of occurrence.

Phylum Arthropoda Phylum Mollusca Class Insecta Class Gasteropoda Order Lepidoptera Family Planorbidae Family Pyralidae Genus Gyraulus sp. Genus Parapoynx sp. A, I, 1,606 18.78% A, I, 4,678 71 % Family Physidae Order Odonata Genus Physa sp. Sub-Order Zygoptera A, 602, 7% Family Coenagrionidae I, 88, 1 % Family Thiaridae Genus Melanoides sp. Sub-Order Anisoptera A, 100, 1.2%

Family Cordulidae Family Hydrobiidae Family Libellulidae Genus Lyogyrus sp.1 Family Aeshnidae A, 101, 1.2% I, 10, 0.1 % Genus Lyogyrus sp.2 A, 917, 10.7% Genus Potamopyrous sp. Order Diptera A, 62, 0.73% Family Chironomidae Sub-Family Diamesinae Class Pelecypoda I, 43, 0.5% Family Sphaeriidae Genus Sphaerium Order Coleoptera Species striatum (Lamarck) Family Lampyridae A, 3, 0.04% I, 1, 0.01% Class Monoplacophora Order Ephemeroptera Family Ancylidae Family Baetidae A, 2, 0.02% I, 5, 0.06% Phylum Platelminte Order Hemiptera Class Turbellaria Family Pleidae Order Seriata I, 13, 0.15% Genus Dugesia sp. A, 256, 3% Class Crustacea Order Amphipoda Phylum Annelida A, 71, 0.8% Class Oligochaeta A, 18, 0.21%

Order Decapoda Class Hirudinea Family Palemonidae Order Rhynchobdellidae 76

A, 3, 0.04% Family Glossiphoniidae A, 16, 0.19% Class ArachnidaPl c. Order Acari Phylum Cnidaria Family Hydrachnidae Class Hydrozoa Family Arrenuridae Genus Hydra sp. A, 10, 0.12% A, 44, 0.5%

of Bat shoots. Approximately 30 to 40% of the stem apices were affected Cove, Gamboa, and Salt and Pepper stands during the dry season of 1988 (D. Holness, personal communication, 1988).

Other groups were less conspicuous and their presence (Table did not indicate a negative relation with Hydrilla

18).

Life Cycle

Hydrilla occurred in Gatun Lake in dense and sparse a stands. Horizontally its distribution alternated between

dense canopy of a few meters (ca. 2m) up to several

kilometers of extension with areas of sparse (or completely

absent) shoots. Vertically, the first 1-3 m from the

shoreline are sparse colonized or mixed with Chara sp.

After this depth up to 10 m the stands are monospecific. shown A general description of Hydrilla life cycle is

in Figure 17. The first months of the year are the dry

season in Panama, characterized by strong north winds, high

radiation and little precipitation. A combination of

factors such as decreased water levels, infestation by 77

stands decline in 17. Relative percentage of Hydrilla Fig. during 1988 (A) Salt and Pepper and (B) Lacruces stations 1989, in Gatun Lake, Panama. 78

--MA

601 w z ~40

D 20 z

LL. 0 -m B 8 0

60 0 w 40 CL

20 F

I I I I I I I I I I I I 0 Jan F M A M J J A S ON Dec TIME (Month) 79

the life cycle of Fig. 18. Schematic representation of Gatun Lake, Panama verticillata (L.f.) Royle, in Hydrilla year in relation showing the major events during a typical with the season. 80

C o 00

o "0 co mc l)0z o

E ~~-1 C x0 E T E -'S EEI0

C14)

C - O C" U co cnoE 00 O) (f Cn Co"I

(fl LO

01- LLD.., j:

NOiOlnaGOHd SSVLOIS 81

(Zygnema sp. and Parapoynx sp. larvae, algal blooms of Spirocgra sp.) and wave action diminish standing crops March and May when most Hydrilla. This event occurs between signs of decline of the stands on the lake showed noticeable

(i.e., chlorophyll loss, stem decay). Colorado Decline patterns of stands in Gamboa and Barro areas during the study (September 1987 through March 1989) of stand revealed opposite behavior (Fig. 18). Percentage station decline increased up to 70% in Salt and Pepper in Lacruce during dry season months (February - May) whereas station the maximum decline was recorded in wet season lake follow months (October - December). Other areas of the

Salt and Pepper pattern of stand decline. a In general, old dying shoots appear to provide grow protective cover to the new shoots that begin to underneath. In May the rainy season starts and boosts at the end production until maximum standing crop is reached

of August (Figs. 14, 15). After August, male flowering

shoots begin to appear in the stands and continue cases (Gamboa sporadically to the end of the year or in some

and Chagres areas) until the dry season. The best indicator

of flowering in the field was the amount of floating Figure 19 is perianth (pollen container) above the stands.

a magnified view of these male structures. 82

(L.f.) Fig. 19. Carpellate flowers of Hydrilla verticillata November 1988. Royle, collected at Bat Cove station during Scale bar = 1mm. 83 CHAPTER V

DISCUSSION

Morphology Hydrilla Although, the general external morphology of was noticeable appeared to be uniform in Gatun, there number of variation in leaf surface area, stem diameter, branching. That marginal teeth, leaf form and pattern of ambient variation corresponded closely with differences in stations conditions of transparency, current and depth among throughout Gatun (see Chapter II Study area). The range of that morphological variation in Gatun was similar to of the reported for Hydrilla sampled from different parts world by Verkleij et al., (1983). For example, leaf area

index in plants from Gatun (range from 19 to 38 cm2) was

within that presented in Verkleij's study (range 5.7 to 47

cm2), which included plants from New Zealand to Malaysia.

Although phenotypic plasticity in Hydrilla has been

recognized in several studies (Verkleij et al., 1983; Cook

and Luond, 1982; Pieterse et al., 1984; Pieterse et al., a 1985), my work is the first attempt to quantify it within

single Hydrilla population. Variation in the Gatun

population supports the contention by Cook and Luond (1982)

that morphological characteristics are not valid criteria

84 85

races around the world. for the identification of Hydrilla enabled Cluster analysis of morphometric data groups in the Gatun resolution of four major morphological presented in Figure 4 population. Although the ordination it accords with does not follow any particular trend, Chapter environmental characteristics of each station (see water circulation, II). That is, stations with similar composition cluster transparency, depth, slope and substrate and Cuipo). Separation together (Gatun and Arenosa, Liriois

of Dump 2 station from the others (with less similarity) this area. indicates the unique morphology of plants from of the Discriminant analysis goes beyond a simple ordination (1) which data, by answering two important questions: (Table 3) morphometric characters measured in the population

account for most of the variation; and (2) how these The variables identify the stations sampled in the lake. was therefore hypothesis of no difference among stations with tested and rejected. As expected, variables associated

leaf morphology were significant in the analysis (P

<0.001), surface area index (SAI) and length of the leaf

(LL) with discriminant function 2. A significant with correlation (r=0.65, P <0.05) of stem diameter (SDM)

discriminant function 1 (Table 7) revealed important

differences in stem diameter of Hydrilla within the lake.

Microscopical examinations indicated that there were 86

histological differences in stem among sites, including the texture, which amount of aerenchyma (lacunae) and epidermal River and were clearly noticeable in samples from Chagres could be related to Dump 2. Variation in stem diameter the lake different water current patterns that occur in

(e.g., passing ships, river outfall, wave action) and matter differences in substrate composition or organic available in the lake substrate (Spence, 1982; Barko et al.,

1988).

Based on multivariate analysis (cluster and

discriminant) it is possible to describe, conservatively,

three 'morphotypes' in Gatun. The first occurred in Dump 2

station (Gamboa region). Environmental conditions such as

strong current produced by passing ships and periodic

aquatic weed control are likely the main factors responsible

for Dump 2 morphotype. It appears that the Dump 2 stand is

frequently subjected to drastic conditions causing a

distinctive morphological response. Shoots of Hydrilla are

short and heavily branched, leaves are small, the internode

distance very small, the stem is thick and sturdy compared

with other sites in the lake, and greater dependence on

subterranean tubers for reproduction than other vegetative

mechanisms (fragmentation, above-sediment turions). Tubers

were collected only in Dump 2 stands during this study. 87

Although the first canonical function (leaf morphology, (Fig. Table 10) separates Lacruces and Aguardiente stations small sample 4), proximity of both stations and relatively of more than a size from Lacruces precluded determination morphotype single morphotype in Chagres region. The second (Fig. 1) which occurs in stands at the lower Chagres River is characterized by drastic changes of water level, of periodical runoff and low transparency. The morphology robust with wide Hydrilla under these river conditions is leaves, few branches and thick but softer stem (more aerenchyma tissue). The third morphotype includes plants

from open areas of the lake (e.g., Gatun, Escobal, Cuipo,

Arenosa) which represent localities typical of stable

conditions with similar patterns of water circulation,

transparency, water quality and substrate composition. The

morphology of Hydrilla under the above conditions is

somewhat intermediate between the other two morphotypes. and stem The plant might vary in branch pattern, leaf size,

diameter; but less than the amount of variation found in

Dump 2 and Chagres region.

There is a clear indication that the morphotypes are

environmentally dependent. Significant correlations of

nutrients (N and P) with the morphotypes and distinctive

characteristics of substrate in areas where they occur

(Table 19) suggest that the morphological variation of 88

0 C e H O- 4J ce4.) >)> 0$ z 1 (o4) c o Nce4 0 ONd 05 ) 1) a4J) a-i 0 o 4 (e0 4-40 41 fn) d M 0 -H OH (C H Q) $-4 ro rdtro U) 44 ro 00 4) 4-) Q Ce-H >I 0 .) U)>i 4 4J ) ard C -K- OilH 4-) 40 H- >4 -H Ce fto 0 0 a) a) r4 zO ( o ro : aA) > .4 .- ) o a) 4-4--H a1) N (d 0 4-f-4 44 to O M) 0 .0 z Sco 4-4 (d Q 00) Q0.) 0 >4 U),.i P 4-) C,. ae .-4H 0 4 >I H%%o o0O Sa to0 o U)* OHp Ce 00o -4 0 0)d 44 .,>1 0) a)C MCe 00 a) ro -H 0 (N co Ce O -H O\ z 'o0 0 0 %%oo 0 0 0 0%O -HC-P a) C) o z 4-) -I-i

ar(d U 0 w a) 50 o w a) 4 v rd z MaCe U P4 )4 E-4 ()4-) CeCe4 z 0 H 4JaW 54 04 -C H H 0 ) 0 -H H H H 0 89

are the relative Hydrilla in Gatun is adaptive. What in terms of benefits of these adaptations (morphotypes) a sturdy stem, plant fitness? A heavily branched plant with short internode distance and small leaves (Morphotype I) can reduce the flow rate or turbulence in the stand and keep an shoots. That acceptable pressure gradient around individual tidal strategy has been reported in seaweeds exposed to forces (Chapman, 1987). Presence of subterranean tubers, associated with stressful situations in northern lakes under (i.e., winter), provides reproductive advantage or conditions of frequent surges that can disperse fragments other vegetative structure (axillary turions) out of the stand. Moreover, regular application of herbicides would reduce shoot's 'fitness' to become a reproductive

alternative. Thus, subterranean turions contribute to plant persistence specially during regular herbicide application

that can alter the efficiency of axillary turions and plant

fragments as reproductive mechanism. Wider leaves, and

greater internode distance (morphotype II) enhance light

harvesting efficiency specially under turbid water

conditions. Although Hydrilla photosynthesize at low light

levels, in turbid water (<1.5 m Sechii transparency) its

photosynthetic performance is drastically reduced (Bowes,

1987). Therefore, an increase of surface area and

separation of the leaves (internode distance) might enhance 90

conditions. Thickness photosynthesis under high turbidity presence of of the stem (morphotype II) and increased to withstand aerenchyma tissue (lacunae) improve chances

anaerobic conditions in the sediment since gas exchange (Wetzel, 1983; Smart and compensates for reducing conditions system (wider Barko, 1988). Thus, increase of lacunal gas with high levels stem) can improve resilience in sediments

of organic matter. Profuse branching associated with canopy

formation (morphotype III) has been related to physiological (intraspecific (photosynthesis) and ecological factors reported that competition). Pesacreta and Luu, (1988)

canopy formation in Hydrilla occurs when environmental water conditions (e.g., light, temperature, nutrients, and

flow) are relatively stable. Thus, shoot elongation,

branching, and canopy formation of Hydrilla in Gatun Lake

might contribute to an efficient spatial distribution

(horizontally and vertically) in the lake.

It is likely that the size of Gatun Lake provides broad enough habitat variation for Hydrilla to display the over its range of phenotypes characteristic of the species of axillary geographical range. A recent study on variation

and subterranean turions in Hydrilla by Spencer et al.,

(1987) supports the argument that variations in reproductive

structures are also environmentally dependent. The Hydrilla of population in Gatun Lake illustrates the importance 91

of local adaptation of phenotypic plasticity in the process Pesacreta and a competitive species (Cook and Luond, 1982;

Luu, 1988). recognized Intra and interpopulation variation has been as environmentally related by taxonomists and ecologists trying to identify and characterize aquatic plant MacMillan and populations (Briggs and Walters, 1969;

Phillips, 1981; Bigley and Harrison, 1986; Heard and Semple,

6 1988). Begon et al.,(198 ) described this faculty as it 'somatic polymorphism' (e.g.,heterophylly) and considered success the main reason for the wider ecological range and

of some aquatic weeds (e.g., Salvinia molesta, Eichornia

crassipes).

Isoenzymes

The uniformity of isoenzymes in Gatun Hydrilla (Table

2) accords with observations that aquatic angiosperms 1982; generally exhibit low genetic variation (MacMillan,

Triest, 1986; Van Wijk et al., 1988; Les, 1988). Patterns

of PO (peroxidase) and SOD (superoxidase dismutase) in Gatun et al., Hydrilla were the same as those reported in Verkleij

(1983). Poor resolution of the gels precluded comparison

with the other isoenzymes reported in Verkleij's study which

included PGM (phosglucomutase), AAT (aspartate amino

transferase), and Shk (shikimitate dehydrogenase).

Nonetheless, the five bands that Verkleij et al., (1983)

11111111111,1 11 1 1 mmonow"m 92

from Gatun were never reported for Shk isoenzyme in Hydrilla pattern detected in any of my gels. The common isoenzyme further for Shk in Gatun appears to be one or two bands; matter. analysis is necessary to elucidate this for the two Ryan (1988) in a comparison of isoenzymes States (monoecious biotypes of Hydrilla found in the United

and dioecious), using polyacrylamide gels, indicated reported differences in the bands from PGM and G6PDH. Ryan

three bands for PGM in contrast with the two bands reported

by Verkleij's study. In my study, PGI (phosphoglucose

isomerase) showed a third weak band that was variable in system. expression, suggesting polymorphism in this enzyme

Electrophoresis of samples from the same shoots indicated

that this third band resulted from enzyme concentration

(perhaps caused by a difference in age of the shoot apex),

since some samples in the gel had the third band while

others did not.

Only six of the 16 enzyme systems that have been

surveyed in Hydrilla, are reported as polymorphic (Verkleij

et al., 1983; Pieterse et al., 1984; Pieterse et al., 1985;

Ryan, 1988). The Hydrilla genotype in Gatun accords with

observations of low enzyme variability among aquatic

angiosperms (McMillan, 1982; Silander, 1985). It appears

that high levels of clonal growth in aquatic angiosperms may

account for genetically uniform populations (Les, 1988). 93

in Furthermore, the amount of genetic variation reported plants and aquatic plants is low compared with terrestrial animals (Hamrick et al., 1979). Low genetic variation among aquatic plants is strongly correlated with the high dependence on vegetative apomixis (McMillan, 1982; Silander,

1985; Les, 1988), reduced sexuality, limited outcrossing, and inefficiency of autogamy or geitonogamy.

Based on the few enzyme systems investigated in the

Gatun population, lack of variation suggests that the population is a clone derived from a single introduction that successfuly colonized through vegetative reproduction.

Mass:length relationship

Variation in below and above ground root:shoot biomass

ratios (R/S) for (0.34 to 0.61; Table 13) Bat Cove stand was

large compared with R/S ratios (0.1 - 0.3) reported by Hall

et al., (1984) and Barko et al., (1988) for Hydrilla grown

under optimal culture condition. High R/S biomass ratios in

Hydrilla are characteristic of low sediment fertility since

previous growth can deplete critical elements such as

phosphorous and potassium (Barko et al., 1988).

Asymmetrical distribution of mass along the shoot in Gatun

Hydrilla (i.e., ca. 20% of the total shoot mass is

concentrated in the top 20 to 30 cm; Fig. 9 - 11), accords

with observations by Pesacreta and Luu (1988) that nearly

half of the photosynthetic tissue and biomass of dioecius 94

water surface. In Hydrilla is found within 20 cm of the the first Gatun it is common to find canopy formation within 2 to 6 m meter. Maximum shoot density occurred between Shoot whereas at 9 m depth shoots were small and sparse. of density was affected by slope of the bottom and condition the stand (i.e., decline or productive). For example, steep while slopes had reduced substrate available for Hydrilla, stands in declining stage had less shoot per area than stands in productive stage. Density in aquatic plants has been related to competition for resources (i.e., light, nutrients, substrate). Where individual shoots within a stand compete the result is differential production and mortality, affecting overall density (Noble et al., 1979;

Sutton et al., 1980; Barko and Smart, 1981; Duarte and

Kalff, 1986). Decreased shoot density with increasing depth

in Gatun suggests light dependence (Chambers and Kalff,

1985; Barko et al., 1986).

The biomass:volume ratio (B/V) estimated in Gatun

(450.21 g fresh mass/m3) places Hydrilla as a "Class 1"

aquatic plant with Myriophyllum, and Potamogeton (B/V ratios

from 430 to 1300 g fresh mass/m3). This 'class' includes

tall canopy-forming species that flower above the surface

(Duarte, 1987). The B/V ratio relates plant geometry

(growth form) with the Self-Thinning Rule (Lonsdale and

Watkinson, 1983; Westoby, 1984). The Self-Thinning Rule 95

a function of biomass describes mortality in a stand as independent of any accumulation (intraspecific competition) recently other factor (e.g., time). The rule has been tested in aquatic plants suggesting that weight-density similar environmental relationship of stands growing under (Duarte, conditions is a direct function of growth form

1987; Duarte and Kalff, 1987).

The different patterns of shoot ramification (Figs. 12 - 13), appear to (branching) in Gatun population

be environmentally dependent. Some adaptive advantages branched related with shoot ramification are: a) heavily 2) shoots can reduce water flow in areas of turbulence, and to the shoots with few ramifications can allocate biomass and main stem and grow taller in deeper areas (Goldsborough Chagres and Gamboa Kemp, 1988). Field observations in flow might be regions suggest that transparency and water Plant important variables affecting plant architecture. more form in areas of constant surges were consistently high branched than plants elsewhere; likewise in areas of shoot. turbidity more ramification were noticed in plant's

The variation in shoot architecture has been recognized and as one of the basic morphological expressions in plants

clonal organisms (Harper, 1977; Waller and Steingraeber, Branching 1985; Goldsborough and Kemp, 1988; Porter, 1989). 96

to be a combination of patterns observed in Hydrilla seem a single monopodial (growth by continued activity of lateral meristem) and sympodial (growth from successive The growth meristem) types of growth (Porter, 1989). fit the 'guerilla' pattern and spreading in Gatun Hydrilla that follow model (Harper, 1977; 1980). Apomictic species this model are characterized by extensive spreading of ramets and high levels of interspecific interactions, and occur frequently within habitats having heterogeneous conditions (Harper, 1977; Silander, 1985). Standing crop

Standing crop of Hydrilla in the Barro Colorado region to showed seasonality with a consistantly strong correlation

rainfall and temperature (Table 14). The Salt and Pepper

station had three biomass peaks during the study, suggesting

a more heterogeneous environment than the Bat Cove station.

The range of biomass estimated in Gatun (ca. 110 - 430 dry

mass/m2) corresponds with those from other reservoirs

(Environmental Laboratory, 1985; Harlam et al., 1985;

Westerdahl, 1983). Westerdahl (1983) reported biomasses of

about 6 kg fresh mass/m2 or 370 g dry mass/m2 (using units

of this study) for Frijoles bay area of Gatun.

Unfortunately, Westerdahl did not study a complete annual

cycle, precluding seasonal comparison. Nontheless,

biomass values seem to agree with those found in my study. 97

Associated Fauna showed The invertebrate community found in Gatun in Hydrilla moderate diversity as compared with surveys and Center, 1981; stands elsewhere (Center, 1979; Balciuna 1985). The abundance Balciunas, 1985; Balciunas and Minno, close association of mollusks (Gastropods) suggests their graze on the with Hydrilla. It is likely that mollusk Hydrilla leaves abundant periphyton (diatoms) growing over high and stems. Sheldon (1987) indicated that in some cases Field snail density retards growth in aquatic plants. such a case. observations in Gatun Lake, did not indicate were found For example, Gyralus sp. and Potamopyrqus sp. plants; near roots and less frequently in stems of healthy samples from apparently not causing any damage. Biomass in healthy and declining stands did not show differences

number or species of snails (Y. Aguila, personal

communication, 1989). Moth larvae of Parapoynx sp. (Family of Pyralidae) have potential as a biological control active leaf Hydrilla in Gatun. These aquatic larvae are

eaters, in most cases causing severe defoliation. In

addition to feeding on leaves they use them to build 'cases'

that house the aquatic stage. The larvae move the case was more along the shoot while they eat. Infestation

evident during the night. At points where the shoots were

exposed (out of the water), the stand was practically

m 98

during covered with adults. Larvae were particularly active use of the dry seasons of 1988 and 1989. The potential reported by Parapoynx sp. as a biological control has been in the Balciunas and Center (1981) from field observations of stands was Gamboa region. Yet, at that time, infestation I have noticed minor and not widely spread to other areas. with some that all regions visited in the lake showed stands to limit the damage cause by the larvae. Depth appears are larval action; plants located in more than 3 m depth is severe beyond range of the larvae. When defoliation on the (>40%), the canopy collapses with stems falling bottom. Additional field observations are required to

assess other effects of ParapoynX sp. on Hydrilla. A main the optimal objective for future studies must be to detect

conditions in which larval effects can be enhanced, Hydrilla is especially during periods when standing crop of maximal. Life Cycle

The general life cycle of Hydrilla (with emphasis on Island changes in biomass) observed mainly at Barro Colorado Local appears representative of other stands in the lake.

events such as proximity to river outfall, surge caused by affect passing ships, and chemical or organic pollution can

or alter Hydrilla's life cycle (e.g., as occurred in Chagres

River and Gamboa regions; Table 19). The general growth 99

contrasts model (Fig. 18) proposed for Gatun Lake Hydrilla with the hypothetical model suggested by Westlake (1965) for dieback. The plants with annual growth suffering winter

Hydrilla model depicts a smooth biomass curve partially because of its perennial behaviour in Gatun. My in observations suggest that during the year, dying shoots the stands are continually replaced, keeping the minimal

2 standing crop at ca. 120 g dry mass/m . Wetzel (1983) indicated that in tropical communities aquatic plant biomass remains more or less constant despite continuous growth and mortality, this appears to be the case in Gatun.

Decline of the stands was more pronounced at the end of the dry season when a combination of biotic and abiotic

factors (e.g., defoliation caused by Parapoynx sp., algae blooms over the stand, lower level of water in the lake,

intense solar radiation and strong winds) drastically

affected Hydrilla stands. Kar and Choudhuri (1987)

indicated that light plays an important role inducing

senescence, stimulating Hydrilla leaves to produce cytosolic

enzyme that accelerates chlorophyll loss (photobleaching).

The observed cycle in Gatun differs with productivity

reported in tropical macrophytes, including Hydrilla,

(Rejmankova, 1989). Rejmankova reported that maximum growth

of Hydrilla occurred in late March or April in central

India, the opposite from that in Gatun Lake. Bowes et al., 100

year (1979) indicated that Hydrilla persisted throughout the curve in lake Trafford (south Florida) with a biomass and somewhat similar to that in Gatun Lake. Haramoto

Ikusima (1988) described Eigeria densa's life cycle in Japan where maximum growth was from June to October and minimum Seasonal growth in the winter from January to March. water level are changes in temperature, light and changes in plants key factors in regulating life cycles of aquatic

(Center and Spencer, 1981; Nichols and Shaw, 1986).

Reproduction of Hydrilla in Gatun Lake follows the

'indeterminant' type of asexual reproduction (Environmental

Laboratory, 1985; Chapter II) involving fragments of stems

and rhizome, which can produce a complete plant under normal

ambient conditions. Although plants with axillary turions

occur in Gatun Lake it appears that fragments cut by boats

in the Panama Canal are the principal dispersal units of

Hydrilla (i.e., vegetative reproduction).

Subterranean tubers (i.e., asexual propagule that

serves as perennating structure) have been found at Dump 2

station in Gatun (This study and by R. Gutierrez, Personnal

communication, 1987). These overwintering structures,

typical of populations in higher latitudes (Haller et al.,

1976; Bowes et al., 1979; Steward and Van, 1987), apparently

occur as a consequence of periodical chemical control at

Dump 2. Stands in this station do not mantain above ground 101

biomass for a period of time long enough to permit regular other areas of the lake. vegetative reproduction as occur in a major Management and control of Hydrilla have been concern in infested areas. The literature suggests a variety of methods for controlling submerged vegetation

(Environmental Laboratory, 1985; Joye et al., 1989) including herbicides (chemical), cutting and harvesting

(mechanical), and fishes and microorganisms (biological). because All of these methods have been tested in Gatun Lake and of the severe Hydrilla infestation (Hearne, 1966; Hearne

Pasco, 1972; Custer et al., 1978; Sander et al., 1979;

Westerdahl, 1983). Each of these methods has had only

partial success, since Hydrilla persits even in regularly treated areas. Gatun My.results suggest that regulation of Hydrilla in

Lake could be more efficient if the following are considered

for a management program: (1) Seasonality of biomass

according to micro-habitat type. Timing of treatment in

specific areas must correspond with patterns of growth

(i.e., production) in the stand. (2) Use of regular

sublethal levels of herbicides to weaken Hydrilla and limit in regrowth in critical infested areas. This can be done

Gatun prior to the rainy season when enhanced Hydrilla regular production occurs. (3) Integrate Parapoynx sp. as a

biological control. Effective use of the moth larvae 102

life cycle in the requires a clear understanding of its year in certain lake. Since larvae appear throughout the to infest other areas it might be possible to collect larvae stands that require frequent control. Hydrilla Although the original taxonomic description of Hartog, reported that it was dioecious in Gatun Lake (Den plants 1973; Croat, 1978), I was unable to find female Field observations (carpellate flowers) during this study. that by others in different areas of the lake indicate female plants are extremely rare (R. Gutierrez, personal to communication, 1988). The ability of cultured Hydrilla

shift sexes has been reported by Cook and Luond (1982).

Verkleij et al. (1983) noticed that cultured plants from

Gatun Lake only produced male flower (staminate). These

observations support Cook and Luond's (1982) statement that

the dioecious nature of Hydrilla reported in different

geographical localities (e.g., Panama and Florida) is

disputable.

To what extent specific conditions in the lake could

induce genetic variation is an interesting question. I

believe that areas such as Dump 2, where the periodical

control pressure of herbicides is intense, are likely areas

for future genetic changes to occur. Current success of a Hydrilla in Gatun portends its continued presence as

dominant species in Gatun, unless drastic ecological changes 103

by occur in the lake (e.g., increased rate of eutrophication for Hydrilla. intense deforestation) that make it unsuitable Summary in The Hydrilla verticillata (L.f.) Royle population associated with Gatun displays three distinctive morphotypes different ambient conditions in the lake. Numerical stem diameter as the analysis identifies leaf area index and main morphological characters that differentiate suggests local morphotypes. Variation in these characters adjustment (adaptation). Hydrilla appears to be firmly in Gatun established, dominating almost all regions surveyed

Lake. Its persistance seems related to phenotypic Most of the plasticity rather than genetic variation. with environmental morphological variation can be associated

conditions. The principal phenotypic responses to micro

habitat variation included: (1) changes in width and length

of the leaves associated with levels of turbidity; (2) stem

thickness related to substrate composition and current

velocity; and (3) changes in branching pattern associated

with water flow (surges) and use of light resource. These

responses to different environmental conditions among The aquatic plants is a common trait known as heterophylly. a) the adaptive advantage of these phenotypic responses are:

ability to enhance photosynthesis under a wide range of

water turbidity; b) improve persistence in sediments with 104

and c) reduce turbulance in high content of organic matter; the stand and resist fragmentation. enzyme systems indicates A preliminary survey of eight that a single clone genetic uniformity in Gatun, suggesting of Hydrilla in the was responsible for original dispersion is reported for lake. The triphosphate isomerase phenotype the first time in Hydrilla.

Standing crop estimation from the Barro Colorado region effects. during 18 months revealed striking seasonal

Maximum biomass occured from June through December. that Correlation with environmental parameters indicates variables to rain and temperature were significant (P <0.05) Colorado. explain standing crop seasonality in Barro of Mass measurements in Hydrilla allowed estimations content of water content that range from 82 to 92% with ash

15% of the dry mass. The conversion factor for fresh:dry in mass was 0.078 0.006. Root to shoot ratio was 0.50

Barro Colorado region. Chlorophyll pigments (a + b) ranged in from 0.7 to 1.3 mg/g fresh mass, being relatively higher

stations from open areas of the lake. potential Parapoynx sp. (Lepidoptera, Pyrallidae) has

as a biological control of Hydrilla in Gatun.

Hydrilla strongly dominated the submerged aquatic

macrophyte community in the lake (>95%). Only Chara sp., the occupied the shallow areas (<0.5 m) surrounding

shoreline of the lake. 105

during the High percentage of stand decline occurred factors such as dry season (January to May). Biotic infestation by Parapoynx sp. and algal blooms (periphyton) combined with abiotic conditions such as strong winds, to regulate the intense radiation and wave action appear

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