MORPHOLOGICAL STUDIES OF THE DINOFLAGELLATE KARENIA PAPILIONACEA IN CULTURE

Michelle R. Stuart

A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science

Department of Biology and Marine Biology

University of North Carolina Wilmington

2011

Approved by

Advisory Committee

Alison R. Taylor Richard M. Dillaman

Carmelo R. Tomas Chair

Accepted by

______Dean, Graduate School

This thesis has been prepared in the style and format

consistent with the journal

Journal of Phycology

ii TABLE OF CONTENTS

ABSTRACT ...... iv

ACKNOWLEDGMENTS ...... v

DEDICATION ...... vi

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 5

RESULTS ...... 9

DISCUSSION ...... 14

LITERATURE CITED ...... 22

TABLES ...... 26

FIGURES ...... 28

APPENDIX ...... 36

iii ABSTRACT

Various morphologies of the unarmored dinoflagellate Karenia papilionacea were observed in culture and some forms could easily be mistaken for other toxic species. Using DIC, epifluorescent microscopy and DAPI staining for nuclear DNA, three morphologies were defined in detail, those being a wider than tall butterfly-shape, a wide as tall brevis-shape, and a round spherical shape. The first hypothesis examined was that K. papilionacea develops a K. brevis- like form that can be confused with known toxic species found in the Gulf of Mexico.

Morphological analysis revealed that while the especially wide, clearly butterfly- like cells do occur, the majority of the population was actually very similar in appearance to K. brevis. The second hypothesis was that brevis-shaped cells, as well as other morphologies such as spherical cyst-like cells, represent sexually induced reproductive stages in the K. papilionacea life cycle and therefore would display different DNA contents. There was no statistical difference between the DNA content of brevis-shaped and butterfly-shaped cells supporting the notion that both morphologies represent a natural variation of size with the same ploidy. The third hypothesis was that the butterfly-shaped form was the dominant haploid stage. The majority of K. papilionacea cells observed in culture were not the extremely broad butterfly form that was proposed as the classic representative of this species. These findings emphasize the need for a combination of morphological and molecular evidence when identifying cells in environmental samples. Also, evidence supporting the existence of a pellicle cyst could lead to insight into the bloom dynamics of K. papilionacea.

iv ACKNOWLEDGEMENTS

I am deeply grateful to Carmelo Tomas, Alison Taylor, and Richard Dillaman for inspiration, as well as intellectual and moral support. I sincerely thank Brooke Stuerke, Bob

York, Erika Schwarz, Harris Muhlstein, Tara Haney and Kendra Coles for being such great lab mates and partners in brainstorming. I am grateful to Tyler Cyronak for his generous assistance with flow cytometry. I dearly appreciate all of my friends and family who supported me through this endeavor and helped me to laugh when it was needed. I would also like to thank the

Graduate School and MARBIONC for funding this project.

v DEDICATION

For Dale Trimble and Jackie Prinz.

vi LIST OF TABLES Table Page

1. A summary of descriptive statistical values for the three morphological classifications of Karenia papilionacea...... 26

2. The DNA content in cultured dinoflagellates from non-Symbiodinium genera ...... 27

vii LIST OF FIGURES Figure Page

1. Growth curve of Karenia papilionacea Kp0707-1 including cell morphologies of A) log phase; B) stationary phase; C) decline phase...... 28

2. Frequency distribution of width to height ratios of Karenia papilionacea ...... 29

3. Differential interference contrast images of Karenia papilionacea morphologies in culture; A) log phase butterfly-shaped cell; B) stationary phase butterfly-shaped cell; C) log phase brevis-shaped cell; D) stationary phase brevis-shaped cell; E) stationary phase spherical cell. Scale bar = 20 µm ...... 30

4. Variation within the morphology classifications of Karenia papilionacea: A) butterfly-shaped cells; B) brevis-shaped cells; C) cells losing morphology to become spherical. Nuclei are stained blue with DAPI and viewed with epifluorescent microscopy. Scale bar = 20 µm ...... 31

5. Epifluorescent images of DAPI stained nuclei of Karenia papilionacea progressing through mitosis: A) pre-mitotic nucleus; B) chromosomes beginning to move toward poles; C) nucleus is beginning to take on a hourglass shape; D) hourglass shape has formed with distinct outlines of the two future nuclei; E) two nuclei are evident joined by a small amount of chromatin; F) two nuclei have moved to poles of daughter cells. Scale bar = 20 µm...... 32

6. Differential interference contrast images of Karenia papilionacea undergoing cytokinesis: A) the two daughter nuclei are located in separate lobes of the hypotheca; B) the cleft between lobes of the hypotheca deepens; C) additional lobes form; D) cytokinetic cell appears as two mature cells, attached at a midpoint; E) the final point of attachment at the apical tip. Nuclei are stained blue with DAPI. Scale bar = 20 µm ...... 33

7. Frequency distribution of DNA content in picograms per cell (pg · cell-1) for all cells analyzed...... 34

8. Frequency distribution of DNA content (pg · cell-1) of Karenia papilionacea in A) butterfly-shaped cells; B) brevis-shaped cells, and C) spherical cells ...... 35

viii INTRODUCTION

Dinoflagellates are a large component of primary producers and are estimated to make up about 40% of the total species of marine phytoplankton (Simon et al. 2009). They are key components in food webs, nutrient cycling and the conversion of carbon dioxide to oxygen in ocean systems. A typical dinoflagellate life cycle consists of a motile, haploid (Pfeister and

Anderson, 1987), asexually reproductive cell that undergoes mitosis regularly (Dale 1986).

Sometimes these asexual cells form pellicle cysts (formerly referred to as temporary cysts, see

Bravo et al. 2010) to survive an environmental change. An environmental or internal cue elicits vegetative cells to initiate sexual reproduction by forming haploid homothallic or heterothallic gametes that fuse to form motile, diploid planozygotes, distinguishable by the presence of two longitudinal flagella. The planozygote will undergo reductive division (meiosis) to form planktonic vegetative cells or it will lose motility by absorbing the flagella and undergoing cellular reorganization to form a resting cyst called a hypnozygote. Resting cysts are defined as cells having double walls and a mandatory dormancy period (Bravo et al. 2010). The hypnozygote has the potential to excyst releasing a diploid planomeiocyte that has two longitudinal flagella. Meiosis of the planomeiocyte produces haploid vegetative cells and the cycle continues.

Ecologically these cysts have a function as vegetative propagules and can release motile cells under favorable conditions to reestablish motile populations in the water column. Cysts are thus used as indicators of previous blooms, as a potential for forming motile populations and as a means of disseminating a population to new areas. With both motile and resting (cyst) stages, dinoflagellates express a variety of morphologies, making detailed morphological and molecular studies essential in the accurate identification of the bloom formers.

Relatively few (~ 2%) dinoflagellates are known to cause nuisance or harmful algal blooms

(H.A.B.s) and for some species resting cysts play a key role in bloom formation and dissemination. H.A.B.s can cause millions of dollars in lost revenue to fisheries and tourism

(Anderson et al. 2000). For example, Gambierdiscus toxicus produces ciguatoxin and maitotoxin, causing ciguatera tropical fish poisoning in humans who ingest finfish that have bioaccumulated these toxins from a diet including these dinoflagellates (Bagnis et al. 1980).

Blooms of Noctiluca scintillans form spectacular red water discoloration and produce ammonia that kills fish (Uhlig & Sahling 1990). Various species of Alexandrium, Gymnodinium, and

Pyrodinium produce potent saxitoxins causing paralytic shellfish poisoning in various coastal regions (Anderson et al. 1990, Oshima et al. 1987, Harada et al. 1982). Neurotoxic shellfish poisoning from Florida red tides are also caused by blooms of brevetoxin producing dinoflagellates belonging to the genus Karenia (Watkins et al. 2008).

Historical documents by explorers of the Gulf of Mexico as early as the 16th century describe seasonal fish kills that occurred in areas where modern blooms occur, providing 350 years of anecdotal reports of harmful algal blooms in the Gulf of Mexico (Magaña 2003). From

1954 to 2006, the Florida Fish and Wildlife Research Institute, (Florida Fish and Wildlife

Conservation Commission), maintained a harmful algal bloom monitoring database of samples collected over 29 years in response to fish kills, water discoloration, respiratory irritation to

2 humans and research cruises (Steidinger 2009).

These fish kills, discolored water, and respiratory irritation were associated with dinoflagellate blooms, originally identified as Gymnodinium sp., that formed along the west coast of Florida between 1946 and 1947. Further studies led to the description of the species

Gymnodinium brevis (Davis 1948) which was subsequently changed to G. breve. In 1979, the

Florida red tide organism’s name was changed to Ptychodiscus brevis because of its apical carina

(Steidinger 1979). In 1989, it was agreed that the name Gymnodinium breve would be used conditionally until distinguishing characteristics could be better defined (Granéli et al. 1990). In

2000, using morphological and molecular data, the genus Gymnodinium was split into four genera, Karenia, Karlodinium, Gymnodinium, and Akashiwo. The Florida red tide organism was placed in the genus Karenia and renamed Karenia brevis (Daugbjerg et al. 2000).

Various forms Karenia were observed during blooms. In 1966, Steidinger et al., described three distinct forms of K. brevis: the typical K. brevis shape, a K. brevis shape with a cingulum that was displaced at the sulcus, and a butterfly shape that was almost twice as wide as it was tall. In 1967, Dragovich further elaborated the description to include several variations of the square-like and butterfly-shaped cells found in environmental samples and in the same year

Wilson (1967) presented a full description of the variation of morphology of G. breve observed in culture, including rounded cyst-like cells. Electron microscopy and molecular techniques have now distinguished three new species previously mistaken for K. brevis: K. selliformis, K. bidigitata, and butterfly-like K. papilionacea (Haywood et al. 2004).

3 Karenia brevis is the type species for the genus, and is known as the organism associated with Florida red tides. This species produces potent brevetoxins which can cause fish kills, neurotoxic shellfish poisoning, marine animal mortalities and, when aerosolized by wave action, human respiratory irritation (Backer et al. 2003). Karenia brevis is found throughout the Gulf of

Mexico, into the Caribbean Sea (Lackey 1956), and has been documented in Gulf Stream waters as far north as Onslow Bay, North Carolina (Tester 1991). Despite being studied in environmental samples for over 60 years and being grown in culture for over 50 years, the mode by which this organism forms recurrent blooms has yet to be described. Walker (1982) suggested that K. brevis might be heterothallic because sexual stages were observed in non- clonal cultures and absent in clonal cultures. Walker also observed round forms in environmental samples that were distinct from the spherical forms found in cultures entering the decline phase. Without germination, the proper identification of these cells could not be confirmed. No cysts resulting from sexual reproduction have been observed. Low level but persistent motile populations of K. brevis exist in the Gulf of Mexico year-round (Geesey and

Tester 1993) and could serve as the seed population for bloom formation during favorable conditions.

Karenia papilionacea Haywood et Steidinger was originally described from cells found in New Zealand waters (Haywood et al. 2004) and was identical to the butterfly form described by Steidinger et al., (1966) and Dragovich (1967) from the Gulf of Mexico. It is presently not known to be toxic and neither fish kills nor respiratory irritation have been reported with blooms

4 of this species. Karenia papilionacea is notably wider than K. brevis and has been called the butterfly form (Steidinger et al. 1966). Karenia papilionacea was distinguished as a separate species based on differences in the sequenced large subunit (LSU) D1-2 region (Haywood et al.

2004). This species can co-occur with other cells of the genus in the Gulf of Mexico, Eastern

Pacific, and the Mediterranean. There were reports of cells suspected to be K. papilionacea as far south as Jamaica (Steidinger 2009). In 2007, cells of K. papilionacea were confirmed from live samples taken in the Delaware Inland Bays Region (Tomas, unpublished data), the northernmost occurrence of this species in the USA. Blooms of K. papilionacea have occurred in Delaware coastal waters every summer since 2007 (Whereat 2010), suggesting that there is some presently unknown mechanism allowing populations to develop repeatedly. The objective of this study was to clearly document the different morphologies of K. papilionacea and to better understand the role of these forms in its life cycle. Specifically, the aims of this study are to better describe forms which could potentially be easily misidentified as K. brevis and to determine if the spherical forms are resting cysts.

The hypotheses examined in this study are 1) Karenia papilionacea develops a K. brevis-like form that can be easily confused with the known toxic species found in the Gulf of Mexico, 2) morphological variations such as spherical, cyst-like cells, represent sexually induced stages in the life cycle displaying different DNA contents, 3) the butterfly-like form of K. papilionacea is the dominant, vegetatively-reproducing, haploid stage.

MATERIALS AND METHODS

5 Cell Cultures

Karenia papilionacea clone Kp0707-1 was obtained from the Toxic Algal Culture Collection at University of North Carolina Wilmington’s Center for Marine Science, initially isolated as a single cell by C. Tomas from a surface water sample taken in the Delaware Inland Bays, USA.

This culture was grown in modified L1 medium (Tatters et al. 2010) at 22 ± 1.0ºC with ~ 40

µmol · photons · m-2 · s-1 of cool white fluorescent light on a 16/8 hour light dark cycle. The

1.5 L stock culture was maintained in 2.8 L Fernbach flasks and a portion of culture was transferred to fresh medium every 2 weeks. The stock culture was subcultured in 12 and 96- well tissue culture plates used for detailed morphological observations. Subsamples of the culture were taken by gently swirling the culture three times prior to removal for observations as described below. To culture cells for DNA staining, 12-well plates were inoculated using aseptic techniques with 2 mL of culture per well from Fernbach flasks and grown in the same temperature and light conditions as parent cultures. Subsamples of 1 mL were harvested and used for staining and epifluorescent microscopy. To observe individual cells 96-well plates were filled with 200 µl of modified L1 medium per well and individual cells added to wells and grown in the same temperature and light conditions as parent cultures. These cells were observed using an inverted Olympus CK40 microscope. Cell Counts

Every 1 – 3 days 20 mL aliquots of the 2.5 L Fernbach stock culture flasks were placed into scintillation vials and preserved with two drops of Lugol’s iodine solution (Fisher Scientific,

Pittsburgh, USA). Cell counts of the preserved stock culture samples were enumerated using the

6 Utermöhl method (Utermöhl 1931). Cells were counted with a Motic AE21 inverted microscope.

A minimum of 300 cells were counted in each chamber to guarantee a 95% confidence interval with +/-11.5% accuracy (Guillard 1973).

The total number of cells per milliliter for each culture was determined using the equation

Cells mL-1 = (Nc · fc · n-1) mL-1 where Nc is the total number of cells counted, fc is the number of fields per chamber, n is the number of fields counted, and mL is the volume in milliliter settled in the chamber. Triplicate cell counts were taken and averaged to determine the cell density of the parent culture. Based on these counts, 100 mL of parent culture were added to the experimental Fernbach flask containing

1.5 L of medium to give a log phase lasting between 1-1.5 weeks and a stationary phase that would last 1-1.5 weeks. Cells were taken at these phases for observation and further study. Cell counts were plotted as semi-log plots of densities with time (Guillard 1973) and were used to establish log phase, generate maximum growth rates and follow the general growth curve. Cells from the different phases were transferred to 12 and 24 welled plates for further detailed examination. Detailed Morphological Observations

To observe the different morphologies, cells were examined in 96-well microtiter plates using an Olympus CK40 inverted tissue culture microscope and on standard slides with coverslips using a Zeiss Axio Imager IIe epifluorescence microscope equipped with brightfield, differential interference contrast (DIC), epifluorescence filter set 49 for DAPI stain (excitation: G

365; beamsplitter: FT 395; emission: BP 445/50), filter set 15 for chlorophyll autofluorescence

7 (excitation: BP 546/12; beamsplitter: FT 580; emission: LP 590), an Osram mercury short arc

HBO lamp (Osram, Augsburg, Germany) as the light source, an HRm black and white digital

camera and an MRC5 color digital camera. Morphometric measurements made using the Zeiss

Axiovision Software allowed the calculation of width to height ratios used to quantitatively

define the difference between the brevis-like cells and the butterfly form. On five separate

occasions butterfly-like cells, brevis-like cells, and spherical cells were isolated in single wells of

a 96-well plate. These cells were observed over time until the number of cells grew too plentiful

or necrosis occurred. DAPI staining and epifluorescence microscopy

Live cells for staining were transferred as 1 mL volumes from 12 well plates to 1.5 mL

microcentrifuge tubes and fixed with 2% glutaraldehyde (GTA, final concentration) for 10-15

minutes in the dark at 4ºC prior to centrifugation at 2500 rpm for 10-20 seconds. The

supernatant was removed and the cell pellet was rinsed with phosphate buffered saline (Dulbecco

and Vogt 1954) to a total volume of 1 mL and the cells were resuspended. A 950 µL subsample

of this cell suspension was added to 50 µL of 5 µg · mL-1 (final concentration) 4’,6- diamidino-2-phenylindole stain (DAPI, Invitrogen, Carlsbad, CA, USA) and the cells were incubated in the dark at 4ºC for a minimum of 30 minutes. This was a modification of the method used by Yeung (2005). Cells were observed using a Zeiss Imager IIe as described previously. DIC, DAPI, and chlorophyll autofluorescence images were recorded with an

AxioCam HRm digital camera and stored as digital files. A standard of Gallus erythrocytes

(RBC) (Biosure, Grass Valley, CA, USA) with a mean DNA content of 2.5 pg·cell -1 were

8 measured with every staining sample to calibrate densitometric values for dinoflagellate specimens. Nuclear DNA contents for dinoflagellate samples were estimated by comparison of densitometric mean of fluorescence intensity (If) values for dinoflagellate nuclei and Gallus RBC using the equation (Kapraun 1993):

(RBC If) · (dino If)-1 = 2.5 pg · (x pg)-1

DAPI binds by a non-intercalative mechanism to adenine and thymine rich regions of DNA which contain at least four A-T base pairs (Portugal and Waring 1988). Consequently, nucleated red blood cells are best used as a standard for estimating DNA when A-T contents of both standard and experimental DNA are equivalent (Coleman et al. 1981). Gallus has a nuclear DNA composition of 42-43 mol % G +C (Marmur and Doty 1962). Karenia cells have a similar range and linearity was accepted between DAPI DNA binding in both RBC and Karenia samples

(Lidie et al. 2005). Intensity and consistency of DAPI staining of dinoflagellates and chicken erythrocytes was demonstrated previously (LaJeunesse 2005, Parrow and Burkholder 2003,

Vaulot et al. 1994, Veldhuis et al. 1997). RESULTS Morphological Analysis

A growth curve of K. papilionacea (Fig. 1) consisted of a log phase (day 0 − 8), defined by rapid growth of the culture population, a stationary phase (day 9 − 16) where the cell densities were not changing, and a decline phase (after day 16). Throughout the growth cycle, various morphologies of K. papilionacea dominated the culture. Healthy butterfly-shaped cells and healthy brevis-shaped cells were dominant during the log phase (Fig. 1 A, B) with few to no

9 spherical cells present at the bottom of the culture vessel. During stationary phase, the number of spherical cells on the bottom of the culture vessel increased and the motile population was mixed between healthy and stressed butterfly and brevis-shaped cells (Fig. 1 A-E). In the decline phase, the number of spherical cells on the bottom of the culture vessel increased as well as the debris of burst spherical cells. During this phase, the number of motile cells were less common and appeared to have fewer chloroplasts giving them a colorless appearance (Fig. 1 C-E). Cells from the K. papilionacea culture when observed with DIC microscopy, exhibited motile

(flagellated) cells found throughout the water column and non-motile (cyst-like) cells found at or near the bottom of the water column. The proportions of these two phases depended on the culture’s phase in the growth cycle (Fig. 1).

The classic butterfly-like K. papilionacea form was generally much wider than it was tall.

Those cells that were easily mistaken for K. brevis were as tall or taller than they were wide.

Spherical cells were clearly identified by circular shape and lack of cingulum and sulcus. While spherical cells were easy to identify, a numerical method was needed to quantify the difference between a classic K. papilionacea form and a cell that could be mistaken for K. brevis. Images of cells (n=695) were measured for width and height and the resulting ratio was compared to morphological appearance (Table 1). All non-spherical cells with a width : height ratio (w:h) of

1.091 or greater were grouped visually as the classic butterfly-shaped cell (Fig. 2). All non- spherical cells with w:h of 1.090 and lower could be easily mistaken for K. brevis. For the purposes of this study any cell with a w:h = 1.091 or greater was classified as a butterfly-shaped

10 cell (Fig. 2) and appeared wider than they were tall. The cytoplasm was clear, finely granulated, with distinct, plate-like chloroplasts that gave the cell an appearance of even golden brown coloration (Fig. 3 A). The hyaline membrane was colorless and ruptured easily. Viewed ventrally, a large nucleus was positioned in the left lobe of the hypotheca. The tip of the epitheca formed an apical protuberance called the carina and was usually colorless with a bilobed chloroplast near the medial region. A transverse groove, or cingulum, was centrally located and a sulcus extended into the epitheca. A transverse flagellum and a longitudinal flagellum were readily visible.

Butterfly-shaped cells varied in width from 24.89 to 53.01 µm with a mean of 37.35 µm, width being measured across the widest part of the hypotheca. These cells varied in height from

22.44 to 44.33 µm with a mean of 31.54 µm. They had a w:h (Fig. 2) from 1.091 to 1.522 with a mean of 1.185 (Table 1). As the culture progressed into the stationary phase or when cells were placed in unfavorable conditions more cells with colorless cytoplasm were visible. These cells had condensed chloroplasts that were rounded to reniform and cells became slightly deformed and inflated as the cingulum, sulcus, and cell shape gradually reduced (Fig. 3 B). Red inclusion bodies of an undefined nature were observed in some but not all cells (Fig. 3 A). Butterfly- shaped cells made up 51% of the observed late log phase population.

Brevis-shaped cells were similar to butterfly-shaped cells and could be easily mistaken for Karenia brevis in environmental samples and had a w:h less than or equal to 1.090 (Table 1,

Fig. 2). These cells were as tall as they were wide and not as wide as the butterfly shape (Fig 3

11 C). Brevis-shaped cells varied from 20.23 to 45.85 µm in width with a mean of 30.97 µm and in height from 20.68 to 44.17 µm with a mean of 31.48 µm. The w:h varied from 0.728 to 1.090 with a mean of 0.984 (Table 1). This cell type made up 39% of the population of cells observed during the late log phase. Brevis-shaped cells from stationary cultures or cells placed in unfavorable conditions (Fig. 3 D) follow the same trends as butterfly-shaped cells, containing fewer chloroplasts and losing cell shape.

Spherical cells were circular to ellipsoidal (Fig. 3 E) and had reduced to no motility as their flagella were either shed or reabsorbed. Because of this reduced motility, these cells were found on the bottom of the culture vessel, usually grouped together in what appeared to be a mucoid matrix. Spherical cells were not jostled when a culture plate was nudged during microscopic observation and responded less to the suction of a pipet than their free swimming counterparts suggesting greater adherence to the bottom surface. There was no visible secondary or enclosed membrane. When transferred to fresh medium, these cells reverted to brevis or butterfly-shapes or lysed. Spherical cells varied in width from 26.49 to 50.58 µm with a mean of

36.35 µm and in height from 25.35 to 50.83 µm with a mean of 36.72 µm (Table 1). This cell type made up 7% of the population of cells observed.

Variation within each defined morphology is summarized in Table 1 and Figure 4. Butterfly- shaped (Fig. 4 A-D) and brevis-shaped (Fig. 4 E-H) cells could be difficult to distinguish from each other as there was a gradual increase in size and width to height ratio, yet the largest and widest butterfly-shaped cell was clearly discernible from the smallest brevis-shaped cell. Some

12 cells lost definition in the lobes of the hypotheca (Fig. 4 I-L) prior to losing the cingular groove to become spherical shaped. Some of the cells observed, 3% of the population, were misshapen due to ongoing cytokinesis or rested at an odd angle to the camera so as to be of unidentifiable morphology. These cells were excluded from analysis.

Various nuclear morphologies were also observed (Fig. 5). The standard nuclear morphology was a round nucleus with chromosomes visible in a spotted pattern (Fig. 5 A). Of all nuclei observed, 57 % had this standard, round nuclear morphology. As the nucleus entered mitosis, chromosomes began to spread out and nuclear area increased (Fig. 5 B). The nucleus took on an hourglass shape as the two daughter nuclei begin to separate (Fig. 5 C & D). Of all nuclei observed, 0.6 % had this hourglass morphology. As telophase continued, the nuclei were only joined by a thin thread of chromatin (Fig. 5 E), as demonstrated by 0.3 % of all cells observed.

Eventually, the nuclei took on the round morphology visible in a typical G1 phase cell as cytokinesis completed (Fig. 5 F). A total of 1.7 % of cells had two nuclei within one cellular membrane undergoing cytokinesis.

Cytokinesis was observed when the two daughter nuclei from mitosis were located in separate lobes of the hypotheca (Fig. 6 A). The cleft between lobes of the hypotheca deepened

(Fig. 6 B) and additional lobes formed (Fig. 6 C). The morphology of the two cell halves modified in such a way as to appear as two mature cells, attached at a midpoint, with a ‘quartet of wings’ (Fig. 6 D). The final point of attachment was observed to be the apical tip, or carina, of the epitheca (Fig. 6 E). DNA Content Analysis

13 Densitometric mean data for the various morphotypes were obtained with epifluorescent

microscopy to determine the differences in calculated picograms of DNA (pg·cell-1). The

measured DNA contents for the entire population were analyzed using a frequency distribution

histogram (Fig. 7). The values ranged from 28.1 to 215.3 pg·cell-1 with a mean of 93.3 pg·cell-1. Using frequency distribution histograms (Fig. 8) DNA content of the different forms could be compared. Butterfly-shaped cells varied in DNA content from 35.8 to 215.3 pg·cell-1 with a mean of 94.7 pg·cell-1 (n=333). Brevis-shaped cells varied in DNA content from 28.1 to

197.36 pg·cell-1 with a mean of 92.0 pg·cell-1 (n=280) and spherical cells varied in DNA content from 52.1 to 156.0 pg·cell-1 with a mean of 90.3 pg·cell-1 (n=44). A one way analysis of variance (ANOVA, p<0.001) showed that spherical cells had statistically significantly less

DNA than brevis-shaped cells and butterfly-shaped cells. There was no statistically significant difference in DNA content between butterfly-shaped cells and brevis-shaped cells.

Comparison of fluorescence (If) data from the three morphotypes indicated a large range of

DNA contents within each type (3410-11330). Differences in fluorescence can be explained by variation involved with the fluorescence technique, nuclear orientation in relation to chloroplasts within the cell, background fluorescence, and the fact that the amount of DNA varies depending on which stage of the cell cycle each cell is at for any given time. Therefore, If data should only be considered accurate to +/- 0.1 pg (Kapraun 2005). This DNA content value was compared to other cultured dinoflagellate DNA values in Table 3.

DISCUSSION

14 These results suggest that the butterfly-shaped and brevis-shaped cells are numerous vegetatively reproducing cells in healthy, exponentially growing cultures. This conclusion is further supported by microscopic observations of mitosis. Brevis-shaped cells were observed dividing, suggesting that these cells do not need to reach the size of the butterfly-shaped cells prior to division. Butterfly-shaped cells were also observed dividing. As cultures progress into senescence, chloroplasts appeared to shrink and cell morphology became less defined.

Definition between hypothecal lobes diminished and eventually even the sulcus and cingulum disappeared as the cell became spherical. Similar features were observed in environmental samples of Gymnodinium breve when K. papilionacea was still considered part of that species

(Dragovich 1967), indicating that this is not an artifact of culture but a natural cellular process within this genus.

When isolated, the brevis-shaped cells divided vegetatively over time into both brevis-shaped cells and butterfly-shaped cells. The differences in morphology between these two cell shapes cannot be simply explained by transition of life stage or sexual reproduction as shown by DNA content analysis. Because these two morphologies did not have statistically different DNA contents, they both were considered haploid, the generally assumed ploidy of most dinoflagellates (Pfiester and Anderson 1987). The differences can possibly be explained by variation of size within a population. When this species was originally described, it was defined as 18 - 48 µm wide which is a very large range (Haywood et al. 2004). While butterfly-shaped cells varied in w:h from 1.091 to 1.522, the median value was 1.17. For the entire population of

15 cells observed, 50% were between 1.014 and 1.177, indicating that while the classic butterfly- shaped has been described by Steidinger et al. (1966) as twice as wide as long and by Dragovich

(1967) as several times broader than long, extremely wide butterfly-shaped cells were not the norm. As shown in Figure 4 (B, C, F, G), the typical butterfly-shaped cells and the typical brevis-shaped cells are generally very similar in appearance, with differences apparent only at the extremes (Fig. 4 A, D, E, H). The typical K. papilionacea cell closely resembles K. brevis in morphology and appearance. In fact, the morphological description of K. papilionacea is virtually indistinguishable from that of K. brevis. Haywood (2004) found that the only morphological features K. papilionacea possessed that differed from K. brevis were a more pronounced hypothecal excavation and a more pointed apical carina. This study showed that the both hypothecal excavation and the definition of the apical carina vary throughout the life cycle of the cell and are not good indicators of species. Wilson’s early descriptions (1967) of K. brevis included a laterally extended form, his paper included a micrograph that resembles K. papilionacea. He suggests that the reason that cells become laterally extended (butterfly-shaped) was because they had delayed cell division for some reason, although further study was needed to support this hypothesis. The sequences of the large subunit ribosomal DNA (LSU rDNA) allowed these two species to be differentiated, underscoring the importance of molecular information in combination with morphological observations when identifying potentially harmful species. A sandwich hybridization assay (SHA) was developed to detect K. brevis, K. papilionacea, K. mikimotoi, and K. selliformis (Haywood et al. 2007). This study further

16 emphasized the need to develop and use molecular techniques along with classical morphological studies to resolve cryptic morphologies of Karenia as with cryptic species of the diatom Skeletonema (Kooistra et al. 2008).

The low number of spherical cells compared to the other two morphologies observed can be explained by two factors. Cells for this study were harvested during log phase growth, however, some spherical cells were captured if the log phase growth was transitioning into stationary phase. The number of spherical cells increases throughout the growth cycle as the culture moves toward stationary phase and decline phase. Also, spherical cells adhered to the bottom of the culture container so when cultures were sampled, the planktonic forms were favored by the pipet and the spherical cells resisted suction.

Wilson (1967) described two spherical cells in his culture of Gymnodinium breve (= Karenia brevis). One form was spherical or ellipsoidal in form with a reduced flagella. He observed these rounded cells only after cultures reached a maximum population level and found them in masses on the bottom of the container. These rounded cells reverted to normal cells after they were transferred to fresh medium. The other form he observed less frequently and found that it contained a membrane within a membrane. He did not describe germination from this second form.

The spherical cells observed in the present study closely resemble the description of the first form of K. brevis put forth by Wilson. They did not have a membrane within a membrane.

Spherical cells of K. papilionacea in this study appeared after cultures had completed the log

17 phase and either shed or reabsorbed their flagella, sometimes retaining a portion with which they circled slowly on the vessel bottom. The membranes of these spherical cells appeared to be more adhesive and they tended to group together in masses on the culture vessel bottom. Some spherical cells reverted to normal cells when they were transferred to fresh medium, suggesting that these are pellicle cysts. These findings also suggest that this formation of a spherical cell occurs for more than one species within the genus.

Bravo (2010) defined pellicle cysts as cysts that have a single layered wall and either no required dormancy or one that is shorter than the dormancy of the hypnocyst for that specific species. The spherical cells observed in this study appear to be pellicle cysts that are forming in response to degrading environmental conditions as the population of the culture begins to tax available resources and moves into stationary phase. If environmental conditions do not exceed the tolerable limits of the pellicle cyst, then the cell will remain in that state until conditions become favorable again. However, if environmental conditions do exceed the tolerable limits of the cyst, it will progress into necrosis and lyse. A combination of environmental factors including light, temperature, salinity, nutrient availability, and bacterial load likely contribute to the viability of a pellicle cyst. In culture, temperature is fairly constant but light availability will change as the culture becomes more densely populated, salinity could change as the culture medium ages, nutrient availability decreases as an increasing population of cells utilizes nutrients, and bacterial load increases as the small population of bacteria in culture also grows.

In preliminary experiments, DAPI stain was used on live cells and was not readily taken

18 up by the nucleus. Dead cells stained significantly brighter than live cells. Live cells also had a significantly lower densitometric mean than the chicken erythrocyte standard. This difference can be explained by a cellular mechanism that prevented DAPI stain from reaching the nucleus.

When fixed cells were used along with DAPI (Yeung et al. 2005, Fojtova et al. 2010), this wide variation in stained cells was eliminated and results similar to other published values for dinoflagellate species were observed (Table 2, LaJeunesse et al. 2005). DAPI stain was successfully used to establish a 1C value of DNA content of 75.5 pg·cell-1 for the species K. papilionacea which is considered haploid following the assumption of Pfiester and Anderson

(1987) that all vegetative dinoflagellate cells are haploid with few exceptions. As seen in Table 2 this value is different than other Karenia species but falls within the scale of variation found among species of Alexandrium and Prorocentrum.

The range of observed DNA contents represents a population at various stages of the cell cycle. As with similar studies conducted on K. brevis (VanDolah and Leighfield 1999), the majority of the population is in the G1 phase of the cell cycle with a very low percentage in the

S, G2, and M phases. The standard nuclear morphology of a K. papilionacea cell in the G1 phase of the cell cycle was round, with visible chromosomes that were condensed, arch shaped fibers (Bhaud et al. 2000). The ends of these fibers were oriented toward the exterior of the nucleus which gave it a spotted appearance with a baseline nuclear area and fluorescence. As the cells progressed through the S phase into the G2 phase of the cell cycle, these fibers became more tightly packed and nuclear area was expected to remain similar to G1 phase cells while

19 fluorescence was expected to increase. As the cell cycle progressed into the M phase (mitosis), nuclear area was expected to increase. Several 24 hour cell cycle observations would need to be conducted to observe this pattern over time. At any given sample time, a range of nuclear morphologies was observed suggesting that the population was actively going through the cell cycle and supporting the conclusion that this range of DNA content is due to ongoing DNA synthesis.

The first hypothesis of this study was that K. papilionacea develops a K. brevis-like form that can be easily confused with the known toxic species found in the Gulf of Mexico.

Morphological analysis conducted in this study revealed that while the wide, butterfly-like cells did occur, the brevis-shaped cells reported in natural samples (Tomas, unpublished data) pose a large potential for misidentification particularly in the absence of molecular probes.

The second hypothesis of this study was that the brevis-shaped cells, as well as other morphologies such as spherical, cyst-like cells, represent sexually induced stages in the life cycle displaying different DNA contents. There was no statistical difference in DNA content between brevis-shaped and butterfly-shaped cells supporting the conclusion that both of these morphologies are actually one ploidy with a natural variation of size within a population. There was a statistically significant difference in DNA content between each of these forms and the spherical cells, but these cells had significantly less DNA than the other morphologies. The reduced DNA content as measured by the DAPI staining method remains unexplained. Because vegetative cells are already considered haploid further reduction of DNA content would suggest

20 degradation of DNA as cells entered necrosis.

Spherical cells also lacked a double wall. A true hypnocyst resulting from sexual reproduction would be expected to have a mandatory dormancy period, a double wall, and double the DNA content of the vegetative form (Bravo et al. 2010). Most likely these spherical cells are haploid pellicle cysts responding to unfavorable environmental conditions although further experiments focusing on excystment are needed.

This study also indicated that the nuclear DNA content of K. papilionacea could be determined by DAPI-DNA staining and quantified with epifluorescence microscopy. It was found that when applied to fixed cells, DAPI provided results that are consistent with other published values for DNA content of athecate dinoflagellates. The range of DNA contents observed reflects a population that is actively synthesizing DNA as part of the cell cycle.

The third hypothesis of this study was that the butterfly-shaped form is the dominant, vegetatively-reproducing, haploid stage. Based on the width:height ratios (Fig. 2) the majority of cells in a population of K. papilionacea are only slightly wider than they are tall and are not the extremely broad butterfly form that is the classic representative of this species. For the purposes of this study, cells with a width to height ratio of 1.091 or greater appeared to be wider than they were tall and so were termed butterfly-shaped and cells with a smaller width to height ratio were termed brevis-shaped. However, cells in K. brevis cultures can appear slightly wider than they are tall (Wilson 1967) and 50% of the cells evaluated in this study had measurements that were close to that dividing line (1.01 to 1.17) therefore the term butterfly-shaped does not refer to

21 Steidinger et al., (1966) and Dragovich’s (1967) butterfly form that was at least twice as wide as it was tall. At present there is no clear morphological marker to distinguish the K. papilionacea brevis-like cells from those of a true K. brevis in a natural sample. These findings highlight the need for a combination of morphological observations and molecular evidence when identifying cells in environmental samples. Also, the evidence supporting the possible existence of a pellicle cyst could lead to insight into the bloom dynamics of K. papilionacea and the role it plays in the coastal ecosystem of the Delaware Inland Bays region.

22 LITERATURE CITED

Anderson, D. M., Kulis, D. M., Sullivan, J. J., Hall, S. 1990. Toxin composition variations in one isolate of the dinoflagellate Alexandrium fundyense. Toxicon 28(8): 885-93.

Anderson, D.M., Hoagland, P., Kaoru, Y., & White, A.W. [Eds.] 2000. Estimated Annual

Economic Impacts from Harmful Algal Blooms (HABs) in the United States. Woods Hole

Oceanographic Inst. Tech. Rept., WHOI 2000-11.

Backer, L. C., Fleming, L. E., Rowan, A., Cheng, Y., Benson, J., Pierce, R. H., Zaias, J., Bean, J.,

Bossart, G. D., Johnson, D., Quimbo, R., Baden, D. G. 2003. Recreational exposure to aerosolized brevetoxins during Florida red tide events. Harmful Algae 2(1):19-28.

Bagnis, R., Chanteau, S., Chungue, E., Hurtel, J. M., Yasumoto, T., Inoue, A. 1980. Origins of ciguatera fish poisoning: a new dinoflagellate, Gambierdiscus toxicus Adachi and Fukuyo, definitively involved as causal agent. Toxicon 18 (2):199-208.

Bhaud, Y., Guillebault, D., Lennon, J. -F., Defacque, H., Soyer-Gobillard, M. -O. & Moreau, H.

2000. Morphology and behaviour of dinoflagellate chromosomes during the cell cycle and mitosis. Journal of Cell Science 113:1231-1239.

Bravo, I., Figueroa, R. I., Garces, E., Fraga, S., & Massanet, A. 2010. The intricacies of dinoflagellate pellicle cysts: the example of Alexandrium minutum cysts from a bloom-recurrent

23 area (Bay of Baiona, NW Spain). Deep-Sea Res. II. 57:166-74.

Coleman, A. W., Maguire, M. J., & Coleman, J. R. 1981. Mithramycin- and 4’’,6-diamidino-2- phenylindole (DAPI)-staining for fluorescence microspectrophotometric measurement of DNA in nuclei, plastids, and virus particles. J. Histochem. Cytochem. 29:959-68.

Dale, B. 1986. Life cycle strategies of oceanic dinoflagellates. In Pierrot-Bults, A. C., van der

Spoel, S., Zahuranec, B. J. & Johnson, R. K. [Eds.] Pelagic Biogeography. UNESCO Technical

Papers in Marine Science, Paris, pp. 65-72.

Daugbjerg, N., Hansen, G., Larsen, J. & Moestrup, Ø. 2000. Phylogeny of some of the major genera of dinoflagellates based on ultrastructure and partial LSU rDNA sequence data, including the erection of three new genera of unarmoured dinoflagellates. Phycologia. 39(4):302-17.

Davis, C. C. 1948. Gymnodinium brevis sp. nov., a cause of discolored water and animal mortality in the Gulf of Mexico. Botanical Gazette. 109(3):358-60.

Dulbecco, R. & Vogt, M. 1954. Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99(2):167-82.

24 Dragovich, A. 1967. Morphological variations of Gymnodinium breve Davis in situ. Quart. Jour.

Florida Acad. Sci. 30(4):245-9.

Fojtová, M., Wong, J. Y., Dvorácková, M., Yan, K. H., Sykorova, E., & Fajkus, J. 2010.

Telomere maintenance in liquid cyrstalline chromosomes of dinoflagellates. Chromosoma.

119(5):485-93.

Geesey, M. E & Tester, P. A. 1993. Gymnodinium breve: ubiquitous in Gulf of Mexico waters? In

Smayda, T. J. & Shimizu, Y. [Eds] Toxic Phytoplankton Blooms in the Sea. Proceedings of the

5th International Conference on Toxic Marine Phytoplankton. Elsevier, Amsterdam, pp. 251-5.

Granéli, E., B. Sundström, L. Edler, & D.M. Anderson. 1990. Toxic Marine Phytoplankton.

Proceedings of the 4th International Conference on Toxic Marine Phytoplankton. Elsevier, New

York

Guillard, R. L. 1973. Division rates. In R. J. Stein [Ed] Handbook of phycological methods: culture methods and growth measurements. Cambridge University Press, Cambridge, UK, pp.

289-311.

Harada, T., Oshima, Y., Kamiya, H., Yasumuto, T. 1982. Confirmation of paralytic shellfish toxins in the dinoflagellate Pyrodium bahamense var. compressa and bivalves in Palau. Bull.

25 Japan. Soc. Sci. Fisheries 48:8215.

Haywood, A. J., Steidinger, K. A. & Truby, E. W. 2004. Comparative morphology and molecular phylogenetic analysis of three new species of the genus Karenia (Dinophyceae) from New

Zealand. J. Phycol. 40:165-79.

Haywood, A.J., Scholin, C., Marin, R., Steidinger, K., Heil, C., Ray, J., 2007. Molecular detection of the brevetoxin-producing dinoflagellate Karenia brevis (Dinophyceae) and closely related species using rRNA-targeted probes and a semiautomated sandwich hybridization assay.

J. Phycol. 43:1271–86.

Kapraun, D. F. 1993. Karyology and cytophotometric estimation of nuclear DNA content variation in Gracilaria, Gracilariopsis, and Hydropuntia (Gracilariales, Rhodophyta). Eur. J.

Phycol. 28(4):253-60.

Kapraun, D. F. 2005. Nuclear DNA content estimates in multicellular green, red, and brown algae: phylogenetic considerations. Ann. Bot. (Lond.). 95:7-44.

Kooistra, W. H. C. F., Sarno, D., Balzano, S., Gu, H., Andersen, R. A., Zingone, A. 2008. Global diversity and biogeography of Skeletonema species (Bacillariophyta). Protist 159: 177-93.

26 Lackey, J.B., 1956. Known geographic range of Gymnodinium breve Davis. Quart. Jour. Florida

Acad. Sci. 19:71.

LaJeunesse, T. C. 2005. Symbiodinium (Pyrrhophyta) genome sizes (DNA content) are smallest

among dinoflagellates. J. Phycol. 41:880-6.

Lidie, K. B., Ryan, J. C., Barbier, M. & Van Dolah, F. M. 2005. Gene expression in Florida red

tide dinoflagellate Karenia brevis: analysis of an expressed sequence tag library and

development of DNA microarray. Mar. Biotechnol. 7:481-93.

Magaña, H. A., Contreras, C. & Villareal, T. A. 2003. A historical assessment of Karenia brevis

in the western Gulf of Mexico. Harmful Algae. 2:163-71.

Marmur, J. & Doty, P. 1962. Determination of the base composition of deoxyribonucleic acid

from its thermal denaturation temperature. J. Mol. Biol. 5:109-18.

Oshima, Y., Hasegawa, M., Yasumoto, T., Hallegraeff, G., Blackburn, S. 1987. Dinoflagellate

Gymnodinium catenatum as the source of paralytic shellfish toxins in Tasmanian shellfish.

Toxicon 25(10): 1105-11.

27 Parrow, M. W. & Burkholder, J. M. 2003. Estuarine heterotrophic cryptoperidiniopsoids

(Dinophyceae): life cycle and culture studies. J. Phycol. 39:678-96.

Pfiester, L. A. & Anderson, D. M. 1987. Dinoflagellate reproduction. In Taylor, F. J. R.[Ed.] The

Biology of Dinoflagellates. Blackwell, Oxford, pp 611-48.

Portugal, J., Waring, M. 1988. Assignment of DNA binding sites for DAPI and bisbenzimide

(Hoeschst 33258). Comparative footprinting study. Biochim. Biophys. Acta. 949:158-68.

Simon, N., Cras, A.-L., Foulon, E., & Lemée, R. 2009. Diversity and evolution of marine phytoplankton. C. R. Biol. 332:159-70.

Steidinger, K. A., Davis, J. T. & Williams, J. 1966. Observations of Gymnodinium breve Davis and other dinoflagellates. In Observations of an Unusual Red Tide. A Symposium. Florida Board of Conservation Marine Laboratory Professional Paper Series, Florida Board of Conservation

Marine Laboratory, St. Petersburg, FL, USA. 8:8–15.

Steidinger, K. A. 1979. Collection, enumeration and identification of free-living marine dinoflagellates. In Taylor, D.L., Seliger, H.H. [Eds.], Toxic Dinoflagellate Blooms. Elsevier, New

York, pp. 435–42.

28 Steidinger, K. A. 2009. Historical perspective on Karenia brevis red tide research in the Gulf of

Mexico. Harmful Algae. 8:549-61.

Tatters, A. O., Muhlstein, H. I., & Tomas, C. R. 2010. The hemolytic activity of Karenia selliformis and two clones of Karenia brevis throughout a growth cycle. J. Appl. Phycol.

22:435-42.

Tester, P.A., Stumpf, R.P., Vukovich, F.M., Fowler, P.K. & Turner, J.T., 1991. An expatriate red tide bloom: transport, distribution, and persistence. Limnol. Oceanogr. 36(5):1053–61.

Uhlig, G. & Sahling, G. 1990. Long-term studies on Noctiluca scintillans in the German Bight population dynamics and red tide phenomena 1968-1988. Netherlands Journal of Sea Research

25(1-2):101-12.

Utermöhl, H. 1931. Neue Wege in der quantitativen Erfassung des Planktons. Verh Int Verein

Limnol. 5:567-96.

Van Dolah, F. M., & Leighfield, T. A. 1999. Diel phasing of the cell-cycle in the Florida red tide dinoflagellate, Gymnodinium breve. J. Phycol. 35:1404-11.

29 Vaulot, D., Birrien, J. -L., Marie, D., Casotti, R., Veldhuis, M. J., Kraay, G. W. & Chrétiennot-

Dinet, M. -J. 1994. Morphology, ploidy, pigment composition, and genome size of cultured strains of Phaeocystis (Prymnesiophyceae). J. Phycol. 30:1022-35.

Veldhuis, M. J., Cucci, T. L. & Sieracki, M. E. 1997. Cellular DNA content of marine phytoplankton using two new fluorochromes: taxonomic and ecological implications. J. Phycol.

33:527-41.

Walker, L. M. 1982. Evidence for a sexual cycle in the Florida red tide dinoflagellate,

Ptychodiscus brevis (=Gymnodinium breve). Transactions fo the American Microscopical

Society. 101(3):287-93.

Watkins, S. M., Reich, A., Fleming, L. E., Hammond, R. 2008. Neurotoxic shellfish poisoning.

Mar. Drugs 6(3): 431-55.

Whereat, E. 2010. Harmful Algae Report. Available at: http://citizen-monitoring.udel.edu/ reports/archive.shtml (last accessed 02 August 2011)

Wilson, W. B. 1967. Forms of the dinoflagellate Gymnodinium breve Davis in cultures.

30 Contributions in Marine Science. 12:120-34.

Yeung, P. K., Hung, V. L., Chan, F. C., & Wong, J. Y. 2005. Characterization of a Karenia papilionacea-like dinoflagellate from the South China Sea. J. Mar. Biol. Ass. U.K. 85:779-81.

31 Table 1. A summary of descriptive statistical values for the three morphological classifications of Karenia papilionacea.

DNA content Morphology Width (µm) Height (µm) w:h (pg·cell-1)

Butterfly min/max 24.89/53.01 22.44/44.33 1.09/1.52 36/215 median 37.25 31.46 1.17 87 mean 37.26 31.52 1.18 95 SD 5.44 4.51 0.06 36 n 339 339 339 333

Brevis min/max 20.23/45.85 20.68/44.17 0.73/1.09 28/197 median 30.82 31.10 1.00 91 mean 37.26 31.44 0.98 92 SD 4.81 4.25 0.08 27 n 270 270 270 280

Spherical min/max 26.49/50.58 25.35/50.83 52/156 median 36.25 37.02 84 mean 36.35 36.72 90 SD 6.08 6.80 26 n 47 47 44

32 Table 2. The DNA content in cultured dinoflagellates*. Size in µm (median Taxon (Dinophyceae) CCMP culture no. length) pg·cell-1 (n) Alexandrium andersonii 2222 20-24 (22) 21.8 (2) Alexandrium insuetum 2082 22-28 (25) 30.8 (2) Alexandrium lusitanicum 1888 17-19 (18) 31 Alexandrium tamarense 1598 20-30 (25) 103.5 Amphidinium carterae 1314 12-18 (15) 5.9 (2) Gymnodinium simples 419 8-12 (10) 11.6 (7) Heterocapsa triquetra 449 18-20 (19) 24.1 Heterocapsa pygmaea 1322 9-14 (11.5) 3.8 (2) Plarella glacialis 2088 9-14 (11.5) 7.0 (2) Karenia brevis 2229 20-28 (24) 57.1 Karenia mikimotoi 429 28-30 (29) 100.1 Karenia papilionacea 20-53 (37) 75.5 (16) Karlodinium galatheanum 1974 10-18 (14) 16.9 (2) Karlodinium galatheanum 2282 8-20 (14) 16.3 Katodinium rotundatum 1542 11-16 (13.5) 3.6 Pfiesteria piscicida 1830 10-12 (11) 5.5 (2) Pfiesteria schumwayae 2357 10-18 (14) 19.8 Prorocentrum balticum 1260 10-15 (12.5) 8.3 (2) Prorocentrum dentatum 1517 14-17 (15.5) 6.6 (2) Prorocentrum micans 689 22-38 (30) 115.2; 225.0 Prorocentrum minimum 1329 7-16 (12.5) 6.9 (3) *modified from LaJeunesse et al. 2005

33 34

Figure 1. Growth curve of Karenia papilionacea Kp0707-1. Morphologies A & B dominate the log phase, morphologies A-E are found in the stationary phase, and morphologies C, D, and E dominate the decline phase. Scale bar represents 20 µm. 35

Figure 2. Frequency distribution of width to height ratios of Karenia papilionacea. Cells with a width to height ratio of 1.091 or greater appeared wider than tall and were classified as butterfly shaped cells (A). Cells with a width to height ratio of less than or equal to 1.090 appeared as tall or taller than wide and were classified as brevis shaped cells (B). The dotted line marks 1.09 on the y axis. Scale bar represents 20 µm.

Figure 3. Differential interference contrast images of Karenia papilionacea morphologies in culture; A) log phase butterfly-shaped cell with deep hypothecal excavation (he), visible nucleus (n), chloroplast (chl), red inclusion body (skinny arrow) and cingular groove (broad arrow); B) stationary phase butterfly-shaped cell; C) log phase brevis-shaped cell; D) stationary phase brevis-shaped cell; E) stationary phase spherical cell. Scale bar represents 20 µm.

36

Figure 4. Variation within the morphology classifications of Karenia papilionacea: A-D) butterfly-shaped cells; B) cell is slightly wider and taller than A; (C - D) reduced chloroplasts and less definition in the cingulum and hypothecal excavation. (E-H) brevis-shaped cells; E) cell has reduced chloroplasts; F) cell has reduced chloroplasts and a slight hypothecal excavation but has a defined cingulum; G) cell has reduced definition of the hypothecal excavation and cingulum; H) a large brevis-shaped cell with minimal loss in definition of the cingulum and hypothecal excavation; I) a brevis-shaped cell that lacks hypothecal excavation; J) reduced morphological definition of cingulum and hypothecal excavation; K) extreme reduction in definition of cingulum and hypothecal excavation; L) spherical cell with reduced chloroplasts. Nuclei are stained blue with DAPI and viewed with epifluorescent microscopy. Scale bar represents 20 µm.

37

Figure 5. Epifluorescent images of DAPI stained nuclei of Karenia papilionacea progressing through mitosis: A) pre-mitotic nucleus; B) chromosomes beginning to move toward poles; C) nucleus is beginning to take on a hourglass shape; D) hourglass shape has formed with distinct outlines of the two future nuclei; E) two nuclei are evident joined by a small amount of chromatin; F) two nuclei have moved to poles of daughter cells. Scale bar represents 20 µm.

38

Figure 6. Differential interference contrast images of Karenia papilionacea undergoing cytokinesis: A) the two daughter nuclei are located in separate lobes of the hypotheca; B) the cleft between lobes of the hypotheca deepens; C) additional lobes form; D) cytokinetic cell appears as two mature cells, attached at a midpoint; E) the final point of attachment at the apical tip. Nuclei are stained blue with DAPI. Scale bar represents 20 µm. 39

Figure 7. Frequency distribution of DNA content (pg · cell-1) of Karenia papilionacea.

40

Figure 8. Frequency distribution of DNA content (pg · cell -1) of Karenia papilionacea in A) butterfly-shaped cells; B) brevis-shaped cells, and C) spherical cells.

41 F4::Unit width height µm² pg DensMean w:h morph clock time round nuc tube30_01 47.96 41.07 163.5 6.20 5231.56 1.16776236 pap 10AM tube30_02 163.88 5.04 4251.67 #DIV/0! round 10AM tube30_03 45.85 44.17 220.53 7.20 6077.16 1.03803487 kar 10AM tube30_04 36.11 31.77 131.37 7.23 6104.81 1.13660686 pap 10AM 1 tube30_05 175.44 5.63 4754.51 #DIV/0! round 10AM 1 tube30_06 138.54 6.51 5494.13 #DIV/0! round 10AM 1 tube30_07 34.62 34.35 131.52 7.00 5910.71 1.00786026 kar 10AM tube30_08 31.83 29.21 97.6 8.32 7026.76 1.08969531 kar 10AM 1 tube30_08 33.05 28.3 98.04 8.10 6839.41 1.16784452 pap 10AM 1 tube30_09 32.71 29.87 91.41 7.93 6694.99 1.09507867 kar 10AM 1 tube30_10 125.78 5.77 4868.4 #DIV/0! round 10AM tube30_11 179.59 5.68 4796.58 #DIV/0! round 10AM 1 tube30_12 50.63 43.34 249.64 6.20 5233 1.16820489 pap 10AM tube30_13 43.72 36.71 154.45 4.97 4199.62 1.19095614 pap 10AM 1 42 tube30_14 154.29 5.28 4455.54 #DIV/0! round 10AM 1 tube30_15 37.74 37.32 125.1 4.32 3646.51 1.01125402 kar 10AM tube30_15 148.3 4.71 3976.76 #DIV/0! unk 10AM tube30_16 37.39 35.54 124.89 6.59 5563.56 1.05205402 kar 10AM 1 tube30_17 49.27 41.98 231.35 7.55 6372.3 1.17365412 pap 10AM tube30_18 39.87 37.62 202.25 7.17 6055.33 1.05980861 kar 10AM 1 tube30_18 36.57 29.93 110.66 6.18 5213.99 1.22185099 pap 10AM 1 tube30_18 37.69 31.46 123.14 6.83 5769.02 1.19802924 pap 10AM 1 tube30_19 35.91 31.1 137 6.07 5121.41 1.15466238 pap 10AM 1 tube30_19 35.14 31.84 133.35 6.38 5389.86 1.10364322 pap 10AM 1 tube30_20 50.91 44.21 199.95 6.42 5415.82 1.15154942 pap 10AM tube30_21 41.7 31.84 156.3 5.64 4763.29 1.30967337 pap 10AM 1 tube30_22 34.21 35.44 119.73 5.81 4907.46 0.96529345 kar 10AM 1 tube30_22 37.23 29.06 133.65 6.05 5111.09 1.28114246 pap 10AM 1 tube30_23 42.61 35.38 232.6 6.29 5308.15 1.20435274 dividing 10AM tube30_24 53.01 44.33 237.53 7.12 6010.97 1.1958042 pap 10AM tube30_25 31.8 31.51 123.4 6.55 5526.41 1.00920343 kar 10AM 1 tube30_26 33.76 30.63 128.67 6.93 5853.36 1.1021874 pap 10AM 1 tube30_27 34.2 30.71 125.8 6.49 5480.72 1.11364376 pap 10AM 1 tube30_28 41.2 35.3 110.07 5.47 4619.32 1.16713881 pap 10AM 1 tube30_29 39.3 34.44 104.72 5.41 4569.78 1.14111498 pap 10AM 1 tube30_31 31.86 31.09 127.02 6.84 5775.14 1.02476681 kar 10AM 1 tube30_33 13.92 2.50 2110.54 #DIV/0! CN 10AM tube30_34 40.3 38.18 196.63 8.78 7415.91 1.05552645 kar 10AM tube30_35 36.15 34.83 104.06 6.52 5507.65 1.03789836 kar 10AM 1 tube30_35 43.45 36.65 173.98 6.47 5464.55 1.18553888 pap 10AM 1 tube30_36 34.52 28.31 107.13 5.92 5001.65 1.21935712 pap 10AM 1 tube30_37 36.04 34.77 183.06 7.61 6422.29 1.03652574 kar 10AM 1 tube30_37 37.72 30.91 132.14 6.00 5065.56 1.22031705 pap 10AM 1 tube30_38 36.43 31.27 144.45 7.19 6070.1 1.16501439 pap 10AM 1 43 tube30_39 37.18 35.28 115.05 5.70 4810.84 1.05385488 kar 10AM 1 tube30_40 33.48 29.16 117.9 6.78 5722.34 1.14814815 pap 10AM 1 tube30_41 38.73 33.63 170.98 5.18 4370.16 1.15165031 pap 10AM tube30_42 35.07 28.63 144.48 6.88 5805.31 1.22493888 pap 10AM 1 tube30_42 39.89 33.46 192.09 8.19 6911.71 1.19216975 pap 10AM 1 tube30_43 40.26 34.61 171.49 4.48 3781.73 1.16324762 pap 10AM 1 tube34_01 37.75 32.35 117.92 8.60 8381.99 1.16692427 pap 12PM 1 tube34_02 30.18 24.97 81.09 7.74 7546.21 1.20865038 pap 12PM 1 tube34_03 29.63 26.4 88.18 6.14 5982.55 1.12234848 pap 12PM 1 tube34_04 28.45 25.58 86.84 7.57 7381.55 1.11219703 pap 12PM 1 tube34_05 31.87 27.64 123.34 7.63 7439.31 1.15303907 pap 12PM 1 tube34_06 27.99 24.94 75.17 7.53 7338.06 1.1222935 pap 12PM 1 tube34_07 12.01 2.64 2570.48 #DIV/0! CN 12PM tube34_08 36.88 32.56 118.32 6.46 6293.26 1.13267813 pap 12PM 1 tube34_08 32.94 26.89 94.61 6.30 6138.9 1.2249907 pap 12PM 1 tube34_09 41.98 36.1 216.51 6.90 6721.82 1.16288089 pap 12PM tube34_10 40.61 35.9 111.65 6.71 6541.89 1.13119777 pap 12PM 1 tube34_11 38.46 33.44 116.86 7.13 6953.09 1.15011962 pap 12PM tube34_12 27.28 23.79 124.81 6.43 6263.02 1.14670029 pap 12PM 1 tube34_13 40.13 35.15 153.24 6.09 5940.28 1.14167852 pap 12PM 1 tube34_14 44.32 39.59 231.99 6.31 6151.16 1.11947461 pap 12PM tube34_15 41.41 35.83 196.32 7.15 6972.11 1.15573542 pap 12PM tube34_16 51.86 31.07 261.81 6.01 5859.18 1.66913421 dividing 12PM tube34_17 23.87 23.41 121.21 7.36 7177.09 1.01964972 kar 12PM tube34_18 43.21 34.35 177.15 5.92 5767.54 1.25793304 pap 12PM tube34_19 35.25 28.2 118.41 5.33 5191.21 1.25 pap 12PM tube34_20 29.48 28.24 110.29 6.11 5959.09 1.04390935 kar 12PM tube34_21 41.91 35.66 184.15 6.71 6537.05 1.1752664 pap 12PM tube34_22 37.14 34.3 113.78 7.01 6835 1.08279883 kar 12PM 1 tube34_23 37.58 31.15 209.14 7.04 6863.36 1.20642055 pap 12PM 44 tube34_24 30.55 27.51 140.57 6.29 6131.83 1.11050527 pap 12PM tube34_25 38.77 36.13 128.36 7.13 6946.73 1.07306947 kar 12PM 1 tube34_25 29.81 25.7 105.15 7.68 7484.41 1.15992218 pap 12PM 1 tube34_26 145.65 6.18 6025.65 #DIV/0! round 12PM tube34_27 29.55 27.36 82.5 6.12 5961.99 1.08004386 kar 12PM tube34_28 27.81 24.68 80.16 5.22 5084.89 1.12682334 pap 12PM 1 tube34_29 32.98 27.94 95.01 7.72 7528.31 1.18038654 pap 12PM 1 tube34_30 34.8 32.78 91.62 7.90 7701.19 1.06162294 kar 12PM 1 tube34_31 40.81 34.93 175.63 6.54 6369.63 1.16833667 pap 12PM tube34_32 39.25 34.02 167.34 6.88 6710.54 1.1537331 pap 12PM tube34_33 35.55 28.8 114.81 6.55 6387.17 1.234375 pap 12PM 1 tube34_34 10.52 2.36 2302.95 #DIV/0! CN 12PM tube34_35 35.23 32.72 105.63 7.26 7079.65 1.07671149 kar 12PM 1 tube34_36 37.77 30.42 148.21 7.53 7340.68 1.24161736 pap 12PM tube34_37 37.74 32.21 175.79 5.91 5761.7 1.17168581 pap 12PM tube34_38 33.11 28.61 96.39 7.87 7666.75 1.15728766 pap 12PM 1 tube34_39 28.2 26.61 92.11 8.14 7931.73 1.05975197 kar 12PM 1 tube34_40 30.95 34.02 108.43 6.40 6238.04 0.90975897 kar 12PM tube34_41 32.31 29.62 89.54 6.89 6713.31 1.09081702 kar 12PM 1 tube34_42 38.39 32.9 118.05 6.90 6724.75 1.1668693 pap 12PM tube34_43 22.72 22.8 96.4 7.55 7356.75 0.99649123 kar 12PM 1 tube34_44 27.85 26.94 93.31 6.11 5959.85 1.03377877 kar 12PM 1 tube34_45 39.58 43.73 151.24 6.26 6100.71 0.90509947 kar 12PM 1 tube34_46 30.9 28.6 86.98 6.95 6774.4 1.08041958 kar 12PM 1 tube34_46 38.95 34.47 118.09 7.60 7407.68 1.12996809 pap 12PM 1 tube34_47 46.86 39.87 168.16 7.59 7400.23 1.17531979 pap 12PM 1 tube34_48 33.78 30.99 153.46 5.12 4988.78 1.09002904 kar 12PM tube34_49 31.91 30.27 117.68 7.13 6953.53 1.05417906 kar 12PM tube34_50 44.56 38.27 193.64 7.77 7571.1 1.16435851 pap 12PM tube38_01 30.72 27.86 82.93 6.05 6135.8 1.10265614 pap 2PM 1 45 tube38_02 33.31 28.59 91.02 4.15 4208.27 1.16509269 pap 2PM 1 tube38_03 8.11 2.46 2491.11 #DIV/0! CN 2PM tube38_04 39.47 34.26 134.07 8.96 9085.86 1.15207239 pap 2PM 1 tube38_05 35.58 31.19 91.53 6.47 6555.67 1.14075024 pap 2PM 1 tube38_06 37.06 31.07 108.9 4.98 5043.42 1.19279047 pap 2PM 1 tube38_07 34.81 27.95 99.65 7.14 7233.1 1.24543828 pap 2PM 1 tube38_08 150.69 6.81 6904.49 #DIV/0! round 2PM 1 tube38_09 8.06 2.82 2857.92 #DIV/0! CN 2PM tube38_10 40.48 34.15 168.93 7.50 7600.72 1.18535871 pap 2PM tube38_10 37.65 31.94 156.87 7.80 7903.61 1.1787727 pap 2PM tube38_11 35.07 30.18 96.28 8.34 8450.38 1.16202783 pap 2PM 1 tube38_12 42.63 37.22 178.1 6.97 7063.2 1.14535196 pap 2PM 1 tube38_13 36.54 29.81 99.22 7.95 8054.07 1.22576317 pap 2PM 1 tube38_14 33.46 30.64 99.62 6.69 6782.07 1.09203655 kar 2PM 1 tube38_15 38.29 32.61 109.54 8.02 8131.76 1.1741797 pap 2PM tube38_16 31.25 26.01 87.06 7.53 7634.79 1.20146098 pap 2PM 1 tube38_17 34.84 30.99 154.78 8.39 8499.49 1.12423362 pap 2PM 1 tube38_18 38.6 30.54 238.89 8.67 8788.54 1.26391618 pap 2PM tube38_19 36.13 30.55 150.06 5.54 5619.34 1.18265139 pap 2PM 1 tube38_20 34.13 29.63 106.14 7.44 7539.85 1.1518731 pap 2PM 1 tube38_21 31.59 26.62 111.86 6.43 6520.13 1.18670173 pap 2PM tube38_22 31.24 25.79 101.4 5.02 5084.1 1.21132222 pap 2PM 1 tube38_23 34.35 32 141.94 5.56 5640.83 1.0734375 kar 2PM tube38_24 15.34 28.16 110.79 9.32 9445.09 0.54474432 unk 2PM 1 tube38_24 36.45 29.34 139.88 6.79 6883.41 1.24233129 pap 2PM tube38_25 37.03 32.76 96.48 4.92 4982.55 1.13034188 pap 2PM tube38_26 31.46 27.75 105.18 6.99 7087.48 1.13369369 pap 2PM 1 tube38_27 39.03 33.26 158.09 8.40 8509.84 1.17348166 pap 2PM 1 tube38_28 7.39 2.26 2287.81 #DIV/0! CN 2PM tube38_28 11.88 2.46 2494.08 #DIV/0! CN 2PM 46 tube38_29 10.5 2.46 2497.26 #DIV/0! CN 2PM tube38_30 38.41 33.14 116.48 7.48 7586.61 1.15902233 pap 2PM 1 tube38_31 40.51 34.15 187.08 6.34 6427.16 1.18623719 pap 2PM tube38_32 36.12 32.17 118.94 7.14 7234.06 1.1227852 pap 2PM 1 tube38_33 99.81 5.28 5353.64 #DIV/0! unk 2PM tube38_34 37.37 33.48 121.12 5.87 5948.2 1.11618877 pap 2PM 1 tube38_35 27.21 22.61 86.84 4.15 4211.28 1.2034498 pap 2PM tube38_36 46.52 41.94 191.57 5.61 5683.75 1.10920362 pap 2PM tube38_37 52.04 42.31 207.29 7.06 7157.54 1.22996927 pap 2PM tube38_38 42.66 38.27 169 5.29 5360.86 1.11471126 pap 2PM tube38_39 148.81 5.72 5795.78 #DIV/0! round 2PM tube38_40 38.85 32.92 99.39 4.98 5045.4 1.18013366 pap 2PM 1 tube38_41 34.97 32.69 143.79 5.92 5996.48 1.0697461 kar 2PM tube38_42 30.48 28.7 80.39 3.36 3409.59 1.06202091 kar 2PM tube38_43 29.81 32.62 133.94 5.34 5416.98 0.91385653 kar 2PM tube38_43 39.08 32.61 132.08 5.67 5750.2 1.1984054 pap 2PM tube38_44 41.73 37.78 200.66 5.74 5818.7 1.10455267 pap 2PM tube38_45 39.54 35.21 149.14 7.14 7236.67 1.12297643 pap 2PM tube38_46 11.37 2.33 2358.62 #DIV/0! CN 2PM tube38_47 28.5 28.45 104.61 7.35 7455.22 1.00175747 kar 2PM 1 tube38_48 35.65 29.47 98.63 8.02 8129.93 1.20970478 pap 2PM 1 tube38_49 37.28 32.36 164.78 8.35 8460.23 1.15203956 pap 2PM tube38_50 10.11 2.72 2752.06 #DIV/0! CN 2PM tube38_51 30.36 25.3 111.58 5.36 5434.45 1.2 pap 2PM tube42_01 11.47 2.55 2616.27 #DIV/0! CN 4PM tube42_02 10.53 2.44 2501.76 #DIV/0! CN 4PM tube42_27 10.25 2.50 2566.34 #DIV/0! CN 4PM tube42_20 30.89 28.87 114.35 6.29 6447.26 1.06996883 kar 4PM 1 tube42_30 28.91 27.6 87.32 4.75 4863.91 1.04746377 kar 4PM 1 tube42_35 30.23 29.93 96.87 8.01 8211.95 1.01002339 kar 4PM 1 47 tube42_41 29.42 27.88 131.15 6.28 6430.93 1.05523673 kar 4PM 1 tube42_43 34.53 32.28 109.19 6.90 7066.99 1.0697026 kar 4PM 1 tube42_46 32.39 30.34 98.46 6.90 7066.16 1.06756757 kar 4PM 1 tube42_50 28.83 27.65 115.86 7.31 7492.18 1.04267631 kar 4PM 1 tube42_52 25.84 25.81 100.58 7.41 7596.41 1.00116234 kar 4PM tube42_03 36.27 29.94 112.41 6.44 6599.27 1.21142285 pap 4PM 1 tube42_04 35.77 29.93 128.24 6.15 6300.68 1.19512195 pap 4PM 1 tube42_05 30.58 24.81 99.97 5.63 5772.01 1.23256751 pap 4PM 1 tube42_06 36.56 28.95 126.97 7.21 7386.06 1.26286701 pap 4PM 1 tube42_07 29.93 25.84 78.52 4.90 5025.25 1.15828173 pap 4PM 1 tube42_08 31.49 23.74 89.48 8.90 9118.73 1.32645324 pap 4PM 1 tube42_09 30.68 27.38 91.79 6.92 7092.16 1.12052593 pap 4PM 1 tube42_10 29.88 26.61 111.62 5.88 6028.23 1.12288613 pap 4PM tube42_11 29.39 26.44 85.82 5.99 6139.04 1.11157337 pap 4PM 1 tube42_13 29.91 25.3 94.93 7.90 8098.44 1.18221344 pap 4PM 1 tube42_15 40.22 33.39 175.23 7.89 8086.86 1.20455226 pap 4PM 1 tube42_17 40.62 34.47 187.73 7.95 8147.95 1.17841601 pap 4PM 1 tube42_18 43.25 36.41 156.69 7.47 7657.99 1.18786048 pap 4PM 1 tube42_19 37.78 33.2 123.52 7.90 8097.9 1.13795181 pap 4PM 1 tube42_22 44.81 38.06 217.93 6.21 6359.81 1.17735155 pap 4PM 1 tube42_23 39.73 32.4 107.08 6.19 6340.07 1.22623457 pap 4PM 1 tube42_24 29.55 26.77 108.8 5.76 5905.06 1.10384759 pap 4PM 1 tube42_25 35.78 31.06 136.67 7.49 7676.76 1.15196394 pap 4PM tube42_26 33.32 29.7 101.08 6.05 6194.5 1.12188552 pap 4PM 1 tube42_28 49.02 42.08 189.46 5.22 5344.61 1.16492395 pap 4PM tube42_31 33.03 29.89 110.77 6.56 6721.11 1.10505186 pap 4PM 1 tube42_32 36.98 32.04 126.11 6.58 6739.6 1.15418227 pap 4PM 1 tube42_33 38.74 34.08 125.96 5.07 5194.18 1.13673709 pap 4PM 1 tube42_34 35.83 30.46 115.08 6.05 6194.73 1.17629678 pap 4PM 1 tube42_36 33.55 27.61 103.82 5.53 5661.46 1.21513944 pap 4PM 1 48 tube42_37 35.95 32.05 105.51 7.81 8005.86 1.12168487 pap 4PM 1 tube42_38 44.42 36.45 187.9 8.57 8780.89 1.21865569 pap 4PM 1 tube42_39 47.97 38.64 202.91 7.39 7571.44 1.24145963 pap 4PM 1 tube42_40 46.56 39.01 185.41 8.07 8265.2 1.19354012 pap 4PM tube42_42 36.32 32.17 117.99 7.11 7282.32 1.12900218 pap 4PM tube42_44 34.56 29.71 118.34 6.64 6807.19 1.1632447 pap 4PM tube42_45 40.52 32.69 109.24 6.20 6353.8 1.23952279 pap 4PM 1 tube42_47 42.57 35.77 164.83 6.69 6855.01 1.19010344 pap 4PM tube42_49 33.75 27.62 104.64 6.71 6875.19 1.22194062 pap 4PM tube42_51 26.84 23.93 84.97 6.21 6363.85 1.12160468 pap 4PM tube42_53 35.47 27.53 141.91 6.83 7000.62 1.28841264 pap 4PM tube42_12 115.7 5.84 5986.69 #DIV/0! round 4PM tube42_14 96.83 7.37 7556.23 #DIV/0! round 4PM tube42_16 194.2 8.64 8853.43 #DIV/0! round 4PM tube42_21 149.3 5.89 6031.74 #DIV/0! round 4PM tube42_29 123.72 7.50 7684.53 #DIV/0! round 4PM 1 tube42_48 122.82 7.21 7388.57 #DIV/0! round 4PM 1 tube46_01 33.05 27.2 97.92 5.15 4406.28 1.21507353 pap 6PM tube46_02 32.09 27.48 163.33 8.25 7048.12 1.16775837 pap 6PM tube46_03 26.92 26.16 80.44 6.75 5768.42 1.02905199 kar 6PM 1 tube46_04 26.97 25.3 111.69 6.42 5486.06 1.06600791 kar 6PM 1 tube46_05 27.45 22.45 101.75 7.45 6366.02 1.22271715 pap 6PM tube46_06 97.91 7.29 6232.16 #DIV/0! round 6PM tube46_07 39.88 30.65 165.17 6.50 5555.06 1.30114192 pap 6PM tube46_07 31.03 26.41 105.93 6.97 5956.04 1.17493374 pap 6PM 1 tube46_08 9.1 2.50 2137.05 #DIV/0! CN 6PM tube46_09 25.42 23.36 106.82 6.96 5950.53 1.08818493 kar 6PM 1 tube46_10 27.38 23.94 99.92 7.28 6223.81 1.14369256 pap 6PM 1 tube46_11 27.5 31.98 88.42 7.25 6195.64 0.85991245 kar 6PM 1 tube46_12 28.35 23.37 106.74 7.42 6346.13 1.21309371 pap 6PM 49 tube46_14 136.71 6.91 5908.68 #DIV/0! round 6PM tube46_15 32 29.71 135.93 6.97 5960.52 1.07707842 kar 6PM tube46_16 41.8 34.05 137.51 9.41 8041.4 1.22760646 pap 6PM tube46_17 37.9 31.34 162.52 9.46 8086.54 1.20931717 pap 6PM tube46_18 32.55 27.61 98.09 5.73 4894 1.17892068 pap 6PM 1 tube46_19 30.22 27.99 95.16 9.35 7993.99 1.07967131 kar 6PM 1 tube46_20 37.87 30.03 149.07 9.02 7706.85 1.26107226 pap 6PM tube46_21 34.77 27.86 99.48 9.52 8134.11 1.24802584 pap 6PM 1 tube46_22 139.1 9.40 8036.95 #DIV/0! round 6PM tube46_23 117.36 6.51 5562.41 #DIV/0! unk 6PM 1 tube46_24 26.51 22.77 70.67 7.57 6473.37 1.16425121 pap 6PM 1 tube46_25 24.55 23.43 81.94 12.26 10476.32 1.04780196 kar 6PM 1 tube46_26 32.34 35.92 193.65 6.92 5918.45 0.90033408 kar 6PM tube46_27 94.7 7.59 6492.32 #DIV/0! round 6PM tube46_27 112.39 6.70 5723.55 #DIV/0! round 6PM tube46_28 24.89 22.44 87.61 8.88 7587.93 1.10918004 pap 6PM 1 tube46_29 38.36 30.12 178.27 8.97 7666.73 1.27357238 pap 6PM tube46_30 42 32.2 175.9 10.26 8766.86 1.30434783 pap 6PM 1 tube46_31 33.66 33.12 102.38 6.43 5499.79 1.01630435 kar 6PM 1 tube46_32 32.79 28.62 96.36 8.67 7407.6 1.14570231 pap 6PM 1 tube46_33 32.7 29.83 120.56 8.08 6911.05 1.09621187 kar 6PM 1 tube46_34 42.04 36.45 149.94 8.58 7335.09 1.15336077 pap 6PM tube46_35 39.78 34.44 178.1 7.25 6201.68 1.15505226 pap 6PM tube46_36 32.23 28.07 109.48 7.25 6193.66 1.14820093 pap 6PM 1 tube46_37 26.94 23.25 106.56 8.33 7120.1 1.15870968 pap 6PM 1 tube46_38 39.6 31.17 138.25 6.21 5307.11 1.27045236 pap 6PM 1 tube46_39 43.67 36.32 188.07 8.36 7147.78 1.20236784 pap 6PM tube46_40 34.95 32.56 162.03 9.03 7719.28 1.07340295 kar 6PM tube46_41 36.47 28.21 109.04 7.74 6617.3 1.29280397 pap 6PM 1 tube46_42 37.63 32.45 191.7 7.61 6502.95 1.1596302 pap 6PM 50 tube46_43 34.32 32.21 132.25 7.16 6118.84 1.06550761 kar 6PM tube46_44 104.88 6.33 5414.58 #DIV/0! round 6PM 1 tube46_45 32.94 30.43 131.29 7.62 6511.4 1.08248439 kar 6PM 1 tube46_46 121.97 7.02 5999.89 #DIV/0! round 6PM tube46_47 45.02 38.22 242.99 7.37 6304.29 1.17791732 pap 6PM tube46_48 36.91 32.84 133.03 5.91 5053.79 1.12393423 pap 6PM 1 tube46_49 133.11 7.20 6153.76 #DIV/0! round 6PM tube50_01 29.44 24.34 58.72 5.95 5982.17 1.20953164 pap 8PM 1 tube50_02 38.02 32.48 192.21 7.06 7099.16 1.1705665 pap 8PM tube50_03 24.31 23.34 67.91 7.35 7385.72 1.04155955 kar 8PM tube50_04 28.77 26.75 85.61 8.86 8903.61 1.07551402 kar 8PM 1 tube50_05 29.04 25.54 80.62 5.71 5737.03 1.13703994 pap 8PM tube50_06 29.26 25.64 72.06 5.63 5663.02 1.14118565 pap 8PM 1 tube50_07 35.17 28.94 102.73 7.22 7257.76 1.21527298 pap 8PM tube50_08 39.87 35.6 195.85 8.02 8059.07 1.11994382 pap 8PM tube50_09 40.77 33.35 143.47 6.71 6740.33 1.22248876 pap 8PM tube50_10 33.44 30.5 95.74 6.16 6193.25 1.09639344 kar 8PM 1 tube50_11 28.84 25.69 121.93 7.15 7181.64 1.1226158 pap 8PM tube50_12 31.1 28.54 130.46 5.60 5632.51 1.08969867 kar 8PM tube50_13 33.05 28.09 110.39 4.67 4691.61 1.17657529 pap 8PM 1 tube50_14 42.9 32.77 146.39 6.98 7017.54 1.3091242 pap 8PM 1 tube50_14 38.09 29.59 120.64 7.99 8031.26 1.28725921 pap 8PM 1 tube50_15 29.74 26.27 74.85 7.26 7298.91 1.13208984 pap 8PM 1 tube50_16 29.42 25.69 90.25 6.72 6755.12 1.14519268 pap 8PM 1 tube50_16 29.49 23.9 76.24 6.24 6267.02 1.23389121 pap 8PM 1 tube50_17 8.76 2.77 2785.05 #DIV/0! CN 8PM tube50_18 25.71 25.68 122.96 6.92 6955.58 1.00116822 kar 8PM 1 tube50_19 28.22 25.52 98.37 7.03 7068.27 1.10579937 pap 8PM 1 tube50_20 10.04 2.23 2240.29 #DIV/0! CN 8PM tube50_21 29.29 25.1 118.43 6.29 6323.22 1.16693227 dividing 8PM 1 51 tube50_21 26.3 23.03 104.31 4.85 4875.71 1.14198871 dividing 8PM 1 tube50_22 33.44 27.85 90 7.61 7644.9 1.20071813 pap 8PM tube50_22 30.45 24.05 103.68 4.95 4976.44 1.26611227 pap 8PM tube50_23 34.48 31.78 186.43 7.10 7135.11 1.08495909 kar 8PM tube50_24 28.92 24.58 120.55 6.79 6828.75 1.17656631 pap 8PM tube50_25 29.03 23.73 73.84 5.36 5389.49 1.22334598 pap 8PM 1 tube50_25 31.73 28.1 141.24 5.84 5869.56 1.12918149 pap 8PM tube50_26 32.3 28.71 100.13 7.50 7537.98 1.12504354 pap 8PM 1 tube50_26 32.51 26.9 116.5 7.20 7233.14 1.20855019 pap 8PM 1 tube50_27 23.4 23.84 81.28 7.52 7555.39 0.98154362 kar 8PM 1 tube50_28 133.94 6.87 6904.31 #DIV/0! round 8PM tube50_29 154.15 5.64 5664.75 #DIV/0! round 8PM tube50_30 33.03 28.99 111.22 6.05 6084.34 1.1393584 pap 8PM 1 tube50_31 36.67 30.38 138.76 6.46 6493.49 1.20704411 pap 8PM tube50_32 36.62 38.45 220.29 5.92 5951.68 0.95240572 kar 8PM tube50_33 34.57 31.38 203.13 8.11 8150.98 1.10165711 pap 8PM tube50_34 29.25 27.2 118.29 7.03 7060.77 1.07536765 kar 8PM tube50_35 37.56 32.78 165.49 4.34 4362.5 1.14582062 pap 8PM tube50_36 39.27 32.78 93.9 5.77 5800.9 1.19798658 pap 8PM 1 tube50_37 29.69 25.56 124.91 6.71 6743.85 1.16158059 pap 8PM 1 tube50_37 145.49 6.37 6402.68 #DIV/0! round 8PM 1 tube50_38 48.86 39.07 230.74 6.93 6963.55 1.25057589 pap 8PM tube50_39 46.9 39.46 271.85 5.79 5820.38 1.18854536 pap 8PM tube50_40 32.93 31.43 138.85 5.87 5902.34 1.0477251 kar 8PM 1 tube50_41 38.68 33.7 107.94 5.30 5331.01 1.14777448 pap 8PM 1 tube50_42 40.25 30.68 234.92 5.45 5480.89 1.3119296 dividing 8PM tube50_43 33.9 31.69 151.62 5.55 5573.69 1.06973809 kar 8PM tube50_44 27.8 23.99 90.56 5.16 5185.09 1.15881617 pap 8PM tube50_44 37.12 32.86 117.53 3.88 3904.3 1.1296409 pap 8PM tube50_44 37.04 33.49 187.08 6.19 6218.69 1.10600179 dividing 8PM 52 tube50_45 34.75 32.05 127.85 6.18 6210.49 1.08424337 kar 8PM 1 tube54_01 10 2.14 2176.76 #DIV/0! CN 10PM tube54_02 34.09 34.44 160.75 7.47 7600.84 0.9898374 kar 10PM tube54_03 41.88 31.2 154.2 7.64 7782.34 1.34230769 dividing 10PM 1 tube54_04 31.16 23.3 90.71 7.70 7843 1.33733906 pap 10PM 1 tube54_05 28.21 28.56 92.29 6.89 7012.65 0.9877451 kar 10PM 1 tube54_05 27.47 28.92 92.33 8.40 8551.73 0.94986169 kar 10PM 1 tube54_06 25.33 24.86 107.48 6.30 6418.2 1.01890587 unk 10PM 1 tube54_06 31.34 21.27 104.23 7.10 7223.84 1.47343677 unk 10PM 1 tube54_07 32.64 32.53 116.44 7.13 7261.45 1.00338149 kar 10PM 1 tube54_08 33.21 32.75 93.49 7.22 7351.59 1.0140458 kar 10PM 1 tube54_09 36.35 32.89 135.25 7.47 7601.9 1.10519915 pap 10PM 1 tube54_10 29.43 29.75 117.22 6.65 6770.22 0.9892437 kar 10PM 1 tube54_10 31.88 28.09 99.19 7.83 7973.59 1.13492346 pap 10PM 1 tube54_11 26.23 30.36 106.33 8.04 8187.73 0.86396574 kar 10PM 1 tube54_12 38.93 35.67 143.77 6.66 6785.62 1.09139333 kar 10PM 1 tube54_13 25.07 20.32 111.7 8.06 8201.53 1.23375984 unk 10PM 1 tube54_14 40.57 34.94 124.15 7.79 7929.07 1.16113337 pap 10PM 1 tube54_15 29.46 28.22 126.68 7.05 7173.8 1.04394047 kar 10PM 1 tube54_16 31.21 25.84 80.37 7.10 7225.89 1.20781734 pap 10PM 1 tube54_17 36.35 26.89 114.43 7.35 7486.38 1.35180364 pap 10PM 1 tube54_18 39.9 34.46 109.33 7.96 8104.11 1.15786419 pap 10PM 1 tube54_19 10.75 2.86 2913.76 #DIV/0! CN 10PM tube54_20 39.9 34.19 202.55 8.08 8229.06 1.1670079 pap 10PM tube54_21 38.72 31.71 169.46 6.05 6158.36 1.22106591 pap 10PM tube54_22 31.01 27.33 73.92 6.65 6770.29 1.13465057 pap 10PM 1 tube54_23 49.16 35.42 243.84 6.26 6373.2 1.38791643 dividing 10PM tube54_24 31.46 24.91 109.25 6.10 6205.94 1.26294661 pap 10PM tube54_25 37.18 33.26 119.55 6.76 6878.24 1.11785929 pap 10PM 1 tube54_26 26.95 23.87 130.35 6.40 6513.49 1.12903226 pap 10PM 53 tube54_27 36.52 34.62 134.59 6.56 6679.67 1.05488157 kar 10PM tube54_28 33.31 29.01 125.9 6.47 6586.12 1.14822475 pap 10PM 1 tube54_29 144.01 5.70 5807.57 #DIV/0! round 10PM tube54_30 24.31 28.68 131.77 6.47 6582.99 0.84762901 kar 10PM 1 tube54_30 28.63 28.78 137.84 6.40 6512.23 0.99478805 kar 10PM 1 tube54_31 38.18 39.01 254.32 6.39 6509.21 0.9787234 kar 10PM tube54_32 38.27 32.53 134.11 4.71 4794.98 1.17645251 pap 10PM 1 tube54_33 40 32.13 137.07 6.49 6609.54 1.24494242 pap 10PM 1 tube54_33 40.44 36.2 251.84 6.10 6209.78 1.11712707 dividing 10PM tube54_34 37.7 29.65 119.88 6.08 6187.49 1.27150084 pap 10PM 1 tube54_35 30.01 29.64 109.98 6.97 7099.9 1.01248313 kar 10PM 1 tube54_36 41.09 33.36 128.15 6.45 6566.81 1.23171463 pap 10PM 1 tube54_37 108.63 6.82 6947.87 #DIV/0! round 10PM 1 tube54_38 41.33 33.75 162.42 6.73 6846.82 1.22459259 pap 10PM 1 tube54_39 34.44 34.54 250.78 6.64 6759.41 0.99710481 kar 10PM tube54_40 47.59 40.78 196.4 7.22 7351.79 1.16699362 pap 10PM 1 tube54_41 36.83 26.98 88.77 5.14 5236.62 1.36508525 pap 10PM 1 tube54_42 41.93 37.11 189.54 7.56 7696.13 1.12988413 pap 10PM 1 tube54_43 28.01 26.62 140.54 5.81 5918.13 1.05221638 kar 10PM 1 tube54_44 37.82 36.79 163.83 6.51 6628.2 1.02799674 kar 10PM tube54_45 42.93 37.28 172.82 6.22 6337.19 1.15155579 pap 10PM tube54_46 41.9 33.66 171.44 6.42 6540.68 1.24480095 pap 10PM 1 tube54_47 113.82 6.35 6469.2 #DIV/0! round 10PM 1 tube54_48 41.42 34.82 133.48 6.08 6191.29 1.18954624 pap 10PM 1 tube54_49 41.83 33.8 140.54 5.84 5947.34 1.23757396 pap 10PM 1 tube54_49 35.09 29.99 116.53 6.23 6338.24 1.17005669 pap 10PM 1 tube54_50 164.6 4.80 4891.98 #DIV/0! round 10PM tube58_01 28.17 25.93 78.93 5.26 4734.97 1.08638642 kar 12AM 1 tube58_02 8.92 2.27 2041.45 #DIV/0! CN 12AM tube58_03 29.48 25.86 125.12 6.67 5999.76 1.13998453 pap 12AM 1 54 tube58_04 25.44 22.74 90.77 4.79 4311.7 1.11873351 pap 12AM 1 tube58_05 34.19 29.43 148.24 7.47 6722.18 1.16173972 pap 12AM tube58_05 34.12 28.65 155.02 6.30 5664.99 1.19092496 pap 12AM tube58_06 22.14 28.36 103.7 7.07 6356.41 0.78067701 kar 12AM 1 tube58_07 32.84 25.82 154.83 7.32 6588.01 1.27188226 pap 12AM tube58_07 126.42 6.98 6279.77 #DIV/0! unk 12AM tube58_08 32.85 31 126.75 6.00 5392.78 1.05967742 kar 12AM 1 tube58_09 33.83 30.71 119.39 6.18 5559.72 1.10159557 pap 12AM 1 tube58_10 42.97 39.95 197.63 7.34 6602.58 1.07559449 kar 12AM tube58_11 88.28 7.64 6873.9 #DIV/0! unk 12AM 1 tube58_12 32.87 30.27 103.19 5.98 5382.14 1.08589362 kar 12AM 1 tube58_13 33.84 26.87 107.7 5.92 5328.51 1.2593971 pap 12AM 1 tube58_14 166.74 6.09 5475.28 #DIV/0! round 12AM 1 tube58_15 34.17 25.17 97.04 7.21 6487.31 1.35756853 pap 12AM 1 tube58_16 36.63 35.03 110.05 7.43 6687.5 1.04567514 kar 12AM 1 tube58_17 39.17 32.07 163.71 6.89 6197.6 1.22139071 pap 12AM tube58_18 35.97 29.71 174.92 7.58 6817.69 1.21070347 pap 12AM 1 tube58_19 28.81 29.54 127.96 5.64 5070.35 0.97528775 kar 12AM 1 tube58_19 28.5 32.6 120.84 7.08 6368.14 0.87423313 kar 12AM 1 tube58_20 38.13 37.65 253.31 7.06 6348.92 1.012749 dividing 12AM tube58_21 29.25 26.69 139.85 6.35 5715.34 1.09591607 kar 12AM 1 tube58_22 42.96 35.91 246.03 7.02 6313.55 1.19632414 dividing 12AM tube58_23 36.78 33.68 128.07 7.20 6479.02 1.09204276 kar 12AM 1 tube58_24 75.77 5.15 4634.94 #DIV/0! unk 12AM 1 tube58_25 38.1 39.44 117.18 7.14 6423.54 0.96602434 kar 12AM 1 tube58_26 33.25 29.41 153.88 7.05 6342.06 1.13056783 pap 12AM tube58_27 36.53 31.79 149.96 3.94 3547.56 1.14910349 pap 12AM 1 tube58_28 36.61 35.79 151.48 7.24 6515.57 1.02291143 kar 12AM 1 tube58_29 36.57 32.47 133.2 6.75 6069.79 1.1262704 pap 12AM 1 tube58_30 27.42 29.41 131.84 6.44 5789.02 0.93233594 kar 12AM 1 55 tube58_31 33.84 30.3 176.09 7.02 6314.38 1.11683168 pap 12AM 1 tube58_32 25.8 31.15 127.63 7.27 6539.02 0.8282504 kar 12AM tube58_33 29.05 27.73 117.71 6.47 5815.8 1.04760188 kar 12AM tube58_34 47.97 28.83 319.03 5.12 4608.09 1.66389178 dividing 12AM tube58_35 9.33 2.73 2456.06 #DIV/0! CN 12AM tube58_35 28.88 29.74 121.87 7.22 6489.99 0.97108272 kar 12AM 1 tube58_36 26.44 29.41 94.57 8.28 7452.06 0.89901394 kar 12AM 1 tube58_37 132.45 6.13 5512.55 #DIV/0! round 12AM 1 tube58_38 35.39 34.93 130.74 6.64 5974.25 1.0131692 kar 12AM tube58_39 158.6 6.09 5480.7 #DIV/0! round 12AM tube58_40 236.89 6.57 5906.6 #DIV/0! unk 12AM tube58_41 41.03 33.62 223.81 7.24 6508.46 1.22040452 pap 12AM tube58_42 215.87 4.75 4275.08 #DIV/0! round 12AM tube58_43 38.21 33.4 129.75 6.71 6035.7 1.14401198 pap 12AM 1 tube58_44 37.59 33.64 220.92 5.72 5147.24 1.11741974 pap 12AM tube58_45 126.84 4.18 3764.27 #DIV/0! round 12AM tube58_46 26.38 31.27 125.9 6.86 6169.79 0.84362008 kar 12AM tube58_47 36.58 29.31 126.12 5.08 4566.26 1.24803821 pap 12AM 1 tube58_48 22.31 27.98 92.01 5.70 5126.93 0.79735525 kar 12AM 1 tube58_49 26.66 34.29 119.9 6.76 6083.64 0.77748615 kar 12AM 1 tube58_50 34.11 36.61 126.73 6.59 5923.39 0.93171265 kar 12AM 1 tube58_51 39 36.4 202.27 7.11 6394.86 1.07142857 kar 12AM 1 tube58_52 29.79 27.94 145.26 6.26 5628.99 1.06621331 kar 12AM 1 tube58_54 39.67 37.21 142.45 6.12 5508.52 1.06611126 kar 12AM tube58_54 35.69 33.91 128.09 6.86 6169.21 1.05249189 kar 12AM 1 tube58_54 39.27 36.86 157.12 6.45 5800.3 1.06538253 kar 12AM tube62_01 17.19 23.94 75.94 3.73 3755.32 0.71804511 unk 2AM 1 tube62_02 32.06 28.27 113.76 6.77 6813.32 1.13406438 pap 2AM 1 tube62_03 34.08 39.06 118.72 4.74 4770.14 0.87250384 kar 2AM tube62_04 17.21 20.05 88.58 8.65 8708.54 0.85835411 unk 2AM 1 56 tube62_05 30.33 30.41 140.55 7.95 8006.16 0.99736929 kar 2AM 1 tube62_06 27.6 27.24 73.06 5.38 5420.15 1.01321586 kar 2AM 1 tube62_07 36.7 32.1 193.12 7.22 7268.18 1.14330218 pap 2AM 1 tube62_08 11.5 2.50 2517.24 #DIV/0! CN 2AM tube62_09 40.72 32.61 185.42 7.82 7875.55 1.24869672 pap 2AM tube62_09 184.67 9.28 9343.37 #DIV/0! round 2AM tube62_10 37.71 27.81 158.93 7.90 7954.17 1.35598706 pap 2AM tube62_11 47.64 39.82 165.79 8.98 9044.94 1.19638373 pap 2AM 1 tube62_12 37.25 31.33 119.21 8.05 8108.8 1.18895627 pap 2AM 1 tube62_13 38.46 31 179.56 9.30 9365.98 1.24064516 pap 2AM 1 tube62_14 177.08 5.85 5887.9 #DIV/0! round 2AM 1 tube62_15 29.47 27.32 121.53 7.21 7264.45 1.07869693 kar 2AM tube62_16 38.69 32.46 110.13 7.60 7654.18 1.19192853 pap 2AM 1 tube62_17 22.92 24.66 101.18 7.20 7251.28 0.92944039 kar 2AM 1 tube62_18 31.16 30.6 117.65 6.63 6678.44 1.01830065 kar 2AM tube62_19 35.6 30.57 105.58 6.01 6047.31 1.1645404 pap 2AM 1 tube62_19 37.71 31.36 125.03 7.80 7853.09 1.20248724 pap 2AM 1 tube62_20 30 29.17 136.19 7.14 7188.21 1.02845389 kar 2AM tube62_21 37.66 32.82 123.78 6.11 6152.84 1.14747105 pap 2AM 1 tube62_22 32.71 31.45 136.98 7.46 7509.67 1.04006359 kar 2AM 1 tube62_23 34.32 32.31 141.69 6.92 6969.78 1.06220984 kar 2AM 1 tube62_23 37.02 33.46 151.3 8.64 8695.96 1.1063957 pap 2AM 1 tube62_24 36.66 35.82 125.74 7.83 7885.99 1.02345059 kar 2AM 1 tube62_24 35.36 23.23 167.32 8.93 8994.62 1.52216961 pap 2AM 1 tube62_25 38.69 35.72 117.53 8.17 8222.54 1.0831467 kar 2AM 1 tube62_26 33.23 31.7 130.06 6.86 6909.64 1.04826498 kar 2AM 1 tube62_26 257.33 5.72 5757.44 #DIV/0! round 2AM tube62_27 40.06 30.94 253.58 7.41 7456.7 1.29476406 dividing 2AM tube62_28 40.13 36.34 174.93 8.04 8095.62 1.10429279 pap 2AM 1 tube62_29 28.56 28.74 140.49 7.85 7906.04 0.99373695 kar 2AM 1 57 tube62_29 40.02 34.33 111.67 8.00 8059.49 1.16574425 pap 2AM 1 tube62_30 42.82 35.57 215.09 8.62 8676.35 1.20382345 pap 2AM 1 tube62_31 38.01 39.07 131.38 6.12 6158.75 0.97286921 kar 2AM tube62_32 20.77 20.68 71.26 6.89 6936.23 1.00435203 kar 2AM 1 tube62_33 34.6 27.26 129.84 5.38 5416.91 1.26925899 pap 2AM 1 tube62_34 106.34 6.09 6133.32 #DIV/0! round 2AM 1 tube62_35 31.45 30.92 133 6.60 6640.91 1.01714101 kar 2AM 1 tube62_36 30.79 31.55 119.07 7.12 7165.12 0.97591125 kar 2AM 1 tube62_37 32.7 29.83 120.56 7.16 7207.17 1.09621187 kar 2AM 1 tube62_38 36.76 30.65 131.14 6.79 6836.59 1.19934747 pap 2AM 1 tube62_39 36.37 35.27 163.64 7.22 7273.97 1.03118798 kar 2AM 1 tube62_40 42.64 40.34 252.05 7.30 7345.56 1.05701537 kar 2AM tube62_41 73.55 49.7 437.68 7.09 7140.75 1.47987928 dividing 2AM tube62_42 38.54 33.26 151.2 6.05 6089.32 1.15874925 pap 2AM 1 tube62_43 43.67 38.48 192.36 8.53 8586.24 1.13487526 pap 2AM tube62_44 115.01 7.03 7079.42 #DIV/0! unk 2AM tube62_45 192.82 8.41 8463.96 #DIV/0! round 2AM tube62_46 33.9 35.42 127.33 7.13 7180.64 0.95708639 kar 2AM 1 tube66_01 9.48 2.40 2548.28 #DIV/0! CN 4AM tube66_02 47.07 38 135.25 6.99 7404.75 1.23868421 pap 4AM 1 tube66_02 37.65 31.67 121.11 7.51 7962.21 1.18882223 pap 4AM 1 tube66_03 43.79 36.5 193.87 8.47 8977.23 1.19972603 pap 4AM tube66_04 37.59 29.82 172.62 6.38 6767.36 1.26056338 pap 4AM tube66_04 38.83 28.43 238.5 7.16 7593.69 1.36581076 pap 4AM tube66_05 40.2 33.83 109.25 7.59 8048.06 1.18829441 pap 4AM 1 tube66_06 35.37 33.75 116.37 6.44 6823.19 1.048 kar 4AM 1 tube66_07 37.48 33.27 151.18 6.51 6905.15 1.12654043 pap 4AM 1 tube66_08 31.22 31.27 136.29 6.85 7255.81 0.99840102 kar 4AM 1 tube66_09 38.08 34.79 120.59 7.41 7856.38 1.0945674 kar 4AM 1 tube66_10 22.57 23.84 95.37 7.24 7678.44 0.94672819 kar 4AM 1 58 tube66_10 44.61 39.05 126.36 7.42 7862.39 1.14238156 pap 4AM 1 tube66_11 48.01 38.87 125.62 7.61 8066.38 1.23514278 pap 4AM 1 tube66_12 43.75 33.76 137.2 6.55 6939.54 1.29591232 pap 4AM 1 tube66_13 29.3 31.81 124.47 7.13 7561.72 0.921094 kar 4AM 1 tube66_13 45.23 33.23 126.84 7.28 7717.64 1.36111947 pap 4AM 1 tube66_14 30.98 31.1 124.53 7.02 7446.04 0.99614148 kar 4AM 1 tube66_15 50.03 39.01 207.12 7.97 8449.41 1.28249167 pap 4AM 1 tube66_16 28.24 27.35 123.31 6.89 7306.41 1.03254113 kar 4AM 1 tube66_17 10.5 2.55 2701.37 #DIV/0! CN 4AM tube66_18 40.73 33.79 122.43 5.79 6136.22 1.20538621 pap 4AM 1 tube66_19 40.11 34.53 225.1 6.21 6585.96 1.16159861 pap 4AM tube66_19 44.37 39.37 144.49 6.43 6813.17 1.12700025 pap 4AM tube66_20 49.86 39.13 137.19 6.67 7065.26 1.27421416 pap 4AM 1 tube66_21 45.76 39.04 127.29 6.65 7052.77 1.17213115 pap 4AM 1 tube66_22 31.24 32.98 128.03 6.74 7145.23 0.94724075 kar 4AM 1 tube66_23 56.82 30.78 257.57 6.20 6572.85 1.8460039 dividing 4AM tube66_23 228.3 6.62 7012.45 #DIV/0! round 4AM 1 tube66_23 124.46 6.28 6659.03 #DIV/0! round 4AM 1 tube66_24 35.77 35.72 135.12 6.02 6379.87 1.00139978 kar 4AM 1 tube66_25 37.44 34.81 139.33 6.04 6403.41 1.075553 kar 4AM 1 tube66_26 51.22 41.74 148.04 6.22 6589.76 1.22712027 pap 4AM 1 tube66_27 46.16 48.02 133.93 6.31 6689.14 0.96126614 kar 4AM 1 tube66_28 36.83 35.22 131.16 5.42 5741.52 1.04571266 kar 4AM 1 tube66_29 41.25 37.33 212 5.10 5407.57 1.10500938 dividing 4AM tube66_30 38.99 32.09 268.55 3.59 3800.91 1.21502026 pap 4AM tube66_31 35.87 39.97 222.88 7.05 7469.55 0.89742307 kar 4AM tube66_32 49.74 34.57 230.17 7.17 7599.77 1.43881979 dividing 4AM tube66_33 33.01 36.63 128.4 9.01 9550.23 0.9011739 kar 4AM tube66_33 34.61 40.06 116.16 7.32 7761.99 0.86395407 kar 4AM 1 tube66_34 12.08 2.55 2700.43 #DIV/0! CN 4AM 59 tube66_36 45.81 37.44 122.71 9.64 10223.54 1.22355769 pap 4AM 1 tube66_37 33.24 32.43 138.72 10.69 11329.72 1.02497687 kar 4AM 1 tube66_37 35.6 29.82 158.1 8.78 9301.65 1.19382964 pap 4AM 1 tube66_38 33.2 26.66 119.01 7.02 7436.87 1.24531133 pap 4AM 1 tube66_39 34.58 35.19 109.36 6.46 6847.38 0.98266553 kar 4AM 1 tube66_40 39.29 33.6 109.61 6.60 7000.54 1.16934524 pap 4AM 1 tube66_41 46 37.01 157.29 9.69 10268.24 1.24290732 pap 4AM 1 tube66_42 26.65 26.31 104.17 6.64 7038.44 1.01292284 kar 4AM 1 tube66_43 30.71 26.23 108.16 7.80 8269.96 1.1707968 pap 4AM 1 tube66_44 32.04 34.29 85.18 8.90 9438.54 0.9343832 kar 4AM 1 tube66_45 41.66 35.06 202.18 8.60 9115.73 1.18824872 pap 4AM 1 tube66_46 27.03 25.92 82.5 7.78 8242.55 1.04282407 kar 4AM 1 tube66_47 25.99 25.46 121.96 7.06 7486.31 1.02081697 kar 4AM 1 tube66_48 36.78 30.13 92.31 7.60 8061.31 1.22071026 pap 4AM 1 tube66_49 35.21 30.22 115.36 8.24 8733.36 1.16512244 pap 4AM 1 tube66_50 30.74 29.31 122.38 7.79 8256.77 1.04878881 kar 4AM 1 tube66_51 41.98 34.12 90.72 9.38 9939.44 1.23036342 pap 4AM 1 tube66_52 42.71 37.36 136.69 9.84 10434.68 1.14320128 pap 4AM 1 tube66_53 35.11 31.03 100.76 7.68 8145.53 1.13148566 pap 4AM 1 tube 70_01 10.55 2.50 1773.82 #DIV/0! CN 6AM tube 70_02 35.06 38.03 81.31 11.39 8079.38 0.92190376 kar 6AM 1 tube 70_03 27.06 31.13 104.9 11.33 8039.07 0.86925795 kar 6AM 1 tube 70_04 30.98 31.52 110.52 9.72 6894.55 0.98286802 kar 6AM 1 tube 70_05 36.77 27.24 156.02 9.24 6559.2 1.34985316 pap 6AM tube 70_06 23.58 25.77 119.81 9.73 6903.48 0.91501746 kar 6AM 1 tube 70_07 23.3 26.25 116.46 10.83 7686.27 0.88761905 kar 6AM 1 tube 70_08 22.2 26 118.97 9.91 7033.81 0.85384615 kar 6AM 1 tube 70_09 40.28 37.58 119.24 9.82 6968.13 1.07184673 kar 6AM 1 tube 70_10 46.18 36.3 131.95 8.88 6301.84 1.27217631 pap 6AM 1 tube 70_11 28.67 32.97 119.44 9.22 6542.67 0.8695784 kar 6AM 1 60 tube 70_11 40.39 30.18 174.47 8.49 6027.02 1.33830351 pap 6AM tube 70_12 33.42 31.78 126.42 9.20 6529.6 1.05160478 kar 6AM 1 tube 70_13 37.79 35.43 132.23 9.19 6522.01 1.06661022 kar 6AM 1 tube 70_14 38.15 33.94 131.36 9.11 6467.14 1.12404243 pap 6AM 1 tube 70_15 31.39 30.91 135.24 8.53 6052.12 1.01552896 kar 6AM 1 tube 70_15 38.84 32.05 97.03 7.29 5174.41 1.21185647 pap 6AM 1 tube 70_16 31.07 30.02 146.02 8.07 5724.94 1.03497668 kar 6AM 1 tube 70_17 37.7 35.13 219.04 9.47 6722.11 1.07315685 kar 6AM tube 70_18 39.27 33.6 142.48 9.10 6456.01 1.16875 pap 6AM 1 tube 70_19 39.08 31.64 147.66 8.36 5929.09 1.23514539 pap 6AM 1 tube 70_20 36.01 30.69 126.8 8.76 6217.28 1.17334637 pap 6AM 1 tube 70_21 28.19 30.03 134.51 8.66 6142.78 0.93872794 kar 6AM 1 tube 70_21 28.88 29.76 120.75 9.67 6863.09 0.97043011 kar 6AM 1 tube 70_22 29.6 30.24 130.31 7.98 5665.53 0.97883598 kar 6AM 1 tube 70_23 36.34 35.43 164.17 7.67 5440.39 1.02568445 kar 6AM tube 70_24 27.22 28.75 139.89 7.94 5636.76 0.94678261 kar 6AM tube 70_24 34.47 34.51 143.71 8.52 6048.34 0.99884092 kar 6AM tube 70_25 27.95 28.36 137.41 8.47 6011.13 0.98554302 kar 6AM tube 70_25 48.66 41.95 205.87 9.41 6678.25 1.15995232 pap 6AM 1 tube 70_26 36.24 34.83 155.4 8.01 5686 1.04048234 kar 6AM 1 tube 70_27 43.19 37.35 162.73 7.44 5277.87 1.15635877 pap 6AM 1 tube 70_28 34.44 34 146.57 7.72 5474.85 1.01294118 kar 6AM 1 tube 70_29 34.02 39.68 173.35 7.04 4992.33 0.85735887 kar 6AM 1 tube 70_30 42.78 40.34 158.69 7.51 5327.14 1.06048587 kar 6AM 1 tube 70_31 23.16 26.86 155.68 8.03 5697.45 0.8622487 kar 6AM 1 tube 70_32 47.2 40.87 205.64 8.08 5733.79 1.15488133 pap 6AM 1 tube 70_33 40.86 35.06 146.83 7.76 5507.68 1.16543069 pap 6AM 1 tube 70_34 41.17 34.21 251.22 8.68 6161.92 1.20344928 pap 6AM tube 70_35 41.22 35.01 150.36 8.10 5744.32 1.17737789 pap 6AM 1 tube 70_36 43.08 37.56 141.62 8.11 5751.86 1.14696486 pap 6AM 1 61 tube 70_37 35.44 28.74 177.09 7.44 5280.7 1.23312457 pap 6AM 1 tube 70_38 30.56 28.06 96.3 6.75 4787.44 1.0890948 kar 6AM 1 tube 70_38 35.99 38.17 151.14 7.99 5665.89 0.94288708 kar 6AM 1 tube 70_39 42.24 40.11 230.78 8.61 6106.93 1.05310396 kar 6AM tube 70_40 25.71 29.31 122.92 8.28 5873.62 0.87717503 kar 6AM 1 tube 70_41 17.51 23 85.77 5.72 4056.22 0.76130435 unk 6AM 1 tube 70_42 28.34 29.34 144.34 8.18 5806.02 0.96591684 kar 6AM tube 70_43 39.91 33.34 165.47 6.82 4835.92 1.19706059 pap 6AM tube 70_44 35.46 31.43 135.21 7.24 5137.15 1.12822144 pap 6AM 1 tube 70_45 43.94 37.25 154.49 7.37 5226.9 1.17959732 pap 6AM 1 tube 70_46 47.85 38.44 238.77 8.00 5678.02 1.24479709 pap 6AM 11.05.06_8 13.36 2.23 2161.05 #DIV/0! CN - 11.05.06_13 9.69 2.56 2483.11 #DIV/0! CN - 11.05.06_14 10.31 2.62 2545.46 #DIV/0! CN - 11.05.06_21 10.2 2.59 2509.6 #DIV/0! CN - 11.05.06_3 29.36 28.78 124.43 5.40 5238.36 1.02015288 kar - 11.05.06_4 28.95 28.65 100.02 7.33 7113.54 1.0104712 kar - 11.05.06_5 35.41 34.2 179.73 8.54 8282.22 1.03538012 kar - 11.05.06_6 27.76 29.69 118.75 6.57 6370.4 0.93499495 kar - 11.05.06_7 29.03 28.48 98.44 6.88 6668.97 1.0193118 kar - 11.05.06_10 32.05 30.53 132.92 6.74 6541.43 1.04978709 kar - 11.05.06_11 32.7 30.69 86.71 8.08 7841.74 1.06549365 kar - 11.05.06_17 32.8 29.93 101 7.21 6996.77 1.09589041 kar - 11.05.06_18 27.17 30.48 97.57 7.60 7375.27 0.8914042 kar - 11.05.06_22 29.73 27.93 114.71 8.48 8224.86 1.06444683 kar - 11.05.06_25 41.29 38.41 179.73 8.61 8353.38 1.07498047 kar - 11.05.06_27 35.58 33.32 120.26 7.87 7630.59 1.06782713 kar - 11.05.06_1 33.82 28.75 119.35 5.92 5742.77 1.17634783 pap - 11.05.06_2 38.25 34.74 183.15 8.88 8616.02 1.10103627 pap - 11.05.06_9 32.75 28.44 144.02 5.84 5667.44 1.15154712 pap - 11.05.06_12 40.09 34.52 175.3 7.88 7641.25 1.16135574 pap - 11.05.06_15 36.31 32.18 166.73 9.42 9132.97 1.12834058 pap - 11.05.06_16 31.39 26.91 121.89 7.69 7463.12 1.16648086 pap - 11.05.06_19 38.81 32.36 203.93 9.12 8842.53 1.19932015 pap - 11.05.06_20 26.9 29.77 107.27 8.91 8645.19 0.90359422 pap - 11.05.06_24 45.38 35.91 182.57 7.98 7741.05 1.26371484 pap - 11.05.06_26 31.66 27.01 122.85 7.85 7611.26 1.17215846 pap - 11.05.06_28 34.94 28.41 87.83 10.32 10013.13 1.22984864 pap - 11.05.11_1 29.83 27.64 109.68 7.67 7245.3 1.079233 kar - 11.05.11_10 27.22 29.09 86.82 8.88 8394.73 0.93571674 kar - 11.05.11_11 33.58 31.79 167.59 10.66 10075.52 1.05630701 kar - 11.05.11_12 23.44 30.93 137.36 6.95 6568.14 0.75784028 kar - 11.05.11_12 25.88 28.85 108.74 8.77 8290.99 0.89705373 kar - 11.05.11_12 30.57 27.53 117.41 7.63 7213.19 1.11042499 pap - 11.05.11_13 23.96 25.6 127.45 7.38 6978.84 0.9359375 kar - 11.05.11_14 23.15 26.53 133.31 7.18 6783.51 0.87259706 kar - 11.05.11_15 31.29 29.74 110.68 9.09 8587.7 1.05211836 kar - 11.05.11_16 24.91 24.13 111.84 8.20 7752.79 1.03232491 kar - 11.05.11_17 22.61 25.26 91.7 9.50 8978.88 0.89509105 kar - 11.05.11_18 23.87 30.25 127.71 7.46 7047.91 0.78909091 kar - 11.05.11_19 23.99 24.3 104.4 9.14 8639.44 0.9872428 kar - 11.05.11_2 25.34 25.63 97.52 8.44 7980.36 0.98868513 kar - 11.05.11_20 23.97 25.57 98.66 8.94 8453.83 0.93742667 kar - 11.05.11_20 26.62 26.11 129.63 7.80 7374.05 1.01953275 kar - 11.05.11_21 38.84 33.78 152.3 7.25 6854.17 1.14979278 pap - 11.05.11_22 30.75 29.2 158.25 6.93 6547.36 1.05308219 kar - 11.05.11_23 25.06 27.08 131.94 7.26 6863.17 0.9254062 kar - 11.05.11_23 26.18 29.03 128.89 7.56 7141.51 0.9018257 kar - 11.05.11_24 39.11 33.78 156.94 6.97 6591.22 1.15778567 pap - 11.05.11_25 43.34 30.97 215.49 8.77 8292.89 1.39941879 pap - 11.05.11_26 31.59 32.51 110.97 8.44 7973.13 0.97170102 kar - 11.05.11_27 30.45 34.92 133.17 7.59 7172.24 0.87199313 kar - 11.05.11_27 30.76 32.52 114.04 8.29 7838.2 0.94587946 kar - 11.05.11_27 38.14 32.09 233.68 8.44 7972.98 1.18853225 pap - 11.05.11_28 24.3 24.51 125.11 7.85 7415.35 0.99143207 kar - 11.05.11_29 27.39 29.61 143.09 7.08 6696.51 0.92502533 kar - 11.05.11_30 27.86 29.54 145.2 7.07 6683.43 0.94312796 kar - 11.05.11_31 33.37 34.37 137.06 7.55 7135.43 0.97090486 kar - 11.05.11_31 29.79 31.59 128.51 7.91 7473.46 0.94301994 kar - 11.05.11_32 31.49 35.11 216.75 8.57 8101.95 0.89689547 dividing - 11.05.11_34 27.37 30.17 213.17 4.52 4274.49 0.90719258 kar - 11.05.11_35 28.84 30.36 140.91 7.10 6711.31 0.94993412 kar - 11.05.11_36 32.64 33.39 142.79 7.38 6972.01 0.97753819 kar - 11.05.11_37 33.18 33.69 135.79 7.27 6874.31 0.98486198 kar - 11.05.11_38 38.48 40.97 139.41 6.74 6368.67 0.93922382 kar - 11.05.11_39 22.9 26.77 153.28 6.44 6084.12 0.85543519 kar - 11.05.11_4 38.98 33.78 120.02 8.01 7569.64 1.15393724 pap - 11.05.11_40 31.72 33.94 186.74 7.43 7023.59 0.93459045 kar - 11.05.11_41 33.11 31.28 152.83 6.30 5956.06 1.05850384 kar - 11.05.11_42 32.77 36.2 262.81 6.82 6450.06 0.90524862 kar - 11.05.11_43 39.71 38.82 153.44 6.26 5918.9 1.02292633 kar - 11.05.11_44 39.05 38.73 165.96 6.04 5710.17 1.00826233 kar - 11.05.11_45 117.59 7.27 6870.53 #DIV/0! round - 11.05.11_46 31.64 32.87 139.47 7.01 6627.6 0.96257986 kar - 11.05.11_47 32.54 33.81 150.53 6.56 6200.01 0.96243715 kar - 11.05.11_48 32.82 33.15 153.04 6.61 6249.73 0.99004525 kar - 11.05.11_49 31.06 38.68 168.7 6.25 5910.59 0.80299897 kar - 11.05.11_5 28.19 28.1 107.71 9.48 8957.27 1.00320285 kar - 11.05.11_50 30.85 34.05 149.89 6.87 6493.21 0.90602056 kar - 11.05.11_50 30.05 30.45 160.33 6.61 6244.79 0.98686371 kar - 11.05.11_51 33.44 36.03 139.51 6.93 6551.29 0.92811546 kar - 11.05.11_51 31.21 29.66 152.78 6.63 6265.01 1.05225893 kar - 11.05.11_52 29.67 29.75 144.61 6.58 6216.83 0.99731092 kar - 11.05.11_53 28.81 28.9 149.29 6.73 6357.9 0.99688581 kar - 11.05.11_54 34.37 32.8 162.91 6.23 5888.51 1.04786585 kar - 11.05.11_55 27.92 30.82 155.35 6.19 5846.24 0.90590526 kar - 11.05.11_55 26.97 32.59 141.4 6.00 5671.89 0.82755446 kar - 11.05.11_55 35.58 31.86 166.74 6.25 5908.54 1.11676083 pap - 11.05.11_56 32 39.03 180.83 5.78 5466.01 0.81988214 kar - 11.05.11_56 31.67 31.7 158.68 6.51 6153.53 0.99905363 kar - 11.05.11_57 31.52 30.24 161.42 6.34 5994.06 1.04232804 kar - 11.05.11_57 26.17 35.63 145.01 6.32 5972.44 0.7344934 kar - 11.05.11_58 30.05 33.19 156.96 6.58 6220.33 0.90539319 kar - 11.05.11_59 35.14 35.85 258.67 7.48 7068.45 0.98019526 kar - 11.05.11_59 33.82 31.11 155.88 6.53 6168.57 1.08711025 kar - 11.05.11_6 34.26 30.45 126.86 7.74 7313.13 1.12512315 pap - 11.05.11_60 29.26 22.56 139.18 7.03 6649.44 1.29698582 pap - 11.05.11_61 28.35 34.29 161.89 6.53 6174.93 0.82677165 kar - 11.05.11_61 153.08 6.23 5890.77 #DIV/0! round - 11.05.11_62 38.33 36.5 155.37 6.23 5885.84 1.05013699 kar - 11.05.11_63 36.47 37.13 165.46 6.01 5677.03 0.98222462 kar - 11.05.11_64 31.04 35.49 152.36 6.49 6137.49 0.87461257 kar - 11.05.11_65 28.78 32.78 140.73 7.04 6654.69 0.87797437 kar - 11.05.11_66 31.02 32.51 131.06 7.60 7187.73 0.95416795 kar - 11.05.11_67 32.38 28.03 151.89 7.05 6666.59 1.15519087 pap - 11.05.11_68 95.38 7.50 7090.7 #DIV/0! unk - 11.05.11_69 26.94 28.91 98.77 7.59 7172.47 0.93185749 kar - 11.05.11_7 24.3 25.89 115.65 7.81 7379.12 0.93858633 kar - 11.05.11_7 32.28 29.06 108.86 8.09 7646.41 1.11080523 pap - 11.05.11_70 128.85 5.83 5510.1 #DIV/0! unk - 11.05.11_71 24.78 25.74 78.15 7.51 7103.04 0.96270396 kar - 11.05.11_72 35.83 35.22 188.62 8.82 8336.84 1.0173197 kar - 11.05.11_73 26.39 27.94 114.56 7.93 7499.42 0.94452398 kar - 11.05.11_74 29.73 32.74 113.43 8.49 8023.19 0.90806353 kar - 11.05.11_74 20.7 28.45 97.39 8.71 8232.03 0.72759227 kar - 11.05.11_75 24.71 31.27 124.04 7.48 7070.24 0.79021426 kar - 11.05.11_75 37.42 32.74 109.2 8.66 8184.17 1.14294441 pap - 11.05.11_75 176.18 9.34 8832.1 #DIV/0! unk - 11.05.11_76 25.1 27.39 118.33 7.56 7146.85 0.91639284 kar - 11.05.11_76 27.34 27.18 92.08 8.82 8334.23 1.00588668 kar - 11.05.11_76 27.08 30.35 102.33 8.11 7666.08 0.892257 kar - 11.05.11_77 31.22 37.67 81.67 9.40 8887.84 0.82877621 kar - 11.05.11_77 80.74 9.63 9100.6 #DIV/0! unk - 11.05.11_78 30.52 33.62 98.3 9.59 9067.39 0.90779298 kar - 11.05.11_79 26.27 28.81 134.63 7.71 7284.12 0.91183617 kar - 11.05.11_8 27.37 30.77 111.54 8.35 7895.69 0.88950276 kar - 11.05.11_80 20.23 23.73 119.07 8.18 7731.93 0.85250737 kar - 11.05.11_9 26.13 28.06 130.79 7.15 6755.66 0.93121882 kar - 11.05.11_9 24.94 28.65 110.66 8.02 7582.86 0.87050611 kar -