Evolutionary Trends of the Carboniferous Ostracod Velatomorpha Altilis, Joggins Fossil Cliffs, Unesco World Heritage Site

Home , Carbonita (ostracod), Joggins

EVOLUTIONARY TRENDS OF THE CARBONIFEROUS OSTRACOD VELATOMORPHA ALTILIS, JOGGINS FOSSIL CLIFFS, UNESCO WORLD HERITAGE SITE

Regan Maloney

Thesis submitted in partial fulfillment of the requirements for the Degree of Bachelor of Science with Honours in Biology

Acadia University April, 2016 © Copyright by Regan Maloney, 2016

This thesis by Regan Maloney is accepted in its present form by the Department of Biology as satisfying the thesis requirements for the degree of Bachelor of Science with Honours

Approved by the Thesis Supervisor ______Peir Pufahl Date

Approved by the Head of the Department ______Brian Wilson Date

Approved by the Honours Committee ______Anna Redden Date

I, Regan Maloney, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in microform, paper or electronic formats on a non-profit basis. I, however, retain the copyright in my thesis.

______Regan Maloney

______Date

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Acknowledgements

I received a lot of support while writing this thesis. Neil Tibert was instrumental to this project. He guided me with enthusiasm in the early stages. His passing is a loss well beyond his research. He always had time for my questions and his excitement about ostracods and life was infectious. Melissa Grey and Peir Pufahl were always encouraging along way and helped me a great deal. This project was made possible with a NSERC USRA grant.

Table of Contents

Acknowledgements ...... iv List of Tables ...... vi List of Figures ...... vii Abstract ...... viii 1. Introduction ...... 1 1.1 Study site ...... 1 1.2 Facies Associations ...... 2 1.3 Limestone Lithofacies ...... 3 1.4 Ostracods ...... 7 1.5 Evolutionary Modes ...... 9 1.6 Purpose ...... 11 2. Materials and Methods ...... 12 2.1 Extraction ...... 12 2.2 Measurement ...... 15 2.3 Multivariate Analysis ...... 17 2.4 Evolutionary Mode ...... 17 2.5 Abundance and population structure...... 18 3. Results ...... 20 3.1 Multivariate analysis ...... 20 2.2 Evolutionary Mode ...... 23 3.3 Thin Sections ...... 24 4. Discussion ...... 29 4.1 Evolutionary Mode ...... 29 4.2 Population Structure and Environmental Interpretations ...... 33 5. Conclusions ...... 35 References ...... 36 Appendix ...... 39

List of Tables

Table 1. Location of limestones used for ostracod sampling and thin sectionanalysis…………19

Table 2. Confusion matrix derived from the CVA……………………………………………… 22 Table 3. Results of Hunt’s (2008) “fit 3 models” tests...... 24

Table 4. Results of thin section analysis...... 25

Table 5. Akaike and AICC of Hunt’s (2008) “fit 3 models”...... 39

List of Figures

Figure 1. Dark, organic rich limestone beach outcrop at the Joggins Fossil Cliffs site (Aziz, 2010).Figure 1 ...... 5

Figure 2. Wave rippled limestone outcrop on beach at the Joggins Fossil Cliffs site (Aziz, 2010).Figure 2 ...... 6

Figure 3. Location of the Joggins Fossil Cliffs UNESCO World Heritage Site on the Bay of Fundy, in the Cumberland Basin (blue star).Figure 3 ...... 13

Figure 4. Stratigraphic column of formations at the Joggins Fossil Cliffs ...... 14

Figure 5. Example of V.altilis in left lateral view...... 16

Figure 6. Example of V.altilis in dorsal view...... 16

Figure 7. Principal component analysis results ...... 20

Figure 8. Canonical variate analysis results ...... 21

Figure 9. Example of a thin section from Boss Point 2 ...... 26

Figure 10. Joggins Formation thin section (Joggins 4t) ...... 27

Figure 11. Thin section from the Joggins 5T (Table 1) limestone ...... 28

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Abstract

The ostracod Velatomorpha altilis thrived in brackish coastal environments of the

Carboniferous. Especially well preserved examples occur in strata exposed at the Joggins

Fossil Cliffs World Heritage Site, Cumberland Basin, Nova Scotia, providing an opportunity to investigate the link between evolutionary mode and temporal changes in depositional environment. The relationship between the evolutionary mode of V. altilis and its environment is especially important because few other studies have examined the evolutionary mode of organisms living in marginal environments. The evolutionary mode of V. altilis was analyzed by measuring the length, height, width, area, and perimeter of valves from 332 specimens from limestone at five stratigraphic levels in the Boss Point and overlying Joggins formations. Interpretations of depositional environment in this study were based in part on ostracod population structure with no statistical treatment of the data. After quantitative model-based analysis, an unbiased random walk was supported as the evolutionary mode when ostracods from all five stratigraphic levels were included. Stasis was the suggested evolutionary mode only when ostracods from each formation were considered separately. This is consistent with previous research on V. altilis from the Joggins Formation. Stasis is expected in fluctuating, stressed environments, such as the brackish coastal paleoenvironments of the Boss Point and

Joggins formations. This is because organisms that inhabit those environments are usually tolerant to changing conditions. It is possible that between the formations an environmental threshold was reached that triggered rapid change in shell size. Ostracod population structure analysis supports this interpretation and suggests shell size may record a difference in hydrologic regime of limestone between the Boss Point and

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Joggins formations. This represents a classic example of punctuated equilibrium model of evolutionary change wherein long periods of stasis are punctuated by geologically short periods of drastic morphological change.

1. Introduction 1.1 Study site In 2008, the Joggins Fossil Cliffs site was inscribed on the UNESCO World

Heritage List because of its unrivaled fossil record of the Pennsylvanian (320 to ca. 286 mya). Located on the Bay of Fundy in the Cumberland Basin (Figure 1), the site has yielded significant discoveries such as the world’s earliest-known confirmed reptile,

Hylonomous lyelli, and the earliest-known land snail, Dendropupa vetusta (Falcon-Lang et al. 2010). The Joggins Fossil Cliffs site is also home to upright lycopsid trees that have been preserved in situ and contain the remains of other organisms such as reptiles and amphibians within the inner layers of the trees (Falcon-Lang et al. 2010). Research at the cliffs has been undertaken since the early 1800s, with several prominent scientists including Sir William Logan, Sir William Dawson, and Sir Charles Lyell conducting studies there. Joggins is notably mentioned in Darwin’s On the Origins of Species. The site has also made important contributions to the understanding of coal formation; the presence of coal seams with terrestrial flora such as the lycopsid trees demonstrated that coal is formed from terrestrial and not aquatic environments (Rygel and Shipley 2005).

The extensive outcropping at Joggins make for an exceptional study site, particularly for evolutionary research, with 15 kilometers of conformable sedimentary rocks ranging from ca. 325 mya to ca. 310 mya (Utting et al. 2010). The collision of

Euramerica and Gondwana during the Carboniferous caused the uplift and subsidence of crustal blocks to allow such rapid deposition of sediment. The weight of the accumulating layers caused tilting of underlying halite, which uplifted and exposed the sedimentary layers forming the cliffs at Joggins (Falcon-Lang et al. 2010).

The present study focuses on the Boss Point and Joggins formations of the

Joggins Fossil Cliffs site. Based on palynology, Boss Point is Yeaondian- Langsettian in age, ca. 315 Ma (Utting et al. 2010), and the Joggins Formation is Langsettian and accumulated from ca. 314 to 313 Ma (Falcon-Lang et al. 2011; Utting et al. 2010). Thus, strata described herein span a 2 to 3 million year interval (Grey et al. 2012; Utting et al.

2010).

1.2 Facies Associations The Boss Point and Joggins formations contain similar facies associations. Three facies associations are recognized in the Joggins Formation: poorly drained floodplains, well-drained floodplains, and open-water (Davies and Gibling 2003; Davies et al. 2005;

Falcon-Lang et al. 2006). The Boss Point Formation contains four facies associations: channel bodies; poorly-drained floodplains; well-drained floodplains; and open-water

(Rygel et al. 2015). This research focuses on the open-water facies.

While the open-water facies of the Boss Point and Joggins formations are similar, there is almost a complete lack of current generated sedimentary structures preserved in the Boss Point limestones. This suggests accumulation of sediments in calm embayments with little wave and tidal reworking (Rygel et al. 2015). The Boss Point limestones record increasingly shallow water and a transition to a muddy floodplain environment whereas the Joggins limestones record a freshening upward succession from a more open marine to a fluvial dominated system (Grey et al. 2011; Grey et al. 2015; Rygel et al. 2015).

1.3 Limestone Lithofacies The limestone lithofacies are found throughout the Boss Point and Joggins formations. Lithofacies are genetically related subdivisions of a sedimentary succession.

They are linked by depositional history and subsequently their composition. The limestone lithofacies preserves the open-water facies environment through time at the

Joggins Fossil Cliffs (Aziz 2010; Grey et al. 2011). The limestones are ostracod-bivalve packstones (Aziz 2010; Grey et al. 2011). The low diversity assemblage indicates the environment was likely stressed in some way. Ostracod species are almost exclusively restricted to the brackish water Velatomorpha altilis and non-marine bivalves to

Naiadites and Curvirimula (Aziz 2010; Grey et al. 2011). At the base of the Joggins

Formation echinoderm fragments indicate strong marine influence, while increasing abundance of Naiadites and Curvirimula near the top of the formation indicate increasingly non-marine conditions (Aziz 2010; Grey et al. 2011). Recent work on the limestones of the Joggins Formation has shown preservation of a marginal marine setting

(Grey et al. 2011). Less is known about the carbonate deposits of the Boss Point

Formation, but they are also likely coastal marine (Rygel et al. 2015).

The relatively low diversity of fossils within the limestones indicate that only euryhaline organisms would have inhabited shallow coastal environments. The low magnesium calcite shells of the ostracods are unrecrystallized, indicating a low oxygen environment (Rygel et al. 2015; Grey et al. 2011). The combination of fluctuating salinity and low oxygen levels means that the open-water facies represent a stressed environment.

The mixing of freshwater bivalves and brackish water bivalves in the sediment indicates that salinity fluctuated.

The limestones are conformably underlain by coal, of the well-drained flood facies and overlain by carbonaceous siltstone (Aziz 2010; Grey et al. 2011). They mark the onset of cyclothem deposition. Cylcothems are sedimentary successions that record higher order sea level cycles (Aziz 2010; Grey et al. 2011). In outcrops, the limestone beds are between 15 and 100cm thick. The limestones are often dark and organic rich

(Figure 1). Wave ripples are present on some indicating a high energy depositional environment (Figure 2) (Aziz 2010). Though there are some differences between the depositional environments of the Boss Point and Joggins formations both preserve a record of marginal marine deposition (Aziz 2010; Grey et al. 2011; Rygel et al. 2015).

Figure 1. Dark, organic rich limestone beach outcrop at the Joggins Fossil Cliffs site (Aziz, 2010).

Figure 2. Wave rippled limestone outcrop on beach at the Joggins Fossil Cliffs site (Aziz, 2010).

1.4 Ostracods Ostracoda is an order in the class Crustacea. Although they are metazoans

(multicellular) they are often classified as microfossils because of their small size.

Ostracods are enclosed in a dorsally hinged carapace that is often formed of low magnesium calcium carbonate. Ostracods, like all arthropods, molt their carapace multiple times during a lifetime. It is typical for ostracods to go through eight instar phases (molts) (Whatley 1988). The calcium carbonate carapace of ostracods readily fossilizes which has led to a robust fossil record of the lineage. Ostracod fossils can be found throughout most of the Phanerozoic and into recent times (Yamaguchi and Endo

2003). Ostracods are significant microfossils in brackish water environments, and may be the most abundant group of animals preserved within them (Frenzel and Boomer 2005).

Ostracods are commonly used in modern and Quaternary studies as indicators of environmental change in brackish water and other aquatic environments (Frenzel and

Boomer 2005). These modern studies can help us interpret environmental conditions in the fossil record.

This study focuses on the ostracod species Velatomorpha altilis. Velatmorpha is a recently described genus that includes reassigned ostracods from Carbonita, Healdia, and

Microcheilinella genera (Tibert and Dewey 2006). Velatomorpha thrived in the estuarine paleoenvironments of the Joggins and Boss Point formations and is known to occur only in the Pennsylvanian (Tibert and Dewey 2006). It is found in the open-water facies environments that alternated between brackish and freshwater. Ostracoda is an adaptable class of animals which has allowed them to thrive in changing environments; the behavior of Velatomorpha in particular was likely opportunistic, which may be why they

7 thrived in fluctuating conditions (Tibert and Dewey 2006). Ostracods in general have the ability to live in a wide range of environments given sufficient food, oxygen, and water.

Their adaptability has allowed them to colonize fresh-saline transition zones such as lagoons, estuaries, and deltas. The variation of salinity in these zones often leads to low diversity in ostracod species and such is the case in the Boss Point and Joggins formations (Grey et al. 2011; Rygel et al. 2015). These brackish water zones are usually dominated by marine species that are euryhaline tolerant and can survive in non-marine water with sufficient Na+ and Cl- (Carbonel 1988). Although the ostracod Carbinata pungens is also present in the limestones, they are much less abundant (<5%) and were likely washed in from further in the basin (Tibert and Dewey 2006). Monospecific ostracod assemblages such as this are common in modern brackish water environments and been linked to unstable and stressed environments (Frenzel and Boomer 2005).

Examination of population structure can also be used to understand depositional environments. Ostracod valves disarticulate after molting but when an ostracod dies its valves often remain articulated. The relative abundance of articulated valves, disarticulated valves, and instar phases can reveal meaningful information about the depositional environment, such as hydrologic regime (Whatley 1988). Environmental factors such as hydrologic regime and the stability of physical and chemical conditions impact the lifecycles of ostracods. The population structure preserved in the rocks can be used to interpret environmental conditions at the time of deposition. The abundance of each instar phase can also help to interpret whether the ostracods have undergone sorting by grain size or transportation. It is essential that the ostracods have not been transported

8 if one is interested in using population structure to help determine depositional conditions for a specific location (Tibert and Scott 2006; Whatley 1988).

Velatomorpha altilis is numerous in the limestone facies found throughout both the Boss Point and Joggins formations (Rygel et al. 2015; Grey et al. 2011; Tibert and

Dewey 2006). This allows for the study of evolution in a marginal marine species, which has been relatively understudied in comparison to organisms in other, more stable, environments such as distal deep-water environments (Grey et al. 2012). It also allows environmental interpretations based on physical and chemical data to be tested against ostracod population structure, so interpretations of how elements of the entire ecosystem interacted can be reinforced.

1.5 Evolutionary Modes Evolution was once thought to occur exclusively by a slow process in which small incremental adaptations accumulated over long periods of time to gradually form a new species. This is the evolutionary model known as phyletic gradualism. According to the gradualistic model, as one species evolves to the next there are many intermediate forms that occur between the two. The lack of many fossils displaying intermediate forms is attributed to the incomplete nature of the fossil record. This is a reasonable assumption since ideal conditions are needed for an organism to be fossilized. Thus, the overwhelming majority of organisms are not preserved because of the complexity and nature of taphonomic processes (Eldredge and Gould 1972). Gradualism was the widely held model of how evolution occurred until punctuated equilibrium was proposed.

Punctuated equilibrium indicates that speciation happens through a relatively quick burst of change followed by long periods of stasis in which few adaptations accumulate;

9 intermediate forms are not expected as the change would have occurred too rapidly to be preserved in the fossil record (Eldredge and Gould 1972). While it is now commonly accepted that gradualism and punctuated equilibrium occur, their relative frequency and importance is still debated (Hunt 2007). It is difficult to test whether a species evolved by one mode or the other because it is rare to find a succession of thick fossiliferous rock in which one species is found throughout (Eldredge and Gould 1972). The in situ preservation of fossils, along with the continuous and conformable sedimentary layers forming Joggins Fossil Cliffs, provide an excellent natural laboratory for such an evolutionary study.

This research will utilize the ideal conditions at the Joggins Fossil Cliffs and examine whether V. altilis fossils display evidence of gradualism or punctuated equilibrium. A quantitative model-based analysis is an objective way to test between the two. There are three main modes or models of evolution that can be tested: directional change (gradualism), stasis (punctuated equilibrium), and unbiased random walk (Hunt

2007). Unbiased random walk is the result when the trend does not favour either directional change or stasis. Directional changes and unbiased random walks display a significant net change in a character, while in stasis there is no such change. Directional change differs from an unbiased random walk in its pattern of change. A directional change means that the character in question changes at a relatively constant rate, indicating phyletic gradualism. There is no inherent pattern to the character change in a random walk but this can still lead to species divergence or speciation given enough time

(Hunt 2007). A species that evolves through punctuated equilibrium will likely be observed in the fossil record as stasis because characters change so quickly that it is more

10 likely to observe the periods in which little or no change occurred. Stasis can result from stabilizing selection in stressed environments (Hunt 2007; Sheldon 1996). Species that could survive through initial environmental fluctuations are not likely to change after continuous fluctuations because they already have the adaptations needed to survive

(Sheldon 1996). Directional change is also thought to be present in more stable conditions such as the deep sea (Sheldon 1996). The relative frequency of the three modes has long been debated .A recent meta-analysis found that directional change is only found in about 5% of the fossil record whereas unbiased random walks and stasis equally divide up the other 95% (Hunt 2007). As the depositional environment in both the study formations would have been stressed and fluctuating it is hypothesized that the ostracods from the Joggins Fossil Cliffs World Heritage Site, Cumberland Basin, Nova

Scotia, would display stasis.

1.6 Objectives Study sites such as the Joggins Fossil Cliffs are rare. The extensive exposure of layers and the exceptional preservation of fossils make the site ideal for evolutionary studies. The objectives of this study are:

1) To determine the evolutionary mode of V. altilis using size and shape measures.

Results will be compared to previous work on V. altilis to test for consistency

between different methodologies.

2) To determine whether recently described differences in hydrologic regime of the

depositional environment between the Boss Point and Joggins formations

coincides with changing ostracod morphology.

3) To test past interpretations of the depositional environments against

interpretations based on ostracod population structure.

2. Materials and Methods 2.1 Extraction Limestone hand samples were taken from the beach and cliff-face at low tide from the Joggins Fossil Cliffs site (Figure 3). Three limestones from the Boss Point

Formation and two from the Joggins Formation were selected for analysis (Figure 4,

Table 1). Other limestone samples from the Joggins Formation were collected but not used as the low concentrations of ostracods did not allow for easy extraction of the numerous specimens required for rigorous quantitative study. For the samples that were used, the limestone was broken into roughly pebble-sized pieces with a hammer and then ground into a coarse sand with a porcelain mortar and pestle. The samples were then placed in 150 ml beakers with water and about 5g of Calgon washing powder. The beakers were placed on a warm heating stir plate with gentle stirring for

3-4 days, allowing the sediment to separate from the ostracod carapaces. The samples were sorted by grain size with 500um, 250um, and 125um sieves. Whole ostracods with intact carapaces of the species V. atilis were picked out for measuring from the

250um and 500um size class. The 125um size class was not used as it would not contain late instar (adult) phases (Tibert and Dewey 2006).

Figure 3. Location of the Joggins Fossil Cliffs UNESCO World Heritage Site on the Bay of Fundy, in the Cumberland Basin (blue star).

Figure 4. Stratigraphic column of formations at the Joggins Fossil Cliffs UNESCO World Heritage Site according to IUGS time scale (modified from Boon and Calder 2007; Utting et al. 2010). *Indicates formations sampled in this study.

2.2 Measurement Isolated ostracod carapaces were mounted onto a glass slide using Scotch© brand double-coated tape. They were then positioned in the left lateral view and photographed with a Nikon Infinity 1 microscope camera. The shell area and perimeter were obtained using NIS-elements image software with an “automatic outline function”. The maximum feret diameter and minimum feret diameters (which equate to length and height, respectively) were also measured using the NIS-elements image software (Figure 5).

Afterwards, the ostracods were placed in the dorsal view and the process was repeated

(Figure 6). The minimum feret diameter was used in this view as it represents width.

Growth plots were created in Microsoft Excel using length and width data to view instar phases. Instar phases will group together so the mature or late instar phases could be distinguished. Only the last grouping (length greater than 900um) from each stratigraphic level was used as these contain the last instar phases, allowing meaningful comparison of adult morphologies. This ensures that juveniles from one stratum are not being compared with adults from another.

Figure 5. Example of V. altilis in left lateral view. Outline traced with NIS-elements image program in green. The red line indicates the length and the blue line indicates the height.

Figure 6. Example of V. altilis in dorsal view. Outline traced with NIS-elements image program in green. The red line indicates the width.

2.3 Multivariate Analysis A principle component analysis (PCA) and canonical variate analysis (CVA) were performed using the statistical program PAST (Hammer et al. 2001). All variables were log transformed to standardize. The PCA was used to explore the morphospace (shape and structure) of the sampled, adult ostracods, while the CVA was used for predictive classification (Grey et al. 2007). The ostracods were grouped a priori based on the limestone layer from which they were extracted. Both of these multivariate tests use all the available measurements and summarize them into two axes. A discriminate analysis was also performed where the ostracods were grouped by formation. Discriminate analysis (DA) and CVA are similar except in CVA there are multiple groups whereas in

DA there are only two groups. The groups in the PCA and CVA are the five limestone layers that are used to test for shape changes over time. A so-called “confusion matrix” derived from the CVA analysis was made to determine the amount of ostracods that can correctly be classified into their respective strata based on their character traits. The confusion matrix was jackknifed to assess confidence in the resulting patterns. The groups in the DA are the two separate formations that are used to test for shape changes between formations that may be a result of environmental change.

2.4 Evolutionary Mode Evolutionary mode for the ostracods was tested using the PaleoTS package in the statistical program R (Hunt 2008). The “fit3models” function determined evolutionary mode for the first canonical variate (CV1), area, perimeter, and length to width ratio. The tests were completed including all five stratigraphic levels. Separate tests were performed using the first canonical variant and length to width ratio of the ostracods only within the

Boss Point Formation to test evolutionary mode confined within the formation. The tests could not be carried out within the Joggins Formation as more than two stratigraphic levels are required.

2.5 Abundance and Population Structure Ostracod assemblages are useful environmental indicators (Whatley 1988).

Uncovered thin sections were prepared from the three in the Boss Point Formation as well as five randomly selected limestones from the Joggins Formation (Table 1).

Ostracod abundance in each limestone was estimated from studying percent grain coverage of the thin sections. Population structure was also examined in each limestone from thin section analysis to interpret depositional environment and determine the degree of transport of the specimens (after Whatley 1988). Stagnant water is indicated by large numbers of all instar phases (Whatley 1988). If the assemblage is comprised of mostly late instars then it is likely current activity removed the smaller early instars (Whatley

1988). A large number of articulated early instars indicate changes in physical or chemical parameters, as juveniles would have died before maturing (Whatley 1988).

Table 1. Location of limestones used for ostracod sampling and thin section analysis from the Joggins Fossil Cliffs UNESCO World Heritage Site. Formation Relative Name Ostracod Thin Distance Stratigraphic Sampling Section from base Level Analysis of Formation Boss Point 1 Boss Point Yes Yes 098m

Boss Point 2 Boss Point Yes Yes 780m

Boss Point 3 Boss Point Yes Yes 940m

Joggins 4 Joggins 1T No Yes 035.5m

Joggins 5 Joggins 1 Yes No 067m

Joggins 6 Joggins 2T No Yes 074m

Joggins 7 Joggins 3T No Yes 148.5m

Joggins 8 Joggins 4T No Yes 189m

Joggins 9 Joggins 2 Yes No 448m

Joggins 10 Joggins 5T No Yes 872m

3. Results 3.1 Multivariate Analysis The morphospace of the ostracods overlaps considerably but differentiation based on formations is apparent (Figure 7), indicating that the morphology of V. altilis has significantly changed among the formations.

0.12

0.1

0.08

0.06

0.04 Boss Point 1 Boss Point 2 0.02

Boss Point 3 PC2(10%) 0 Joggins 1 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 Joggins 2 -0.02

-0.04

-0.06

-0.08 PC1 (86%)

Figure 7. PCA results showing overlap of V. altilis morphology within the Boss Point and Joggins formations. PC1 is composed of all measured traits (length, width, height) while PC2 is composed of all measured traits with the exception of width.

A similar pattern is revealed by the canonical variate analysis but the differentiation between the formations is much clearer (Figure 8).

1 Boss Point 1 Boss Point 2 0 -6 -4 -2 0 2 4 6 Boss Point 3 CV2 CV2 (24%) Joggins 1 -1 Joggins 2

-2

-3

-4 CV1 (73%)

Figure 8. CVA results for predictive classification of the V. altilis. Seventy-three percent of ostracods could be correctly classified. CV1 is largely a function of area and CV2 is a combination of width, area and perimeter.

The “confusion matrix” from the CVA quantifies the predictive classification.

Percentage of correct classification for individual strata varied from sixty-two to eighty- three and overall seventy-three percent of ostracods were classified into their correct strata (Table 2).

In both the CVA and PCA there is significant overlap of data points of ostracods within the same formation. There is also overlap of data points of ostracods between formations but to a lesser extent. The discriminant analysis was performed to examine differentiation based on formation. Ostracods were classified into the correct formation with ninety-seven percent accuracy in the DA.

Table 2. Confusion matrix derived from the CVA. Rows delineate known groups of ostracods (stratigraphic layer) and columns are the predicted groups from the analysis. Bolded numbers indicate ostracods classified into correct their stratigraphic layer. Confusion Matrix Boss point Boss point Boss point Joggins 1 Joggins 2 Total 1 2 3

Boss point 59 0 17 0 3 79 1

Boss point 1 64 16 0 3 84 2

Boss point 17 12 55 0 5 89 3

Jog 1 0 0 2 44 10 56

Jog 2 1 0 0 3 20 24

Total 78 76 90 47 41 332

Percentage 73 correctly classified (%)

2.2 Evolutionary Mode The lowest AICC and highest Akaike weight indicates the supported mode, as they are indicators of goodness of fit in the “fit three model” test for evolutionary mode (Hunt

2006). An unbiased random walk was supported as the mode of evolution when all five strata were considered for the first canonical variate, area, and perimeter (Table 3;

Appendix). The first canonical variate was used as it best summarizes the variation between ostracods in different stratigraphic layers. This indicates that there has been a net change (but not directional) in ostracod morphology and size from the base of the Boss

Point Formation to the top of the Joggins Formation. However, when testing evolutionary mode within just the Boss Point Formation, stasis is the supported evolutionary pattern when considering the first canonical variant (Table 3). It has already been shown that stasis is the supported mode of evolution for ostracods within the Joggins Formation based on CV1, area, and perimeter (Grey et al. 2012).

There is a lower length to width ratio in the Joggins Formation ostracods compared to those from the Boss Point Formation. From personal observation it appears that this is the case. The “fit 3 models” test was repeated using the length to width ratio of the ostracods to determine if there had been a significant change in morphology and the evolutionary mode. Using the ratio data, an unbiased random walk is still supported when all five layers are considered (Table 3). Stasis is supported within the Boss Point

Formation based on ratio data (Table 3).

Table 3. Results of Hunt’s (2008) “fit 3 models” tests for CV1, area, perimeter, and length to width ratio. Separate tests were done with data from both formations and just from the Boss Point. Character Supported Evolutionary Location data Mode CV1  Random walk  All strata from both formations

Area  Random walk  All strata from both formations

Perimeter  Random walk  All strata from both formations

CV1  Stasis  Boss Point

Area  Directional  Boss Point

Perimeter  Directional  Boss Point

Length to width ratio  Random walk  All strata from both formations

Length to width ratio  Stasis  Boss Point

3.3 Thin Sections Thin sections were analyzed for percent ostracod coverage and population structure. Interpretations of depositional environment were made based on the population structure (Table 4). The Boss Point Formation has a large concentration of shells and shell fragments (Figure 9). Most thin sections in the Joggins Formation showed a variety of instars with fewer shell fragments than in the Boss Point Formation (Figure 10). The last sample (Joggins 5T) has mostly late instars (Figure 11). There is also a general trend of decreasing ostracod abundance through time.

Table 4. Results of thin section analysis. Thin sections from the Boss Point and Joggins Formation where examined for percent coverage and population structure. Environmental interpretations were made using the methods described by Whatley (1988). Strata Coverage Population Structure Interpretation (%) Boss 15  High proportion of  Stagnant Point 1 ostracod fragments conditions  High proportion of all instar phases

Boss 30  High proportion  Stagnant Point 2 ostracod fragments conditions  High proportion of all instar phases

Boss 20  High proportion  Stagnant Point 3 ostracod fragments conditions  High proportion of all instar phases

Joggins 10  All instar phases  Possible sorting 1T  Higher proportion of  Current activity early instars  Some shell fragments

Joggins 5  All instar phases  Some current 2T  Approximate equal activity proportions  Some shell fragments

Joggins 15-20  All instar phases  Some current 3T  Approximate equal activity proportions  Some shell fragments

Joggins 15-20  All instar phases  Some current 4T  Approximate equal activity proportions  Some shell fragments

Joggins 5  Mostly late instars  Moderate 5T  Very few shell current activity fragments

Figure 9. Example of a thin section from Boss Point 2 limestone dominated by complete shells (CS) and shell fragments (SF) of V. altilis.

Figure 10. Joggins Formation thin section (Joggins 4t) showing early instars (EI), middle instars (MI), and late instars (LI).

Figure 11. Thin section from the Joggins 5T (Table 1) limestone. Mostly complete late instar (LI) ostracods are found in this thin section, with few early instars or ostracod shell fragments.

4. Discussion 4.1 Evolutionary Mode Although an unbiased random walk is the statistically supported mode when comparing all data across both formations, punctuated equilibrium (in the form of stasis) might more accurately describe the pattern. This is because stasis is supported within each respective formation. The apparent break in stasis could be due to a period of rapid morphological change between the formations, making the overall pattern appear as a random walk. Punctuated equilibrium is the mode of evolution where morphological stasis is punctuated by rapid change (Eldredge and Gould 1972). In this study it is not possible to precisely quantify the rapid rate of morphological change as biostratigraphic estimates (using palynomorphs) did not yield precise enough dates for when the formations began and ended (Utting et al. 2010). The Little River Formation is found between the Boss Point and Joggins formations. The Little River Formation has no limestones preserved in it, and therefore no open-water facies associations (Calder et al.

2005). Since punctuated evolutionary change is often too rapid to be preserved in the rock record (Eldredge and Gould 1972), a lack of limestones from the Little River

Formation does not hinder our understanding of the evolution of V. altilis.

The break in stasis between the formations could be explained by:

1) a narrowly fluctuating environment in the open-water facies association during

the time period of the Little River Formation; or

2) an environmental shift that was great enough to break the threshold within which

stasis is expected (Sheldon 1996).

When an environment is widely fluctuating, stasis is expected because organisms that survive through these environments have adaptations to different extremes and are

29 not likely to evolve (Sheldon 1996). When there are fewer fluctuations in the environment, changes can accumulate and lead to speciation, according to Sheldon’s

(1996) plus ca change model. Because the Little River Formation does not contain any limestones (Calder et al. 2005) it is not possible to study the open-water facies that would have been between the Boss Point and Joggins formations. The Boss Point and Joggins limestones probably formed in brackish water and salinity and oxygen stresses were most likely similar (Grey et al. 2011; Rygel et al. 2015). Though the limestones in the Boss

Point and the Joggins formations both correspond to open-water facies associations, the environments differ in important respects. The key difference is the lack of wave action associated with the Boss Point limestones compared to the Joggins limestones. The Boss

Point limestones formed in low energy, calm water and the environment would be best described as a bay or lagoon (Rygel et al. 2015). There is insufficient evidence to be certain of a marginal marine environment in the Boss Point Formation, though it is likely

(Grey et al. 2011; Falcon-Lang 2005; Rygel et al. 2015; Tibert and Dewey 2006). The

Boss Point limestones record increasingly shallow water and a transition to a muddy floodplain environment where the Joggins limestones record a freshening upward sequence from more open marine to fluvial dominated system (Grey et al. 2011; Grey et al. 2015; Rygel et al. 2015). It is possible that the change in morphology of V. altilis occurred in response to the environmental changes between the Boss Point and Joggins formations; these changes could be evolutionary (a change in genotype) or they could be due to ecophenotypic variation (a change in phenotype). Environmental conditions such as salinity and climate can cause phenotypic change (Frenzel and Boomer 2005). There is still much debate about the importance of phenotypic plasticity in ostracods (Frenzel and

Boomer 2005) but it is important to note that the morphological change may not necessarily be a result of changes in genotype but of changes in gene expression.

The open-water facies in the Boss Point Formation was likely brackish (Rygel et al. 2015), while the same facies at the base of the Joggins Formation is brackish but has decreasing marine influence through time (Grey et al. 2011). It is possible the change in body form is influenced by differences in salinity (Ruiz et al. 2006). V. altilis is a sexually dimorphic species, meaning the males and females have different morphological characteristics; females are stout while males are more elongate (Tibert and Dewey

2006). Sex ratios in ostracods can shift in response to changes in ecological variables such as salinity (Yousef 2014). The increase in bulbous forms (lower length to width ratios) in the Joggins Formation could be because of an increase in the relative number of females. However, it was not possible to sex the ostracods in this study to test this hypothesis as the morphological characters needed to do so are not visible from the outer carapace (Tibert and Dewey 2006).

Ostracods from only two strata were used for analysis from the Joggins Formation because, while many other limestones contain V. altilis, they were in much lower densities. Ideally, ostracods from more limestones would be used but time constraints prevented enough ostracods from other limestones to be collected for this study.

Previous work, however, indicates that there was not a significant change in size and shape throughout the formation; stasis was the supported evolutionary mode for both perimeter and area measurements (Grey et al. 2012).

It is important to note that only overall shell morphology was examined in this study. Other characteristics, for example pore shape and size, have been linked to

31 environmental factors such as salinity (Frenzel and Boomer 2005). Although morphological stasis was observed, it is possible that there were physiological adaptations not captured by this work.

The findings of this study are similar to the previous study of ostracods in the

Boss Point and Joggins Formations in many respects. Grey et al. (2012) found that there was an increase in the size of the ostracod carapace from the Boss Point to Joggins

Formation but the overall evolutionary trend was stasis. This could be due to different measuring and sampling techniques; width measurements were used in this study while a

Fourier (outline) analysis was performed in Grey et al. (2012). Future studies should take width measurements into account as it can be used as an indicator of how bulbous an ostracod is. Only using measurements taken in ventral view (length, height, area, and perimeter) can miss this shape character. The overall number of ostracods measured in each study are similar but in Grey et al. (2012) fewer ostracods from many strata were used, which is an accepted method in similar studies of evolutionary mode (Hunt et al.

2010). The emphasis in this study was to collect large sample sizes within strata to test for the evolutionary mode of V. altilis throughout both the Boss Point and Joggins formations. This may account for why a random walk was not statistically supported in

Grey et al. (2012) though it did include ostracods from one Boss Point Formation limestone (corresponding to Boss Point 2 in this study). Ideally, more ostracods from all strata would be collected for analysis. Both studies support punctuated equilibrium with this study capturing the rapid change and Grey et al. (2012) the period of stasis in the

Joggins Formation.

The findings of both studies on evolution of V. altilis in Joggins differ from other similar ostracod studies in different environments. The open-water facies is an example of a stressed and fluctuating environment (Grey et al. 2012; Tibert and Dewey 2006).

Body size evolution in this type of environment has not been studied as extensively as more stable environments such as the deep sea (Grey et al. 2012). For instance, ostracods in the deep sea environment of the Indian Ocean were found to undergo directional change (Hunt et al. 2010). It is important that future studies on body size evolution continue to expand the types on environments studied as trends can differ significantly.

This will be a challenge as ideal study sites, such as the Joggins Fossil Cliffs, which preserve a single species over millions of years, are rare (Eldredge and Gould, 1972), and study sites that preserve marginal environments are even more so.

4.2 Population Structure and Environmental Interpretations Ostracod population structure preserved in the rock record can be used to understand depositional environment (Tibert and Scott 1999; Whatley 1988). This study used V. altilis to study depositional environments at the Joggins Fossil Cliffs site. As previously discussed, the open-water facies of the Boss Point Formation likely preserved a brackish water bay or lagoon environment with little current activity (Rygel et al.

2015). The lack of current activity means that there is less sorting by grain size and that all sizes of ostracods (all instar phases) and shell fragments would be expected (Whatley

1988). The population structure in all three Boss Point Formation limestones followed this pattern (Table 8). The Joggins Formation has current- generated sedimentary structures in its limestones, especially at the base of the formation (Grey et al. 2011).

When there is current activity it is expected that shell fragments, and possibly the

33 smallest instar phases, will be transported away. This was indeed the case in most of the

Joggins Formation limestones (Table 8). The only limestone that did not coincide with expectations was Joggins 5T, the oldest limestone analyzed (Table 1). Only large instar adults were found in the thin section analysis of this limestone. This suggests an increase in current activity in comparison to the other Joggins limestones examined. This does not coincide with previous depositional interpretations of that limestone wherein sedimentary evidence did not support a large amount of current activity (Aziz 2010; Grey et al. 2011).

There is also a relatively low density of V. altilis in this limestone (Table 8) and this makes it difficult to examine population structure. It is possible the sample size of ostracods was too small to make interpretations of depositional environments.

There is an anticlinal relationship between the abundance of V. altilis and freshwater bivalves at Joggins (Grey et al. 2011). The decrease may be due to V. altilis’ inability to cope with increasingly freshwater conditions. Velatomorpha are part of a marine superfamily, Healodia. The genus went extinct at the end of the Pennsylvanian as a result of their inability to adapt to fresh water environments (Tibert and Dewey 2006;

Tibert et al. 2013). As the Maritimes Basin became increasingly restricted throughout the

Pennsylvanian, many groups of ostracods adjusted from brackish to a fully non-marine lifestyle (Tibert et al. 2013). Clearly, Velatomorpha was unsuccessful in this transition.

The decrease in V. altilis throughout the Joggins Formation is consistent with the decline of other species within the genus (Tibert et al. 2013); this decline is not accompanied by morphological change within the Joggins Formation (Grey et al. 2012).

5. Conclusions This study of the evolution and population structure of V. altilis at the Joggins

Fossil Cliffs UNESCO World Heritage Site found that:

1) While an unbiased random walk is the supported evolutionary mode of V. altilis

throughout the Boss Point and Joggins formations, stasis is well-supported within

each of the formations. This could indicate that the ostracods underwent

punctuated equilibrium. These finding are similar to previous studies of V. altilis.

The major finding in this study is that the differences between the ostracods of the

Boss Point and Joggins Formation.

2) The differences in morphology of V. altilis between the formations may coincide

with changes in salinity and hydrologic regime.

3) Interpretations of depositional environment based on the population structure of

ostracods preserved in the limestones in both the Boss Point and Joggins

formations are mostly consistent with previous environment interpretations of the

open-water facies in both formations with few exceptions.

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Appendix

Table 5. Results of Hunt’s (2008) “fit 3 models” tests for CV1, area, perimeter, and length to width ratio. Separate tests were done with data from both formations and just from the Boss Point. The lowest AICC and highest Akaike weight indicates the supported mode.

Character Evolutionary AICC Akaike Location data Mode Weight

CV1 Directional 32.695 0.003  All strata Random Walk 20.998 0.994 from both Stasis 32.456 0.003 formations

Area Directional 114.260 0.003  All strata Random Walk 102.913 0.984 from both Stasis 111.592 0.003 formations

Perimeter Directional 65.729 0.003  All strata Random Walk 54.279 0.978 from both Stasis 62.187 0.019 formations

CV1 Directional -0.750 0.148  Boss Point Random Walk Infinite 0.000 Stasis -4.265 0.852

Area Directional -36.355 1.000  Boss Point Random Walk Infinite 0.000 Stasis Infinite 0.000

Perimeter Directional -40.667 1.000  Boss Point Random Walk Infinite 0.000 Stasis Infinite 0.000

Length to Directional 13.135 0.003  All strata width ratio Random Walk 1.215 0.992 from both Stasis 11.771 0.005 formations

Length to Directional -9.227 0.135  Boss Point width ratio Random Walk Infinite 0.000 Stasis -12.940 0.865