The Ecology of Developmental Timing in a Neotropical Turtle

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THE ECOLOGY OF DEVELOPMENTAL TIMING IN A NEOTROPICAL TURTLE,

KINOSTERNON LEUCOSTOMUM

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Brian D. Horne

August 2007

Brian D. Horne

This dissertation titled

THE ECOLOGY OF DEVELOPMENTAL TIMING IN A NEOTROPICAL TURTLE,

KINOSTERNON LEUCOSTOMUM

BRIAN D. HORNE

has been approved for

the Department of Biological Sciences

and the College of Arts and Sciences by

Willem M. Roosenburg

Associate Professor of Biological Sciences

Benjamin M. Ogles

Dean, College of Arts and Sciences

Abstract

HORNE, BRIAN D., Ph.D., August 2007, Biological Sciences

THE ECOLOGY OF DEVELOPMENTAL TIMING IN A NEOTROPICAL TURTLE,

KINOSTERNON LEUCOSTOMUM (136 pp.)

Director of Dissertation: Willem M. Roosenburg

I studied how the expression, timing, and duration of embryonic diapause (ED),

morphogenesis, and embryonic aestivation (EA) relate to variation in environmental

conditions experienced by turtle embryos. ED and EA are putative mechanisms that

increase embryonic survivorship. ED arrests development before main morphogenesis

and is induced before the onset of adverse environmental conditions. EA prolongs

incubation by depressing the metabolism of the embryo after completion of

morphogenesis. I tested two hypotheses with regard to the environmental conditions that

stimulate ED and EA based on climatic patterns. My first hypothesis suggested a single suitable developmental period (SDT; periods when soil moisture and temperature of

nesting substrate are within the physiological tolerances of developing embryos) during

the dry seasons, and my second hypothesis predicted two SDTs at the transition from the

rainy and dry seasons. Incubation experiments using white-lipped mud turtle,

Kinosternon leucostomum, embryos confirmed that temperature is an important factor in

determining the duration of both ED and morphogenesis, and that morphogenesis occurs

during the dry season SDT.

Female size generally correlates positively with egg and offspring size. However,

when embryos experience prolonged incubation periods, females may alter their

reproductive investment strategies to offset potential additional embryonic energy

expenditures. When accounting for female size, larger clutches had eggs with greater mass than smaller clutches; and egg size increased with female size. Thus in K. leucostomum the typical relationship between egg size and clutch size as it pertains to division of maternal resources per propagule is weakened by the embryo’s ability to arrest development during extended incubation periods.

Nearly 50% of all turtle taxa are undocumented as to the expression of ED. I used categorical data modeling to create probabilities for predicting ED expression. Results indicate that greater than one-half of the undocumented species have a high probability of expressing ED. Conservation projects that alter incubation conditions of eggs may alter life history patterns and create unforeseen implications for population viability. Thus, it is critical for biologists performing ex-situ chelonian conservation projects with species which posses prolonged incubation periods to understand the role of ED and EA has in chelonian developmental ecology.

Approved:

Willem M. Roosenburg

Associate Professor, Biological Sciences

Dedication

For my parents,

Dick and Rama, who gave me my first pet snake when I was four,

and my siblings,

Mike and Jen, who have always been my best friends.

Acknowledgments

I have a great many people to thank for their assistance in completing my

dissertation, without their help, I would not been able to accomplish this project. Willem

Roosenburg, my dissertation advisor was gracious in his willingness to indorse my endeavors and he did not shy away from supporting my decisions to conduct fieldwork in

Mexico. I am thankful for his insights into chelonian reproductive ecology, but more

importantly, I am thankful that he believed in my abilities as a scientist when others

doubted. My departmental committee members Geoff Buckley, Kelly Johnson, Don

Miles, and Matt White provided guidance during the early phases of designing my

dissertation proposal and willingly gave of their time during the both my proposal

defense and comprehensive exams. Steve Reilly was also instrumental in helping

formulate the model of facultative developmental timing. The Department of Biological

Sciences provided funding for me to attend national and international meetings, as well as

to participate in an Organization of Tropical Studies graduate course.

The late Mike Ewert guided me throughout the design of this project. I truly wish

he could have seen the results; I know he would have enjoyed them. My great friend, the

late Abigail Sorenson, shared my enthusiasm for tropical turtles and assisted in the field;

I wish she too could have seen the results of this project.

Special thanks must be given to my collaborators in Mexico: Ottho Aquino,

Gustavo Aguirre León, Erasmo Cazares Hernández, Miguel de la Torre Loranca,

Veronica Espejel González, Nora Lopez, Marco Antonio Lopez Luna, Basillio Sanchez,

and Eladio Velasco; without their assistance I would never have been able to make the

cultural conversion necessary to accomplish my field research. Rosamond Coates,

Gonzalo Perez, and Martin Ricker, were instrumental in providing a base for my field research. I am indebted to Dick Vogt for his invitation to work at his previous field sites and his assistance in getting me started in Mexico. His extensive knowledge of the region’s turtles laid the groundwork for my research. Peter Pritchard and Tim Walsh of the Chelonian Institute and Jamie Pena of the Glady’s Porter Zoo are owed a debt of gratitude for facilitating the importation of my samples from Mexico. Additionally, John

Iverson shared unpublished reproductive data on Geoemydids.

My fellow graduate students both current and past provided immeasurable assistance throughout my time at OU; I will remember them fondly. My lab mates: Phil

Allman, Dawn Ford, Tomek Radizo, and Laura Stadler were truly great sounding boards during the development of this project and with its analysis. I thank them for being some of my toughest critics. I must thank Noah Anderson and Tom Lorenz for their numerous discussions on turtle nesting ecology; and many thanks are due to Jonathan Willis for endearing me to the nuisances of survivorship analyses. My oldest friend, Bob Stone, encouraged my appreciation of life beyond academia.

My brother, Mike, and my sister, Jen, have always been ready to lend an ear, offer a hand, or give word of encouragement; for this, I am humbly grateful. My parents Dick and Rama not only encouraged me to chase my dreams but also generously provided the extra financial resources that made this project a possible. Words cannot express my true gratitude towards my family; they have and always will be the principal factor of my success. No one could ask for greater family support.

Table of Contents

Page

Abstract ……………………………………………………………………………….4

Acknowledgements …………………………………………………………….……..7

List of Tables …………………………………………………………………………10

List of Figures ………………………………………………………………………...11

Chapter 1: An Evolutionary Context for Embryonic Diapause …………...……...…13

Chapter 2: Embryonic Diapause in Turtles: Its Geographic Distribution Defined…..18

Chapter 3: Strategies for Seasonal Synchronization of Morphogenesis and Increasing Embryonic Survivorship: Modeling Developmental Timing in a Neotropical Turtle………………………………………………………………………………….33

Chapter 4: The Effect of Female Size on Egg/Clutch Size and Hatching Success in a Turtle (Kinosternon leucostomum) with Embryonic Diapause ………………………62

Literature Cited ………………………………………………………………………76

Appendix A. ED Categorical Modeling Database…………………..………………..100

Appendix B. Calculated Probabilities of Expressing ED …………………………….110

Appendix C. Calculated Probabilities of Not Expressing ED ………………………..112

Appendix D. Tables…………………………………………………………………...113

Appendix E. Figures…………………….………………………………………….….118

List of Tables

Table Page

3.1: Summary of Constant Temperature Equivalents (CTE) based on timing of oviposition and timing of developmental event. ……………………..……………113

3.2: Percent mortality by stage per month ………………………………………….114

4.1: Gravid female body size, clutch size and hatchlings per clutch………...... 115

4.2: Morphological differences (averaged by clutch) between population egg parameters and eggs that produce hatchlings………………………………………...116

4.3: Relationship between hatching success and egg parameters……………………117

List of Figures

Figure Page

2.1: Geographic distribution of the ED trait…………………………………………..118

2.2. Phylogenetic tree of extant turtle families with incidences of embryonic diapause evolution rooted with the extinct turtle Proganochelys; modified from Schafer et al., 1997……………………………………………………………………………………119

2.3: Frequency of ED expression of turtles within the two broad geographic bands…120

2.4: Frequency of ED expression amongst turtles whose distribution is outside the two broad geographic bands………………………………………………………………..121

2.5: Kinosternon phylogeny modified from Iverson, 1991…………………………….122

3.1: Latitudinal changes in seasonal precipitation duration and timing, modified from

Savage, 2004…………………………………………………………………………...123

3.2: Ontogenetic model for facultative developmental timing…………………………124

3.3: Soil temperature profile from the semi-natural incubation experiment……………126

3.4: Relationships between soil moisture, soil temperature, and seasonal changes in duration of ED and morphogenesis…………………………………………………….127

3.5: Seasonal Trends in Length of Developmental Stage……………………………....128

3.6: Survivorship trends based on timing of oviposition………………………..……..129

3.7: Embryonic diapause (ED) and morphogenesis (MORPH) survivorship analysis

………………………………………………………………………………………..…130

3.8: Embryonic diapause (ED) and morphogenesis (MORPH) hazard function analysis

……………………………………………………………………………………..…...131

3.9: Relationship of rainfall with hatchling events ………………………………..…...132

3.10: Modified ontogeny model……………………………………………..…………133

4.1: Relationship amongst female size, clutch size, and number of hatchlings per clutch…………………………………………………………………………………..134

4.2: Relationship of female carapace length to egg parameters……………….……...135

4.3: Relationship of clutch size to mean per clutch egg wet mass (g)………….…….136

Chapter 1

An Evolutionary Context for Embryonic Diapause

The regulation and manifestation of developmental process have long been

integral areas of research in the study of phenotypic development (e.g., phenotypic

plasticity (Sultan, 1987; Harvell, 1990; Freeman and Heron, 1998; Kingsolver and Huey,

1998; Seigel and Ford, 2001)). However, only recently has the link between

development and ecology been used to identify selection on these traits. Beginning in

1990’s with the rise of neo-embryological studies, the new field of developmental

biology emerged (Andrews and Donoghue, 2004), synthesizing the previous more

descriptive embryological studies (e.g., the works of Agassiz, Haeckel, Kowalevsky, and

von Baer) with modern molecular techniques (Gilbert, 1998). New directions within this

discipline include investigations into how developmental processes, particularly the role

of regulatory genes, play an important role in evolutionary processes creating the

research area referred to as “evo-devo” (Gilbert, 2001). Expounding upon the

evolutionary importance of developmental biology, Gilbert et al. (1996) created this

analogy “To go from functional biology to evolutionary biology without development is

like going from displacement to acceleration without dealing with velocity”.

Van Valen (1976) succinctly stated, “evolution is the control of development by

ecology” yet, the ecology of development has received the least attention in the field of

developmental biology. Investigating environmentally induced variation in evolutionary

biology is not novel, yet ecological developmental biology focuses on this variation

unfolding during embryogenesis and early ontogeny (Sultan, 2003). Much as how the

field of ecology was a relatively new field in the 1950-60’s, today ecological

developmental biology is in its infancy. This transition within developmental biology

parallels early ecological questions that were modeled and tested in the lab (e.g., Lotka,

1925; Volterra, 1926; Gause, 1932; and Huffaker, 1958), which then transitioned into

field research (e.g., MacArthur, 1958; Lack, 1966; Pianka, 1969; and Tinkle, 1967).

My studies link development and ecology with specific ontogenies in turtles; and

centered on the variable expression of two traits, embryonic diapause (ED) and

embryonic aestivation (EA). My work expands upon earlier laboratory studies of ED

expression in turtles by Ewert, 1985; 1991; and, Ewert and Wilson, 1996 by investigating

the adaptive significance of how ED and EA influence the facultative developmental

timing in the white-lipped mud turtle, Kinosternon leucostomum. The first objective of

the study was to determine how these two traits synchronize the timing of development

with suitable environmental conditions thereby improving embryonic survivorship. The

second objective was to determine how energetic efficiency by the embryo has impacts

beyond the egg stage (e.g., hatchling size). Integrating the results of the first two

objectives the third objective was to third determine how these traits influence parental

fitness with respect to reproductive potential and propagule size (i.e., is the trade-off

between number of eggs and egg size altered by in a species that express ED and EA).

I begin in chapter 2 by summarizing the current knowledge of the geographic

distribution of turtles expressing ED and elaborate on possible undocumented species

capable of expressing ED. Species that express ED are capable of reproducing over a

greater part of the year than species that do not. Yet, these species appear to be limited in

distribution to areas that subsurface nest temperatures remain above freezing throughout

the year. Although not the prevalent mode of development within turtles, the expression

of ED has been derived independently a minimum of four times within turtles. ED is

found in six of the ten families of freshwater turtles and tortoises with an increased

prevalence of the trait’s expression in two broad geographical bands with distinct

seasonal rainfall cycle, approximately 15-37 degrees north and south latitude.

Using categorical data modeling, I was able to create probabilities for predicting

the expression of ED based on eggshell type, distribution, and presence of ED in sister

taxa. Sixty-one species have a 70% probability of ED expression, and an additional 14

species have a 66% probability of expressing ED. Greater than one-half of the species

undocumented for the expression of ED have a high probability of expressing the trait,

whereas only 15.3% (21 species) have greater than a 96% probability of not expressing

the ED trait.

There is limited knowledge on the life history evolution of ED expressing turtles.

The preponderance of all turtle developmental studies has been on North American pond

turtles (Emydidae) during summer months, a time and region, where ED is unlikely to be

advantageous (e.g., freezing nest temperatures during the coldest months of the year).

Thus, it is perhaps inappropriate to infer evolutionary patterns from these studies to all

turtle species in general. The possibility that nearly a half of all turtle species have a

different developmental biology than the well-studied species encourages a plethora of

future comparisons.

In Chapter 3, I present a model that illustrates potential developmental trajectories

that incorporate ED and EA in K. leucostomum and how these two traits synchronize the

timing of development with suitable environmental conditions. The model makes

predictions based on variation in ontogeny and on the biophysical parameters

experienced in the nesting environment. My first hypothesis suggests a single suitable

developmental period (SDT; a period when soil moisture and temperature in the nesting

substrate is within the physiological tolerances of developing embryos) during the dry

seasons. My second hypothesis predicted two SDTs at the transition from the rainy and

dry seasons. Central to this chapter is the paradigm of nesting ecology; that variation in

environmental parameters relates via a “black-box” of physiology, (i.e., un-described

physiological mechanism) to variation in nesting ecology (timing of nesting and nest site

choice). Results from a semi-natural incubation experiment modify the model and

support the hypothesis that there is a single SDT restricting morphogenesis to the dry

season. Field and laboratory incubation experiments demonstrate that cool temperatures

influence the onset and duration of ED. The laboratory experiment confirmed that ED is

facultative and that temperature is an important regulatory factor in determining the

duration of both ED and morphogenesis. Thus, ED synchronizes morphogenesis to

conditions better suited for development and EA allows neonates to remain in the egg

until environmental conditions are suitable for post-emergence survival. Thus, the

combination of ED and EA may increase embryo survivorship when there are multiple

non-suitable periods for embryogenesis during ontogeny. In addition, these traits are

good examples of physiological mechanisms for germ banking, a more accurate term

than the commonly misused term bet hedging.

In chapter 4, I studied the relationship of female size to egg parameters,

egg/female size to clutch size, and egg/female size to hatching success in K. leucostomum

to determine if females alter their reproductive investment strategies to offset the

potential additional embryonic energy expenditures during prolonged incubation. Egg

length, width, volume, and wet mass increased with female carapace length with egg

mass increasing the most. Female K. leucostomum are not altering their reproductive

investment strategies, as there are minimal costs to prolonging incubation via ED. When

accounting for female size, larger clutches had eggs with greater mass than smaller

clutches. This positive relationship of egg mass and clutch size is atypical of the more

commonly observed trade-off between clutch and egg size (Nager et al 2000). Thus in K.

leucostomum the typical relationship between egg size and clutch size as it pertains to

division of maternal resources per propagule is weakened by the embryos ability to arrest

development during extended incubation periods by expressing ED.

The study of ED and EA in turtles is a complimentary approach to studying the

physiological and morphological aspects of turtle development. Studies on temperature

dependent sex determination in reptiles have focused on specific events (i.e., the

documentation of the critical period in development when sex is differentiated (Ewert,

1985; Ewert and Nelson, 1991)) during development but have not linked entire

ontogenies to environmental parameters. By providing a plausible evolutionary context

for the adaptive significance of ED, I have laid the groundwork for future ecological

studies in over half of all turtles species, which coincidentally are the least studied.

Chapter 2

Embryonic Diapause in Turtles: Its Geographic Distribution Defined

Abstract Currently 33 species of turtles are known to prolong incubation by embryonic

diapause (ED). These turtles that express ED commonly lay brittle shelled eggs in

vegetated habitats with loamy soils during periods of both decreasing and increasing day

length (e.g. fall and spring nesters in temperate regions). Additionally, 32 of the 33

species known to express ED are found in two distinct latitudinal bands between 15o-37o

north and south latitude. I used these correlated traits to categorize all turtle species as to

the expression of ED. Three species were categorized as very likely to express ED

(matching all four criteria but lacking detailed embryological studies). Additionally, 63

species were identified as probable expressers of ED (matching one or more of the

criteria for ED expression while matching none of the criteria for non-expression (i.e.,

turtles distributed outside the latitudinal bands that lay pliable shelled eggs in open sandy

habitats during temperate spring and summer)). Seventy-one species, more than a quarter

of all turtle species, were data deficient (i.e., lacking basic life history data for one or

more of the categories). I used categorical data modeling to create probabilities for

predicting the expression of ED based on eggshell type, distribution, and presence of the

trait in sister taxa. I found that 61 turtle species have a 70% probability of ED

expression, and an additional 14 species to have a 66% probability of expressing ED.

Greater than one-half of the species undocumented for the expression of ED have a high

probability of expressing the trait, whereas only 15.3% (21 species) have greater than a

96% probability of not expressing ED. Of the top 25 most endangered freshwater turtles

and tortoises one species expresses ED and 10 species have > 66% probability of ED

expression. Integrating species-specific information and developmental studies with

conservation information will be a critical component of any future comprehensive

management plan of endangered and threatened turtles.

Introduction Diapause is a broadly used term for down-regulation of metabolism at various life

stages due to adverse environmental conditions, whereas embryonic diapause (ED) is

specific to arresting development during environmental conditions when embryogenesis

would otherwise normally proceed. ED arrests development before main morphogenesis

(e.g. somite formation) and is induced well before the onset of adverse environmental

conditions. Resumption of active morphogenesis occurs after prolonged exposure to an

eco-physiological stimuli (e.g., cool temperatures, Tauber et al., 1986; and low humidity,

Seymour and Jones, 2000). ED differs from quiescence; an immediate depression of

metabolism brought on by conditions that directly alter the physiological state of the

organism, such as anoxia, desiccation, or extreme hot or cold temperatures (Hand and

Pobrasky, 2000). Quiescence ends when favorable environmental conditions resume.

The preponderance of all turtle developmental studies has been on North

American pond turtles (Emydidae) during summer months, a time and region, where ED

is unlikely to be advantageous (e.g., freezing nest temperatures during the coldest months

of the year). The prevailing data suggest that the mean incubation period for Emydids is

50 - 75 days. Consequently, there has emerged the assumption that incubation periods in

turtles that extend beyond two and half months are exceptional. Yet, reported incubation

periods (defined here as the time between oviposition and hatching) range widely. The

Chinese softshell, Pelodiscus sinensis, has the shortest recorded incubation period of 23

days (Kuchling, 1999); while the longest recorded incubation period, of the common

Australian sideneck, Chelodina longicollis is 2.5 years (Cann, 1998).

All but one species of turtle that express ED occur within two broad geographical

bands between 15o-37o north and south latitude (Figure 2.1, Appendix A). In general,

these habitats are seasonally variable and have distinct wet-dry periods where eggs below

the nesting substrate do not freeze. The high latitude limit is probably determined by

what I herein define as the “line of lethality”, often associated with an average annual

minimum surface soil temperature between -9.5 and -12.2 ◦C (Cathey, 1990). When

minimum nest temperatures (i.e., subsurface soil temperatures) surpass the freezing point,

over-wintering survivorship may be greater for fully formed embryos that remain viable

even after ice crystals form in their bodies (Packard and Packard, 2004) rather than as

early staged embryos with a high water content inside the egg that lack super-cooling

abilities. Turtle species known to express ED at the upper latitudinal limits include the

chicken turtle, Deirochelys reticularia in South Carolina (Gibbons and Nelson, 1978;

Ewert, 1991), Kinosternon sonoriense in Arizona (Ewert, 1991; Cameron, 2004),

Acanthochelys pallidipectoris in the Gran Chaco of Northern Argentina (Horne,

unpublished data), and Chelodina expansa in New South Wales, Australia (Booth, 2000;

2002). Year-round conditions suitable for embryonic development characterize the lower

latitude limit of ED expression (see chapter 3). Although there is seasonality near the

equator, environmental extremes may not exceed the physiological limits of

embryogenesis. Thus, for species near the equator that nest year round it may not be

necessary to arrest development at any stage. Rhinoclemmys pulcherrima (Horne,

personal observation), Kinosternon scorpioides (Ewert, 1985) in Costa Rica, and

Kinosternon angustipons (Legler, 1966) in western Panama appear to define the lowest

latitudinal limits of ED.

Additionally, most species that have ED have brittle shelled eggs (thicker

eggshells provide greater resistance to desiccation than thinly shelled eggs (see chapter

3), nest in vegetated (e.g., woodland) habitats with loamy soils and nest during periods of

both decreasing and increasing day length (e.g. fall and spring nesters in temperate

regions). Many of these reproductive characteristics are associated with prolonged

incubation periods. On the other hand, species that lack ED proceed through

embryogenesis relatively quickly. They typically have flexible-shelled eggs, nest in open

sandy areas adjacent to water bodies, and nest during periods of increasing day length

(e.g., temperate spring) and the beginning of temperate summer when day length starts to

decrease.

The goal of this study was to identify those species that potentially have ED. I

generated a predictive model to identify species for which ED is likely based on duration

of incubation, species distribution, nesting habitat, the presence ED in sister taxa, and

eggshell type. Understanding ED has important management implications for threatened

and endangered turtles, especially when captive rearing is a conservation strategy and

where correct incubation methodology is vital to hatching success.

Methods

I used two methods to summarize the occurrence of ED in turtles. First, I used the

correlated geographic range, eggshell type, nesting habitat and incubation duration

(obtained from published species accounts) with ED expression. Due to the paucity of

reported nesting times for tropical species; this criterion was excluded from the analysis.

My first criterion identified whether part or all of the species geographic range

overlapped the range where ED occurs (15o-37o north and south latitude). Species for

which ED is not known are considered highly probable of expressing ED when they

matched all the characteristic of species known to express ED (geographic overlap, brittle

shelled eggs, long incubation, and nesting in loamy soils in close proximity to their

aquatic habitats when applicable). Possible ED expressers were species that matched one

or more of the criteria while other criteria remained unevaluated. Species labeled as

unlikely to have ED matched one or more of the criteria for not expressing ED. Data

deficient species lacked published accounts of one or more criteria.

The second method I used to categorize ED expression was to generate predicted

probabilities for the occurrence of ED based on the reproductive traits and turtle

phylogeny. All species were categorized three ways: (1) within or outside of the

geographical boundaries for expression of ED (see above), (2) the presence or absence of

brittle-shelled egg, and (3) the occurrence of ED in sister taxa, see Appendix A. I used

the Mesquite© (Version 1.12) to map the number of times ED has evolved within turtles

using maximum likelihood algorithms to determine the minimum number of times ED

evolved within turtles. I determined the occurrence of ED in sister taxa using the

hypothesized phylogeny presented by Shaffer et al. (1997) and species level phylogenetic

information from Iverson (1992) and Le (2006). When a species was within a polytomy

(i.e., more than two immediate descendants), the sister group to the polytomy was used to

score for the presence or absence of ED. Although this method is problematic because of

the resolution of the hypothesis, I suggest that it does contribute to information about

phylogenetic distribution of ED. I used categorical modeling (CATMOD; SAS, 1996)

based on an analysis of the weighted least square estimates to create statistical

probabilities on the expression of the ED trait based on the species that either express ED

or not (n = 127). Categorical predictions were compared to each other based on the Chi-

square distribution, and were reported + one standard error. Alpha levels were set at

0.05. I used the results of this analysis to classify species with undetermined ontogenies

with regard to the likelihood of ED expression.

Results

Because there is not a consensus of the species numbers within Geoemydidae

(Van Dijk et al., 2000; Stuart and Parham, 2006), the genus Geochelone (Ernst et al.,

1997), and the genus Trachemys (Ernst and Barbour, 1989; Legler, 1990) I

conservatively estimated that there are 273 historically well-recognized species of turtles.

Of these 273 species, only 33 species are confirmed as ED expressers and 96 species are

definitively known not to express ED, Appendix A. One hundred forty species (>51%)

remain untested, Appendix A.

Six of the 12 families of turtles express ED. Among the genera of turtles for

which there is a phylogenetic hypothesis (all 12 families represented), ED has been

derived a minimum of four times (Figure 2.2). However, 71 genera (currently there are

approximately 95 genera of turtles) are missing from this hypothesis. ED has evolved at

least once within the sub-order Pleurodira, with multiple species within the family

Chelidae expressing ED (e.g., Acanthochelys pallidipectoris and Phrynops hilarii).

However, the presence of ED within the only other Pleurodiran family, Pelomedusidae,

remains undetermined. In the Cryptodira, the super-family Trionychoidea, ED occurs in

the families Kinosternidae (Claudius, Kinosternon, Staurotypus, and Sternotherus),

Dermatemydidae, and Trionychidae (Lissemys and Aspideretes). Additionally, ED has

likely evolved multiple times within the family Emydidae (subfamily Emydinae, genus

Deirochelys; subfamily Geoemydinae, genera Cuora, Hardella, Melanochelys,

Pangshura, and Rhinoclemmys). Lastly, within the family Testudinidae ED occurs in

Geochelone and Malacocherus. ED does not occur in the sea turtles (Cheloniidae and

Dermochelyidae), the monotypic pignose softshell turtle (Carettochelydidae), and the

snapping turtles (Chelydridae, genera Chelydra and Macrochelys). ED remains

undetermined in the family Platysternidae (1 species, 5 subspecies).

The geographic distribution of 201 species occurs between 15o-37o north and

south latitude, the two broad geographic bands where the majority of known ED

expressers occur. Of these species, 65 (32%) are known not to express ED. Seven (3%)

probably do not express ED because their incubation periods are less than 90 days; 32

species (16%) express ED, and 1 (0.05%) has a high probability of expressing ED

(matching all criteria for expression of ED but no detailed embryological studies have

been conducted). Fifty (25%) are possible expressers of ED (based on matching at least

one of the four criteria for ED expression and not matching any criteria for non-ED

expression, yet there exists no published reports on ED expression for these species), and

42 (21%) cannot be categorized because of insufficient data (Figure 2.3). For the 74

species that did not overlap with the latitudinal bands where ED occurs, 32 species (43%)

are known not to express ED, 7 species (9%) had a high probability of not expressing ED,

and 1 species expresses ED. Two additional species have a high probability of

expressing ED, thirteen species (17%) are possible expressers of ED, and nineteen

species (25%) cannot be evaluated concerning the expression of ED, Figure 2.4.

The categorical modeling determined that the best predictive variables for ED

were, in descending order: 1) having a sister group express ED (χ2 = 67.51, P < 0.0001),

2) having brittle shelled eggs (χ2 = 6.53, P = 0.01), and 3) having some part of the species

distribution between 15o - 37o north and south latitude (χ2 = 0.99, P = 0.32). Somewhat

surprisingly, the geographic distribution of the turtles was not a significant predictor.

This probably relates to the high number of species that do not have ED whose range is

both within as well as beyond the two latitudinal bands. Using the categories, I was able

to determine that sixty-one turtle species have a 70% probability of ED expression, and

an additional 14 species have a 66% probability of expressing ED, Appendix A. Thus,

more than half of the untested species have a high probability of expressing ED, whereas

15.3% (21 species) have a 96% probability of not expressing ED, Appendix B. Of the 25

most endangered freshwater turtles and tortoises (Turtle Conservation Fund, 2003) only

Dermatemys mawii (Polisar, 1996) expresses ED and eight species do not express ED.

Of the remaining 16 species, 7 species have a 70% probability and 3 species have a 66%

probability of expressing ED. Yet, only six species have >83% probability of not

expressing ED, Appendix C. Thus, 44% (11 out of 25 species) of the world’s most

endangered turtles have ED or have a high probability of expressing ED, which is

surprisingly similar to the percentage of all freshwater turtles and tortoises that express or

may express ED (43.5 %, 119 out of 273 species).

Discussion

Delayed development remains poorly understood in reptiles, particularly turtles.

Greater than one half of all turtle genera have species that are undocumented for

expression of ED. Thirty genera contain species identified in this study as possibly

expressing ED, whereas the data deficient species are from an additional 26 genera.

Expression of ED varies widely taxonomically, yet when present it is generally expressed

multiple times amongst closely related species. An exception is within the Cryptodiran

subfamily Emydidae, one of the most species rich clades of turtles, where the single

species the chicken turtle, Deirochelys reticularia, expresses ED. In contrast, its sister

subfamily Geoemydidae, ED occurs in at least five genera. Similarly within the sub-

order Pleurodira (the sideneck turtles), the family Chelidae has six species that express

ED, whereas none of the species within the family Pelomedusidae expresses ED. Why

ED occurs in the Chelidae and not in the Pelomedusidae is unclear and the limited life

history studies of sidenecks make comparisons difficult. Both families originated during

the Cretaceous, when Gondwanaland was a super-continent (De La Fuente, 2003).

Chelids however are currently restricted to South America and Australia, whereas

Pelomedusids are found in both South America and Africa, but not Australia (Ernst et al,

1994). Chelids are an average sized turtle, whereas some Pelomedusids species are some

of the largest river turtles (e.g., adult female Podocnemis expansa, range 70 to 80 cm in

carapace length and 23 to 26kg; Ernst et al, 1997). It is perhaps this divergence in size

and the associated habitats and reproductive strategies, which underlie the evolutionary

pattern with Pleurodirans.

Even with in Kinosternidae, one of the most well resolved turtle clades, where ED

has been documented, the patterns of ED expression remain unclear, see Figure 2.5.

Cameron (2004) using phylogenetic comparative methods examined the variation of ED

expression within Kinosternidae and found no clear patterns due to “scarcity of

information regarding diapause and other over wintering strategies”. Currently no family

with high species diversity (i.e., greater then ten species) besides the Kinosternidae is

known to express ED as the predominant mode of development, see Figure 2.1.

Furthermore, within North American Kinosternons, a potentially undocumented case of

ED expression exists. Anderson and Horne (2007) documented nesting of the common

mud turtle, Kinosternon subrubrum, in southern Louisiana in February, a region and

period where ED expression may be advantageous because continuous temperatures

suitable for incubation probably do not occur until April. The widespread lack of

information across turtle taxa indicates the need for additional ED studies. Further

identification of species expressing ED and an accurate interpretation of seasonal

variation in ED expression and duration, along with phylogenetic analyses will better

elucidate evolutionary patterns of ED within turtles.

The current distribution of species with ED demonstrates that there are certain

geographies that promote ED expression, however distribution alone is not a good

predictor of ED expression. Turtle species distributed between 15o - 37o north and south

latitude are more likely to have ED than species outside the geographic bands. The bands

are not exclusively tropical nor do they cover all of the tropics (the area between 23.5°

N&S where there is less seasonal variation in solar expression as the sun is generally high

above the horizon year round). There is a known (Malacocherus tornieri) and two highly

probable ED expressers (Chelodina parkeria and Orlitia borneensis) that do not occur

between the two bands (15o - 37o north and south latitude). The pancake tortoise, M.

tornieri has ED and is an equatorial species found in the highly seasonal grasslands in

central Kenya and southern Tanzania (Iverson, 1992). This area is conducive to ED

expression, an annual climate cycle with distinct periods for embryogenesis and

subsurface nest temperatures that have no prolonged periods of freezing temperatures;

see Chapter 3). There is a possibility that ED expression by M. tornieri is an ancestral

trait and that its expression is an artifact of an earlier evolution of the trait with the clade,

however this species has one of the most derived morphology of any tortoise (Ireland and

Gans, 1972).

The distribution of two Asian turtles Chelodina parkeri (a newly described

species from southern Papua New Guinea) and Orlitia borneensis (a large Malaysian

river turtle) lay outside the southern 15o - 37o band. Results from artificial incubation at

constant temperatures suggest ED occurs in C. parkeri (personal communication, Paul

Van der Schouw). Orlitia borneensis nests in piles of plant debris and has extended

incubation periods (Ernst and Barbour, 1989; Moll and Moll, 2004) but no other detailed

reproductive data is currently available. Both are highly probable of ED expression but

lack published accounts on the incubation duration and/or expression of ED.

It is interesting to note that species distributed near the upper latitudes

(approximately 37°) required longer and cooler temperatures to stimulate halting of ED

expression (Ewert and Wilson, 1996). For example, Buhlmann, (1998) recorded

minimum nest temperatures of 4 ◦C for populations of Deirochelys reticularia, in South

Carolina, USA near the theoretical “line of lethality”. Deirochelys reticularia oviposits

in the fall and express ED through the temperate winter, starting morphogenesis in the

spring (Gibbons and Nelson, 1978; Congdon et al., 1983a, Buhlmann, 1998). Another

example that differs from the tropical pattern is the Sonoran mud turtle, Kinosternon

sonoriense, of southern Arizona, US, which oviposits in the fall and hatchlings emerge in

the rainy season the following year, an incubation period up to nine months (Ernst et al.,

1994). Numerous species known to express ED occur at greater latitudes than the Tropic

of Cancer and Capricorn, thus it is incorrect to generalize ED as a trait specific to the

tropics. Additionally, the longest ED expression occurs at latitudes greater than the

tropics and as species distribution approaches 15°, ED expression becomes shorter or

only occurs seasonally.

The Importance of ED in ex-situ Chelonian Conservation Projects

Due to the rapid declines of turtle populations worldwide, particularly in Asia,

there is an increasing need for ex-situ breeding projects (Ades et al., 2000) and many of

the targeted species possibly express ED. For the rare species that express ED, there is

limited knowledge available on how best to incubate the few eggs so that ED is

successfully halted (active resumption of embryogenesis). Failure to provide the correct

environmental stimuli (changes in temperature and substrate moisture) during artificial

incubation may greatly reduce hatching success as embryos may perish after extended

periods in ED (see chapter 3). I have provided a list of the species that have ED

(Appendix A) and those for which the trait is likely to occur, thereby facilitating the basis

for future research that integrates developmental biology with conservation programs of

endangered and threatened turtles.

Identifying the life history traits that are most commonly associated with ED

(brittle shelled eggs, nesting in vegetated habitats with loamy soils during periods of both

decreasing and increasing day length (e.g. fall and spring nesters in temperate regions),

and a 15-37° latitudinal distribution) is an important first step in understanding the

developmental biology of the species in question. Yet determining the presence of the

traits often correlated to ED is not enough to understand the nuances of the

developmental timing nor is it sufficient to determine the expression of ED. Under

laboratory conditions, embryonic aestivation can prolong hatching of the mud turtle,

Kinosternon arizonense (previously the subspecies Kinosternon sonoriense arizonense)

for greater than 230 days (Ewert, 1991). Additionally, Castano-Mora, 1986 reported

prolonged incubation periods (e.g. greater than 100 days) in Podocnemis lewyana.

However, P. lewyana extends its incubation by embryonic aestivation. Thus, it is

important to follow the incubation procedures for ED determination as outlined by Ewert

and Wilson (1996), where embryos are challenged to begin morphogenesis during

varying incubation conditions.

To artificially incubate eggs properly once ED expression has been confirmed

investigators need to replicate field nest temperatures and moisture levels during artificial

incubation to the best of their ability. Preferably temperatures and moisture levels can be

recorded via miniature data loggers (e.g. Onset Corporation’s HOBO® Weather Station

S-SMC Soil Moisture Smart Sensor and 12-bit Temperature Smart Sensor), but in their

absence microclimate data recorded close to the species origin such as mean monthly

temperatures and rainfall can be substituted. Fluctuating incubation conditions should

include monthly, weekly, as well as daily variation. Such variation can be accomplished

relatively economically by the use of commercial wine coolers (a thermoelectric cooling

unit), which can maintain temperatures in the low to mid 20’s °C with an additional thermostat

and heating element (e.g., ceramic terrarium heater) to raise temperatures into the 30’s °C. Such

incubators should then be maintained inside an air-conditioned room as the solid state electronics

of the cooler can only lower the temperature inside the device approximately ten degrees below

ambient air temperature.

Within the twenty-five most endangered freshwater turtles and tortoises (Turtle

Conservation Fund, 2003) sixteen species have no detailed developmental studies, ten are

predicted to express ED, and six species are predicted not to express ED. With captive

breeding being a tool to assure the survival of these critically endangered species that

have prolonged incubation, it would aid biologists to better understand the role of ED in

the developmental biology of such species. It is crucial to integrate this knowledge into

comprehensive management plans with the goals of increasing hatching success and

reducing any long-term effects of these manipulations. Seigel and Dodd (2000) draw

attention to conservation projects that alter incubation conditions of eggs due to

unforeseen population viability implications these manipulations may have on unknown

life history features. Previous conservation projects that did not account for the

developmental biology of turtles may not have accomplished their goal, for example the

sea turtle hatcheries that incubated eggs at constant cool temperatures producing all male

hatchlings (Frazer, 1992) because temperature-dependent sex determination was

unknown (Mrosovsky and Yntema, 1980; Mrosovsky, 1982). We do not have the luxury

of time and must avoid replicating these same mistakes. It is preferable to semi-naturally

incubate eggs (e.g., maintaining eggs at the nest site with anti-predator protection) of

endangered species within their natural range with minimal manipulations, but when

conservation methods must rely on ex-situ breeding and/or incubation all efforts should

be put forth to replicate the natural daily and seasonal cycles of nest conditions.

Chapter 3

Strategies for Seasonal Synchronization of Morphogenesis and Increasing Embryonic

Survivorship: Modeling Developmental Timing in a Neotropical Turtle

Abstract

Embryonic diapause (ED) and embryonic aestivation (EA) are putative

mechanisms to increase survivorship during prolonged development. Both synchronize

morphogenesis and hatchling emergence with appropriate yet unpredictable

environmental conditions. I tested two hypotheses and their predictions with regard to

the environmental conditions that stimulate ED and EA based on an annual climatic

pattern characteristic of a tropical biome at 18° north that has distinct warm dry seasons

and cool rainy seasons. My first hypothesis has a single suitable developmental period

(SDT; periods when soil moisture and temperature in the nesting substrate are within the

physiological tolerances of developing embryos) during the dry seasons whereas; my

second hypothesis predicted two SDTs at the transition from the rainy and dry seasons.

Field and laboratory incubation experiments tested the model using embryos of the white-

lipped mud turtle, Kinosternon leucostomum. Field incubation times varied from 99 to

209 days, depending primarily on the time embryos diapaused. Evidence suggested that

cool temperatures influence the onset and duration of ED. The laboratory experiment

confirmed that ED is facultative and that temperature is an important regulatory factor in

determining the duration of both ED and morphogenesis in K. leucostomum. Of the three

developmental events (ED, morphogenesis, and EA), mortality was greatest during ED. I

supported the hypothesis that there is a single SDT restricting morphogenesis to the dry

season. Embryonic diapause is a mechanism by which female can increase the length of

the reproductive season and thus their reproductive output because the embryos remain

dormant until environmental conditions suitable to morphogenesis occur.

Introduction

Organisms inhabiting unpredictable, seasonally variable habitats encounter the

challenge of synchronizing reproduction with suitable environmental conditions (Baker,

1938; Stearns, 1992). Many organisms require precipitation as a proximate ecological

factor in order to reproduce. However, the unpredictability of the timing and amount of

precipitation means conditions favoring growth may come early, late, intermittently, or

not at all within the year (Davidowitz, 2002). Stearns (1976) introduced “bet-hedging”

reproductive strategies as mechanisms to increase lifetime reproductive success despite

low offspring survival probability after each reproductive episode (Menu et al., 2000).

One strategy is to lengthen the adult life span thereby compensating for unpredictable

environmental variation by spreading reproduction over numerous events (Stearns and

Crandall, 1981). Another strategy is to delay the start of life (i.e., a reframe from

propagules hatching, a form of “germ-banking” (Evans and Dennehy, 2005)) until

environmental conditions are favorable (Stearns, 1992). The latter strategies most often

are associated with plants where seeds remain dormant until suitable environmental

conditions occur, e.g., desert annuals. However, the energy limitations of many animals

cannot maintain viable embryos through several seasonal cycles that are common to

plants; therefore, animal development should occur during annual favorable

environmental conditions.

Diapause occurs across a wide range of animal taxa and is a physiological

mechanism to minimize energetic costs during challenging environmental conditions.

Insects reduce metabolism during harsh environmental conditions by diapausing in the

egg, larvae, or adult stages (Tauber et al, 1986; Denlinger 1985, 1998). Embryonic

diapause in annual killifish ensures population survival in ephemeral aquatic habitats

(Hand and Pobrasky, 2000). Other examples of species with early developmental

diapause include brine shrimp (Hand, 2000), copepods (Hairston and Munns, 1984),

freshwater snails (Elmslie, 2001), silk moths (Fuduka, 1952; Hasegawa, 1952), field

crickets (Menu et al, 2000), killifish (Wourms, 1972), marsupials (Delaney, 1997), turtles

(Ewert, 1985), chameleons (Blanc, 1974; Ewert, 1991), and possibly one species of snake

(Nadzhafou and Iskenderov, 1994).

A key characteristic of embryonic diapause (ED) in oviparous organisms is that

embryos survive periods when reproduction is separated from the emergence of the next

life cycle stage by sub-optimal or lethal environmental conditions (Wourms, 1972). ED

and embryonic aestivation (EA) are mechanisms by which animal embryos can survive

environmental conditions that are unsuitable for development. ED halts development

before somitogenesis and resumes morphogenesis after prolonged exposure to an

environmental cue (e.g., cool temperatures, Tauber et al., 1986). ED is not a form of

quiescence; rather, it maintains developmental arrest during environmental conditions

when embryogenesis normally could proceed (Ewert, 1991; Andrews and Donoghue,

2004). Quiescence is an immediate physiological response initiated by conditions that

alter metabolic states such as anoxia, desiccation, or extreme temperature changes (Hand

and Pobrasky, 2000) and that ends when favorable environmental conditions resume.

Embryonic aestivation prolongs incubation by depressing the metabolism of the embryo

after completion of morphogenesis (Ewert, 1991), and is a form of quiescence.

Turtles are the only amniote with a cleidoic egg (i.e., enclosed in a calcareous

shell that limits embryonic interaction with the environment) that express ED. Their

embryos are fully provisioned at oviposition with generally no post-ovipositional parental

care. Most temperate turtles initiate morphogenesis 24 to 48 hours after oviposition, yet

many sub-tropical and tropical species can arrest development for up to ten months in

laboratory-controlled incubation environments (Ewert, 1985). Environmental conditions

that initiate and break diapause and aestivation and their effects on embryo fitness are not

well understood in turtles. Furthermore, there are no field studies of EA and the single

field study of ED in turtles tested only for the presence or absence of the trait (Ewert and

Wilson, 1996). Even within a clutch, ED can range from zero to over 300 days (Ewert,

1991). Herein, I present two graphical hypotheses that identify developmental

trajectories and the environmental correlates along with lab and field based experiments

to understand ecological significance of ED and EA in the white-lipped mud turtle,

Kinosternon leucostomum.

The Hypotheses

Some tropical turtles live in environments that allow extended reproductive cycles

in which oviposition could occur during any month of the year. Although some nesting

seasonality is evident, some species can oviposit up to 10 months of the year, spanning a

wide range of environmental conditions that may differ substantially with regard to their

suitability for embryonic development. Due to the wide range of environmental

conditions encountered by embryos, different developmental strategies incorporating ED

and EA may increase embryonic survivorship. For example, many species of tropical

turtles that oviposit during the rainy season arrest development until the dry season with

hatchlings emerging at the end of the dry season or at the onset of the following rainy

season (Moll and Moll, 2004). During the rainy season, cool temperatures and saturated

soils are likely to promote ED because of restricted gas exchange and reduced

development rate. However, in some tropical regions the peak of the dry season may

become unsuitable for development because temperatures are too hot or water becomes a

limiting resource. Additionally, during the dry season, ephemeral wetlands dry out and

emerging hatchlings risk desiccation and low resource abundance. I present two

hypotheses that identify how neotropical turtles use embryonic diapause and aestivation

to avoid developing during environmental conditions that may adversely affect neonate

survivorship or energetics. The models are based on the assumption that precipitation

increases soil water content, decreases soil and atmospheric temperature, and solar

radiation correlate positively to soil temperature and negatively to soil moisture.

My first hypothesis, based on the observation by Moll and Legler (1971),

suggested that conditions suitable for morphogenesis occur during the dry season and

results in three developmental trajectories. First, eggs deposited during the rainy season

diapause until the dry season and then commence morphogenesis. After the completion

of morphogenesis, neonates can either emerge, or aestivate until suitable conditions for

emergence arrive. Second, eggs laid during the dry season directly enter morphogenesis

and emerge upon its completion or delay emergence via aestivation. Finally, eggs laid

during the transition from rainy to dry season facultatively enter diapause depending on

the environmental conditions at the time of oviposition. Those embryos entering

diapause at this time would diapause for shorter periods than eggs laid during the rainy

season.

My second hypothesis is based on the seasonal variation in the timing and

duration of the rainy season(s) in the tropics as outlined by Savage (2002; Figure 3.1).

Interpreting these weather patterns I predicted two times of the year when changing soil

moisture and temperature create biophysical conditions that may be suitable for

development; during the transition from the rainy to dry season and vice versa (Figure

3.2). This pattern of seasonal wet-dry cycles (e.g., warm dry season and cool, wet rainy

season) is characteristic of a tropical biome at 18° north latitude (Figure 3.1). I assumed

that suitable conditions must persist for 75 days, the approximate time required by an

embryo to complete morphogenesis. Based on the criteria of a minimum 75-day

developmental period (Horne, personal observation), two suitable development times

(SDT) are separated by two periods, one too cold and wet for development and the other

too hot and dry for development (Figure 3.1 and 3.2). Furthermore, hatching and

emergence should coincide with environmental cues that reflect the availability of aquatic

habitats and high resource abundance but that also may occur at the conclusion of

morphogenesis. Based on the time of oviposition and the biophysical constraints (e.g.,

soil temperature and moisture) that affect development, I hypothesized six different

developmental trajectories that predict the timing of ED, morphogenesis, EA, and

emergence (see Figure 3.2).

In both hypotheses, flexibility is provided after morphogenesis by aestivation,

which would allow the synchronization of emergence when resources are abundant.

Embryos that aestivate through the dry season and emerge at the beginning of the rainy

season when ephemeral aquatic habitats refill should encounter increased food

availability (e.g., aquatic insects and amphibian larvae). However, embryos that delay

emergence until the onset of the rainy season likely incur a greater energetic cost relative

to individuals that emerge at the conclusion morphogenesis. Hatchlings emerging at the

conclusion of morphogenesis must survive until the rainy season when resources become

abundant. These hatchlings may use their energy reserves to initiate growth that results

in larger body size relative to individuals that aestivate. Other factors that potentially

influence when emergence occurs includes water balance and predation risk.

Methods I investigated the facultative developmental timing in the white-lipped mud turtle

(Kinosternon leucostomum) an inhabitant of permanent and ephemeral aquatic habitats

(Berry and Iverson, 2001). Kinosternon leucostomum has facultative ED (Ewert, 1991)

and facultative EA with incubation periods from 75 to over 300 days (this study and R. C.

Vogt, personal communication). In addition, K. leucostomum lays multiple small

clutches (1 - 6 eggs; Moll and Legler, 1971; Berry and Iverson, 2001; this study) during a

ten month nesting season (Vogt, 1990) from late August (early rainy season) to late May

(dry season).

Field research was conducted at multiple sites within the Los Tuxtlas region of

southern Veracruz, Mexico. The primary study site was Laguna Zacatal, an ephemeral

crater lake on the property of the United Autonomous University of Mexico field station,

Estación de Biología Los Tuxtlas [see Morales-Verdeja and Vogt (1997) for further

details]. Additional field sites included the wetlands in the Rio Papaloapan drainage near

the town of Lerdo de Tejada, and wetlands adjacent to the town of La Margarita on the

northern shore of Laguna Catemaco. Gravid female K. leucostomum were captured using

fyke-nets and baited funnel traps. All gravid females were processed at field camps,

where I injected oxytocin into the inguinal region (0.3cc per kilo) to induce oviposition

(Ewert and Legler, 1978). Eggs were collected for two experiments, one in 2001, and

another in 2003.

Environmental Parameters

Several data sources were used to evaluate how the environmental conditions in

the Los Tuxtlas region matched the two hypotheses. First, the 25-year average daily

temperature and rainfall data collected at the Estación de Biología Los Tuxtlas were used

to identify the beginning and end of the rainy season as well as potential SDTs. Second,

from December 2000 to November 2001, five temperature loggers (Onset Tidbits®) were

placed in the substrate in a known nesting area to record soil temperatures (subsurface

(2.0 cm) soil temperatures recorded every 15 minutes). Average soil temperature was

converted at one-week intervals into consecutive 75-day periods, then into constant

temperature equivalents (Georges et al., 1994, Georges et al., 2005) to identify suitable

development times. During 2003, soil moisture probes were placed at the same nesting

area. Unfortunately, the probes were stolen before the data was recovered. Therefore,

the data obtained in Experiment 2 (see below) was used to evaluate seasonal variation in

soil moisture.

Experiment 1

In 2001, a laboratory experiment was conducted to evaluate the relationship

between incubation temperatures, ED, and morphogenesis. One hundred ninety one eggs

were collected from 13 October through 12 December 2001. Eggs were assigned

randomly to treatments; however, because of the small clutch size of K. leucostomum,

clutch was not replicated within treatment. The eggs were incubated in vermiculite in

groups of ten (five per container) at 17°C, 21°C, 25°C, and 29°C. Furthermore, each

temperature treatment had four hydric conditions of approximately 0.0 mPa, -0.15 mPa,

-0.40 mPa, and -0.75 mPa manipulated by water to vermiculite mass ratio (see Filoramo

and Janzen (2002) for details on the ratio of vermiculite and water and to the

corresponding soil water potential). Experimental conditions “challenged” embryos to

develop (Ewert and Wilson, 1996). On 1 December 2001, challenged embryos were

“switched” to a standardized hydric environment of -0.15 mPa and randomly split

between two incubation temperatures 27°C and 31°C. Two control “unchallenged”

groups of 30 eggs each were maintained at -0.150 mPa and incubated at either 27°C or

31°C during their entire development. Because eggs could not be gathered over a short

time period, exposures to challenge conditions were 30 and 60 days; eggs collected early

in the nesting season had longer exposure to challenge conditions than those collected

later in the season. Eggs were monitored bi-weekly to check soil moisture,

developmental progress, and survival. During evaluation, eggs were candled with a

fiber-optic light and developmental stage was recorded using digital photography.

Hatchling survival, time in ED, morphogenesis, and EA as well as total developmental

time were recorded.

Experiment 2

During 2003, I performed a field experiment to evaluate the developmental

trajectories used by K. leucostomum. To control for microhabitat variation, a raised

wooden box (2m X 0.8m X 0.3m) was constructed and filled with a soil profile

resembling natural nesting habitat, a thin (5 - 10 cm) organic layer atop sandy soil

comprised of loose volcanic material. Drain holes in the bottom of the box prevented

pooling of excess rainwater. Elevating the box and placing its legs in plastic buckets

partially filled with mineral oil provided protection from ants and vertebrates. In

addition, a securely fastened screen lid prevented both predation from vertebrates and

escape of hatchlings. The box was placed in a fenced area near the Estación de Biología

Los Tuxtlas to accurately mimic natural fluctuations in temperature and soil moisture

caused by local macroclimates.

Environmental data from the experimental setup was collected to test the general

fit of the model to the soil moisture and temperature regimes of the region from 12

December 2002 to 23 November 2003. Six ECH2O dielectric aquameter probes

(Decagon Devices, Inc.) and six thermistor probes attached to two Em5 datalogger

(Decagon Devices, Inc.), recorded soil moisture and temperature every fifteen minutes.

Moisture probes were paired with thermistor probes and placed equidistant from other

pairs within the nest box. Depth of sensors resembled the depth of natural nests (1.0 -

2.0cm below the surface; Horne, personal observation). Data collected by the moisture

probes were converted from millivolts to mPa by using the soil moisture release curve (y

= (0.0011*(percentage soil moisture)) -4.6821; Campbell, 1985), which previously had been

created on a dew point meter (Decagon Devices, Inc.). Field data were compared to

temperature data collected in experiment 2 to verify that the experimental setup did not

alter natural variation in temperature profiles.

From 12 December to 24 April 2003, 143 K. leucostomum eggs were collected.

All eggs were placed within the box less than 12 hours after oviposition. To aid in

accurate determination of hatching and emergence times, eggs were placed in individual

plastic screen cylinders (10 cm in diameter by 30 cm long). Each cylinder contained 25

cm of soil (previously collected from a known nesting site). Furthermore, eggs were

buried to 2/3 of their shell height, leaving 1/3 exposed, resembling natural nests (Horne,

personal observation). In order to simulate light and humidity levels in closed canopy

rainforest, I placed woven plastic sheeting loosely but directly over the cylinders and

suspended by a heavy shade cloth over the nest box; both allowed rainwater to reach the

soil substrate. Daily I visually monitored eggs for mortality, hatching, and emergence.

As in experiment 1, I candled embryos with a fiber-optic light biweekly and the

developmental stage was recorded using digital photography. Hatching time was defined

as the date when a neonate was entirely out of the eggshell because tropical

Kinosternidae can spend extended periods within the eggshell after pipping (Vogt,

personal communication). A neonate leaving the nest defined the date of emergence.

Data Processing and Statistical Analysis

The duration of developmental events was reported in days, mean values per

group were reported ± one SE. Individual averages within clutch minimized clutch

effects. F-tests detected differences in hatching success among groups based on the

timing of oviposition (16 Dec, 19 Jan, 29 Jan, 5 Feb, 13 Mar, 16 Mar, and 24 Apr 2003).

Linear regression analyses tested for seasonal changes in the timing and duration of

developmental events; and, a Kruskal-Wallace one-sample test using a Chi-square

distribution tested the relationship between rainfall events and hatching. A general linear

model MANOVA analyzed for differences in seasonal expression of developmental

traits. Survivorship between stages and the control groups in experiment 1 were tested

using Yates corrected Chi-square analysis. To assure data analysis accuracy, all

statistical tests (excluding the survivorship analyses) were completed using both SAS

(1996) and SYSTAT (Version 10) with identical results. PROC PHREG in SAS was

used to calculate both survivorship curves and the hazard functions for ED and

morphogenesis. Constant temperature equivalent calculations (CTE, sensu Georges et al.

1994; 2005) were estimated using a program written in C+. I log10 transformed data to

meet the assumptions of parametric statistical tests; an α level of >0.05 was set for

significance.

Results

The environmental data and findings of the two experiments suggest that the

morphogenesis phase of development in K. leucostomum occurs during the dry season

consistent with the first hypothesis, see Figure 3.4. Although, I observed some of the

developmental trajectories predicted in both hypotheses, the environmental data clearly

illustrate that temperatures are too cold during the rainy season for development to

proceed and the primary factor contributing to active development are the increasing nest

temperatures at the start of the dry season, see Figure 3.4.

Experiment 1

Embryos from eggs collected in October were physiologically capable of

beginning development immediately after oviposition. After incubating 59 days, 17.5%

of embryos (n = 7; 1 at 21ºC, 2 at 25ºC, and 4 at 29ºC) oviposited on 13 Oct 2001 were in

morphogenesis. The remaining embryos (82.5%, n = 33) were in ED. All embryos (n =

56) oviposited on 8 Nov 2001 were still in ED after 34 days of incubation. Furthermore,

all embryos at 17°C arrested development until released from their challenge condition,

indicating that the minimal developmental temperature for K. leucostomum is >17°C.

For challenged embryos, temperature was a more important determinant of the

length of the incubation period than soil moisture. Exposure to cooler temperatures

before morphogenesis decreased developmental time (ANOVA, F3, 26 = 5.47, P = 0.01;

data among hydric treatments and exposure times to challenge conditions were pooled

within temperature treatment): 17°C ( x = 91.6, ± 2.8, n = 13), 21°C ( x = 113.2, ± 5.4 n =

10), 25°C ( x = 122, ± 65, n = 2), and 29°C ( x = 132.8, ± 18.3, n = 4). Incubation time

was not affected by soil moisture level (0.00 mPa ( x = 91.1± 6.9, n = 9), -0.150 mPa

( x = 113.5 ± 11.4, n = 8), -0.40 mPa ( x = 108 ± 7.6, n = 8), and -0.750 mPa ( x = 100 ±

5.8, n = 13); (ANOVA, F3,37 = 1.44, P = 0.24 data among temperatures and exposure time

to challenge conditions were pooled within hydric treatment)). The control groups had

similar incubation periods: 27°C ( x = 112.6, ± 12.6, n = 13) and 31°C ( x = 111.0 ± 27, n

= 4); Mann Whitney U-test, U = 0.51, df = 1, P = 0.61. Additionally, pooled control and

individual experimental groups did not differ (ANOVA, F4, 42 = 1.24, P = 0.31).

Overall, survivorship of experimentally challenged hatchlings was low 27.6%

(39/141). Prior to the switch, survivorship of challenged embryos was high 82.2 %

(116/141), but survivorship decreased after the switch 33.6 % (39/116) resulting a in a

difference between pre- and post-switch survival; χ2 = 60.91, df = 1, P < 0.0001). There

was no difference in hatching success between the two post-switch temperatures (χ2 =

0.62, df = 1, P = 0.43). At 31°C post-switch, 17/58 (29.3%) embryos hatched, and 22/58

(37.9 %) hatched at the 27°C. In both temperature treatments, one third of post-switch

embryos died during morphogenesis and an additional third died at six months during

EA. The late-term mortality suggested that an environmental stimulus for

hatching/emerging was absent in this laboratory experiment. Control embryos (n = 60)

had an overall hatching success rate of 21.6% (13/60). Similar to the control embryos,

the post-switch hatching success of challenged embryos did not differ between

temperature treatments, five (16.6%) hatched at 31◦C and eight (29.6%) hatched at 27◦C

(χ2 = 0.35, df = 1, P = 0.56). Additionally, hatching success did not differ between the

pooled control group and the pooled challenged embryos (χ2 = 2.17, df = 1, P = 0.14).

Experiment 2

Ambient air temperatures, 25-year annual rainfall data and the soil temperatures

collected in the field experiment matched the environmental conditions predicted in the

first hypothesis. Soil temperatures were lower than expected during the time predicted to

be the suitable developmental periods by the second hypothesis and thus morphogenesis

appears constrained to the dry season when warmer temperatures occur. As a result, only

a single SDT occurred from early March to late August corresponding to the dry season.

Mean daily soil temperatures ranged from a low of 16.2°C on 20 January 2003 to a high

of 26.1°C on 31 August 2003 with a mean of 22.5°C and a mean daily variance of 0.18ºC

Figure 3.3. Soil moisture decreased on 8 to 11 February 2003 (maximum of -20.6 – mPa

on 10 Feb) when a southerly weather system brought high dry winds marking the end of

the raining season. From 2 March to 5 April 2003, soil moisture decreased again and

reached its lowest level (maximum -32.6 mPa on the 26 March, Figure 3.4). By 9 March

2003, 95% of embryos (40 out of 42, oviposited from 16 December 2002 to 5 February

2003) synchronously initiated morphogenesis when soil temperatures increased to

20.5°C. Two remaining embryos extended diapause and initiated morphogenesis in late

May/June. Embryos oviposited from 3 Mar to 16 Mar 2003 (after the start of the dry

season) diapaused facultatively and broke ED asynchronously, ranging from 4 Mar to 29

Apr 2003. In addition, 17 individuals entered into EA and only nine aestivated for more

than 15 days.

Total incubation period (oviposition to emergence, x = 135.5 + 25.7 days)

2 decreased seasonally (r = 0.54, F1,76 = 87.77, P < 0.001), with incubation periods ranging

from 209 days (oviposited in Dec) to 99 days (oviposited in April). The mean number of

days spent in ED among all groups was 37.7 + 21.5 days (ranging from 2 – 120 days).

2 Length of ED was negatively related to date of oviposition (r = 0.44, F1,76 = 57.92, P <

0.001), ranging from a mean of 97.6 + 19.6 days in Dec to 11.0 + 0.0 in April. However,

there was no relationship between number of days in morphogenesis and date of

2 oviposition (r = 0.04, F1,76 = 3.38, P = 0.17). The mean number of days spent in

morphogenesis was 94.2 + 13.1 (ranging from 57 to 139 days; Figure 3.5). There was no

relationship between timing of oviposition and length of time spent aestivating (r2 = 0.01,

F1,17 = 0.21, P = 0.65). The mean number of days spent aestivating was 16.5 + 11.5

(ranging from 2 to 36 days). No embryos that proceeded directly into morphogenesis

after ovipositing aestivated. In addition, embryos that aestivated (n = 17) did not differ

from non-aestivating embryos with respect to time spent in morphogenesis (ANOVA,

F1,76 = 1.75, P = 0.19). Time spent in ED had no effect on time spent in aestivation,

greater than 15 days, (ANOVA, F9,62 = 0.50, P = 0.13; 8 cases were excluded due to EA

periods less than 15 days).

Constant Temperature Equivalents

I created seven classes based on the time of year when I collected eggs. Constant

temperature equivalents (CTE) were calculated for the mean duration that embryos spent

in both ED and morphogenesis (Table 3.1). Over the duration of incubation experiment,

CTE values increased by 2.2°C degrees (range 20.5°C during the 16 December 2002 - 24

March 2003 period to 22.7°C during the 24 April – 5 May 2003 period; Table 3.1). CTE

2 values for time spent in ED increased seasonally (r = 0.95, F1,5 = 93.26, P < 0.001). CTE

2 for time spent in morphogenesis did not change (r = 0.55, F1,5 = 6.209, P = 0.06) but

ranged from 23.0°C during 9 March – 9 June to 24.3°C during 5 May – 2 August 2003.

The mean CTE for ED was 21.3 + 0.7°C and the mean CTE for morphogenesis was 23.5

+ 0.4°C.

Survivorship

Overall, hatching success was low, 77/143 (53.8%) embryos hatched, but higher

than experiment 1. Survivorship differed among developmental events (ED,

morphogenesis, or EA) and throughout the year (Figure 3.6). Survivorship was higher

during morphogenesis (77/88, 88%) than ED (88/143, 62%; χ2 = 30.61, df = 1, P <

0.0001). However, embryos in ED survived the coldest periods during the study.

Mortality of diapausing embryos was highest during peak periods of low soil moisture.

Embryos oviposited on 29 January and 5 February 2003 had the highest survivorship to

hatching (86% (12/14) and 90% (9/10) accordingly). Embryos that died during

morphogenesis spent longer time in ED than those that survived morphogenesis

(ANOVA, F1,97 = 0.52, P < 0.05). Only 18 of the 77 (23.3%) embryos that survived

morphogenesis entered into EA. All aestivating individuals survived.

Diapausing embryos died in all months except December and January (Table 3.2)

2 and mortality rates during ED increased as the season progressed (r = 0.76, F1,5 = 19.17,

P = 0.005). Peaks occurred in April (43.5%), June (62.5%), and July (100%) suggesting

that the cue to break ED was missed and therefore morphogenesis never commenced.

Deaths during morphogenesis occurred from April through August were never greater

than 5%.

In several cases, there were environmental conditions at oviposition that

corresponded with peaks in mortality. Embryos oviposited on the 16 March (N = 35) had

the highest post ED mortality rate (57%) were laid during 48hr period with the lowest

mean soil water potential (-15.8 mPa, range -4.3 to -24.5 mPa; Figure 3.4). Mortality

rates of embryos oviposited before or during the lowest observed temperatures (16.2°C

for 2.0 hrs during 19 January, Figure 3.3) were moderate, 50% for the 16 December

group (N = 6) and 37% (N = 27) for the 19 January group. Embryos from 29 January, 5

February, and 3 March 2003 had the highest ED survivorship (86%, 90%, and 76%

respectively). Embryos from these groups were exposed to low temperatures (17°C) for

only 2.25 hours. These findings suggest that embryos are sensitive to environmental

fluctuations shortly after oviposition.

If an embryo survived ED, it had a greater than 80% chance of hatching within

150 days of beginning morphogenesis (Figure 3.7, x = 110 days in morphogenesis).

However, survivorship to hatching decreased to 50% if an embryo spent more than 180

days in morphogenesis. Furthermore, ED had a higher hazard function (or force of

mortality, which is the likelihood that an individual will die during the defined period)

occurring near 40 days of incubation followed by morphogenesis (χ2 = 71.26, df = 1, P <

0.001; Figure 3.8). Whereas the highest hazard function of morphogenesis was at

approximately 110 days, slightly longer than the experimental mean for time spent in

morphogenesis.

Timing of Hatching and Emergence

Hatchlings emerged and were active between 30 May and 5 Sept 2003 after the

onset of the rainy season. The first eight hatchlings were found at the onset of the rainy

season (27 May 2003) when soil water potential increased from -0.30 mPa to -0.18 mPa.

The remaining 69 hatchlings emerged in the first 43 days of the rainy season from 6 June

and 19 July 2003 (Figure 3.9) when soil water potential > -0.18 mPa and before the

heaviest rainfall in early October (>275 mm of daily rainfall). Most embryos hatched

within 48 hours of rainfall ( x = 38.7 hrs + 6.6, range 0 - 144 hrs; χ2 = 16.83, df = 1, p <

0.001, N = 77; Figure 3.9). Fifty embryos hatched within 24 hrs of rainfall. Beyond the

24 hr period, hatching occurred at a mean of 104.7 + 6 hrs (ranging 25 – 144 hrs).

Turtles that aestivated hatched during the beginning of the rainy season over a 23-day

period from 13 June- 6 July 2003, 65 days before the last non-aestivating turtle hatched.

There was no differentiation between time of hatching and time of emergence in this

study. Embryos that aestivated did not differ in hatching and emergence behavior than

those that did not aestivate. An embryo in EA is distinguished during candling by small

pad of shrunken chorioallantois adjacent to the embryo (Ewert, 1985).

Discussion

The biophysical field data and the experimental results indicate that K.

leucostomum is a turtle with facultative development with morphogenesis occurring

during the dry season and emergence corresponding to the onset of the rainy season,

Figure 3.10. This conclusion supports our first hypothesis suggested by Moll and Moll

(2004) that morphogenesis is restricted to the dry season. We have illustrated that our

second hypothesis does not hold for K. leucostomum for the Los Tuxtlas region of

Mexico. However, the second hypothesis may apply to tropical and subtropical regions

where there are two distinct wet and dry seasons (Figure 3.1). Based on the findings of

my study, application of the second hypothesis to regions with semiannual wet and dry

seasons would require that morphogenesis occur during the dry season instead of the

transition for wet to dry season as suggested originally. An example is the African

leopard tortoise, Geochelone paradalis (Cairncross and Greig, 1977), an inhabitant of

highly seasonal grasslands. The reproductive strategy of G. paradalis includes prolonged

incubation periods and asynchronous hatching events (Boycott and Bourquin, 2000), in

an area of highly unpredictable seasonal rainfall. However, neither hypothesis can

explain why some species of turtles incubate for more than a year (e.g., Chelodina

expansa, > 600 days (Cann, 1998)).

All eggs included in both experiments were viable at the beginning of the

experiment as documented by the presence of the white patch on the eggshell surface

(Thompson, 1985). Thus, mortality, particularly during ED, was not related to egg

fertility or embryonic death before oviposition. Yet, overall hatching success in both

experiments was low (53.8%) compared to direct developing species (e.g., 80% for

Graptemys flavimaculata (Horne et al., 2003), 95.2% for Emydoidea blandingii (Gutzke

et al., 1987), and 89-92% Malaclemys terrapin (Burger, 1977)). Embryonic survivorship

during EA in experiment 2 was 100%. This was much higher than experiment 1 (65%

survivorship during EA, N = 47), where soil moisture remained constant at -0.15 mPa

and soil temperature was constant at either 27°C or 31◦C. Experiment 1 lacked the

environmental conditions that simulate the beginning of the rainy season thus possibly

the cue to end EA. Doody et al. (2001) reported that the aestivating pignosed softshell

(Carretochelys insculpta) embryos hatch shortly after the first rains of the wet season;

and embryos of the chicken turtle (Deirochelys reticularia) may die from dehydration

awaiting a precipitation cue to indicate the end of the dry season (Gibbons and Nelson,

1978).

Interestingly, reptile embryos that do not arrest development have longer

incubation times when incubated at lower temperatures (Deeming and Ferguson, 1991).

Cooler challenge temperatures during ED reduced the overall incubation period in K.

leucostomum. Perhaps this increases survival of embryos oviposited before or during the

coldest parts of the year by providing longer neonate foraging time between emergence

and their first post-embryonic quiescent period (i.e., induced reduction in metabolism due

to ambient temperature below the minimal temperatures required for activity).

My study also suggests that diapause and the initiation of morphogenesis are

initiated by environmental factors encountered by the egg after oviposition. Cool

temperatures seem to initiate and maintain diapause and increasing temperature combined

with drying break diapause and initiate morphogenesis. Most embryos in our study broke

diapause when soil temperatures rose above 20◦C in March. Additionally, both hatching

and emergence correspond with rainfall at the termination of the dry season.

The variation in embryonic response to environmental cues for breaking

developmental arrest appears seasonal. Embryos oviposited in December spend on

average 98 days at a CTE of 20.5°C compared to only an average of 12 days at a CTE of

22.7°C for embryos oviposited in April: a difference of 86 days and 2.3 CTE degrees.

Ewert and Wilson (1996) suggested that a chilling period of 25 days at 22.5°C degrees is

necessary to break ED in the Giant Mexican mud turtle, Staurotypus salvinii, during

artificial incubation. This species is sympatric with K. leucostomum in Chiapas, Mexico

but not in the Los Tuxtlas region. The shorter minimal time of 12 CTE days to break ED

perhaps is related to the fluctuating temperatures the embryos experienced in this

experiment. Embryonic metabolic rates are different between fluctuating temperatures

and constant temperatures (Birchard, 2004). Many of the embryos used in Ewert (1985

and 1991) and Ewert and Wilson (1996; excluding embryos from Kinosternon baurii)

came from captive females generally maintained under a constant temperature regime

and a 12-hour light cycle. Furthermore, the seasonal differences in average time spent in

ED observed could be related to hormonal differences between clutches that could be

induced by changing light cycles experienced by the female prior to oviposition.

The SDT of 150 days ranging from late February to late June, exceeds the mean

observed time for morphogenesis by 58 days, yet spans variability from the beginning to

completion of morphogenesis (i.e., 57 to 139 days). The SDT included periods with very

low soil moisture, yet embryos survived these conditions. This perhaps is due to the

brittle shelled eggs of K. leucostomum (Morales-Verdeja and Vogt, 1997) being

desiccation-resistant (reviewed in Packard, 1999). However, in experiment 2 embryos

oviposited in March had the highest monthly mortality rate when the surface soil water

potential was below the permanent wilting point (-150 mPa) for most herbaceous plants

(Braudeau et al, 2005). This suggests that there may be a period of increased

vulnerability to desiccation immediately after oviposition and that the formation of

membranes during early development contribute to water movement and desiccation

resistance. The ability of an embryo to resist desiccation is a function of water reserves

within the albumen (reviewed in Packard 1991 and 1999; Packard et al., 1981), the

surface-to-volume ratio of the egg (Iverson and Ewert, 1991), and the rate of diffusion

across the eggshell (Ewert, 1979, 1985, 1991; Ackerman, 1991). Packard et al. (1982),

Thompson (1987), Packard and Packard (1988), Packard (1991; 1999), and Booth (2002)

have reported on the desiccation resistance of brittle shelled eggs (see Gutzke et al, 1987

for an example of how hydric conditions affect pliable eggs) but they did not expose

embryos to the low soil moisture potentials observed in this study. The levels observed

in this study were 44 times greater than the upper limits in Booth’s (2002) experiments.

Prolonged incubation increases the likelihood of eggs desiccating in dry

substrates. Pliable shelled eggs can uptake additional water during the course of

incubation but the trade-off is that they more readily lose water, whereas brittle shelled

eggs must rely on maternally provided water during incubation. The slider turtle,

Trachemys scripta, also inhabits the Los Tuxtlas region and lays pliable eggs during a

three-month period (June - August) (Vogt, 1990). This species lacks ED and EA,

possibly constraining the turtle to a shorter nesting period due to the desiccation

susceptibility of its eggs. Ackerman et al. (1985) reported that pliable shelled eggs

desiccated under conditions of -0.30 mPa, where as Einem (1956) reported the eggs of

brittle shelled eggs of Kinosternon subrubrum are resistant to desiccation and that eggs

left completely dry will have normal embryonic development. In the laboratory and

without any surrounding incubating substrate, K. leucostomum eggs lose on average

◦ 0.0003 + 0.00001g H20 per min at 27.2 C and 29.3% relative humidity (egg volume, x =

6.0 + 0.1 mm3, N = 4; Horne, personal observation). In comparison the smaller pliable

eggs of the painted turtle, Chrysemys picta, loses 0.001 + 0.0002g H20 per min (egg

volume, x = 3.7 + 0.2 mm3, N = 5), whereas the larger pliable eggs of the slider turtle,

Trachemys scripta, loses 0.0007 + 0.0004g H20 per min (egg volume, x = 10.7 + 0.4

mm3, N = 3; Horne unpublished data). The small C. picta eggs not surprisingly had the

greater evaporative water loss rate. Interestingly, the smaller brittle shelled eggs of K.

leucostomum lost water at a rate of 0.0004 grams per min less than the larger pliable T.

scripta eggs, which are on average 3.7 mm3 larger. The smaller K. leucostomum eggs are

more desiccant resistant.

Variation in the embryonic sensitivity to environmental conditions may be an

adaptive response that reflects characteristics of natural nests (Flatt et al., 2001). Species

differences in the nest depth may indicate their sensitivity to changes in environmental

conditions. Species that dig deep nests may have eggs that are more sensitive to water

loss than shallow nesting species (Flatt et al, 2001). Morales-Verdeja and Vogt (1997)

described K. leucostomum as a surface/subsurface nester (defined as the lack of a well-

defined nest chamber, usually just a slight scraping in the soil with eggs sometimes

covered with leaf litter), similar to two other tropical surface/subsurface nesters, Claudius

angustatus ((Flores-Villela and Zug, 1995) and Rhinoclemmys funerea (Moll and Moll,

1990). Thus, it is likely that the embryos of K. leucostomum are less sensitive to

variation in dry soil conditions, and their nesting behavior and desiccation resistance may

explain why few embryos were found in EA, a physiological response perhaps induced

by low levels of metabolically available water. It also is possible that the embryos of K.

leucostomum are sensitive to the high moisture content of the incubating substrate and

that their nesting behavior reflects this physiological constraint. Brittle shelled eggs may

be more rigid and lack the flexibility to expand preventing additional water uptake.

The hatching events observed in experiment 2 occurred before seasonal wetlands

filled with rainwater. Morales and Vogt (1997) found 20 K. leucostomum nests within 0 -

5 m of the shoreline and the remaining four nests within 5 - 10 meters of the shoreline of

an ephemeral lake in Los Tuxtlas. Perhaps hatching in June and July before the heaviest

rains (see Figure 3.9) is related to the time necessary for hatchlings to transverse the

distance between nesting sites and the lake. A likely scenario is that hatchlings arriving

at the lake before its filling would then reside under the leaf litter and dense herbaceous

plant layer until the wetland filled (the lake generally fills in mid-to-late August). The

lake does not fill until the surrounding soil is saturated and there is surface water runoff.

Previous studies on temperate turtles indicate that turtle embryos develop longer

in wetter substrates (Morris et al., 1983; Packard and Packard, 1986; Packard 1991). The

role of soil moisture as a cue for emergence is of considerable interest in the development

of K. leucostomum. Embryos may need additional metabolic water reserves to activate

metabolism after long periods of metabolic depression due to dehydration during

morphogenesis or aestivation. It also is possible that a saturated substrate is necessary for

the eggshell to become structurally weakened thus facilitating hatching, as evident by

cracks or slits in tropical Kinosternon eggs months before hatching (R.C. Vogt, personal

communication). Thus, dehydration could be a cue for inducing EA (Ewert, 1985;

Kennett et al., 1993; Booth, 1999) and rehydration caused by rainfall and increases

relative humidity a cue for emergence (Long, 1986; Kuchling, 1999). Our findings

suggest a strong association between increased moisture and emergence similar

Kinosternon subrubrum (Burke et al., 1994) and Carretochelys insculpta (Doody et al.,

2001) In experiment 1, the lack of stimulus (e.g., increased hydric conditions) to induce

emergence when embryos were in EA may have resulted in lower hatching success. This

may be due to embryos perishing from dehydration awaiting an increase in moisture as a

cue to indicate the end of the dry season (Gibbons and Nelson, 1978). In our study,

candling also may have stimulated hatching; four embryos in EA hatched within three

days of candling. Minor handling of the eggs of the pig-nosed softshell, C. insculpta will

cause them to hatch (Sean Doody, personal communication).

Although the current study presents evidence for the timing of embryogenesis

being environmental influenced, it is important to account for maternal and genetic

influences. Denlinger (1985; 1998) demonstrated the maternal influences in the diapause

of the larval stage of the flesh fly, Sarcophaga bullata and suggested that the interaction

between environmental and maternal influences may be crucial to understanding

facultative developmental timing in this study system. Similarly, multiple-paternity may

explain phenotypic variation of developmental timing within clutches (Valenzuela, 2000;

Pearse and Avise, 2001; Pearse et al., 2002). Finally, crossbreeding experiments between

two subspecies of Indian black pond turtles (Melanochelys trijuga trijuga and M. trijuga

thermalis) demonstrated a strong genetic component to ED. M. trijuga trijuga normally

expressed ED whereas the more equatorially distributed M. trijuga thermalis did not;

hybrids expressed ED that was approximately half the duration of M. trijuga trijuga in

constant temperature laboratory incubation (Steve Freedberg, personal communication;

Mike Ewert, unpublished data).

The facultative capability to initiate morphogenesis is a mechanism to increase

the potential reproductive season of turtles. Because of the facultative capability, K.

leucostomum can be reproductive during the rainy season when embryos diapause and

continue to reproduce into the dry season. Females can oviposit well into the dry season

when a higher proportion of embryos initiate morphogenesis immediately. Using the

facultative development capability increases the number of clutches that an individual

female can produce during the year. Throughout the rainy season and into the dry season

K. leucostomum are reported to produce between four and five clutches per year (Vogt,

1990). In a sense, turtles that express ED and EA are manipulating the timing of

hatching/emergence in a similar manner to many plants that create seed banks in order to

delay germination (Grime, 1977, 1979). These traits are good examples of physiological

mechanisms for germ banking, a more accurate term than the commonly misused term

bet hedging, reviewed in Evans and Dennehy (2005). Additional multi-year life history

studies on individuals with known ontogenies will better elucidate the cost benefit

relationship for ED expression.

Diapause in insects is highly variable (Denlinger, 1985) and its expression can be

modified according to changes in latitude (Masaki, 2002). This cline of ED expression is

associated with a gradient of selection processes that result from latitudinal variation in

environmental conditions (Masaki, 2002). Although in this study there were no tests for

either stabilizing or directional selection on the ED trait, it probably is weak and highly

variable among populations due to a broad suitable range for trait expression (Ewert and

Wilson, 1996). This variation is perhaps why there has been confusion over which

species express ED. For example, there were conflicting reports on whether chicken

turtles (Deirochelys reticularia) from the southern US express ED. Jackson (1988)

reported embryos proceeded directly into morphogenesis. However, Ewert (1985)

reported that these embryos express ED. It was not until Ewert and Wilson (1996)

confirmed that the embryos in Ewert’s 1985 study were from the fall and the embryos

from Jackson’s 1988 study were from the spring that the issue was clarified. It is now

understood that D. reticularia embryos oviposited in the fall enter ED to over-winter,

with main morphogenesis starting in the spring and those oviposited in the spring

generally proceed directly into morphogenesis (Gibbons and Nelson, 1978; Congdon et

al., 1983a, Buhlmann, 1998).

Why both ED and EA occur in turtles and how environmental conditions

experienced by the embryo affect fitness is beginning to be understood. During the

coolest parts of the year, temperatures may drop below the thermal minimum for

development, resulting in morphological abnormalities (e.g., scute abnormalities; Ewert,

1985), reduced fitness, or death of actively developing embryos. ED can synchronize

morphogenesis to conditions better suited for development and EA allows neonates to

remain in the egg until environmental conditions are suitable for post-emergence survival

(e.g., ephemeral habitats containing water). Thus, the combination of ED and EA may

increase embryo survivorship when there are multiple non-suitable periods for

embryogenesis during ontogeny.

Chapter 4

The Effect of Female Size on Egg/Clutch Size and Hatching Success in a Turtle

(Kinosternon leucostomum) with Embryonic Diapause

Abstract

In oviparous organisms, female size generally correlates positively with egg and

offspring size. However, when developmental arrest of embryos results in prolonged

incubation periods, females may alter their reproductive investment strategies to offset

potential additional embryonic energy expenditures. I studied the relationship between

female size, egg size, clutch size, and hatching success in the white-lipped mud turtle

(Kinosternon leucostomum), a species with embryonic diapause (ED) that can extend

incubation up to ten months. Hatching success was low (51%) and was not affected by

egg or clutch size. Clutch size, egg length, width, volume, and wet mass increased with

female carapace length with egg mass increasing the most. Thus, K. leucostomum is

unusual in that the traditional inverse relationship between clutch size and egg size is not

upheld even when the effects of female body size are removed. I evaluate how this

reproductive strategy may be advantageous for species that can use ED to extend the

length of the nesting season thereby increasing their annual reproductive output.

Introduction

Females with a limited amount of energy available for reproduction (i.e., parental

investment) (Trivers, 1972; 1974; 1985; Godfray and Parker, 1991) should differentiate

between the amount of energy allocated to individual offspring and the number of

offspring (Howe, 1978; Congdon and Gibbons, 1990). As consequence of this parent-

offspring conflict (Parker and Mock, 1987; Godfray and Parker, 1991), there is an

optimal egg size (i.e., the size of egg that maximizes offspring survival and female

reproductive potential (Smith and Fretwell, 1974; Parker and Begon, 1986; Stearns,

1992)). Falconer (1965) documented the first maternal influences on offspring phenotype

leading to additional discoveries across a wide range of taxa.

The effect of maternal condition and environment variation during development

contributes to offspring phenotype (Rhen and Lang, 1995) and has become an active area

of research (reviewed in Lacey, 1998). In many reptiles, particularly turtles, variation in

nest temperature influences hatchling sex (Pieau, 1974; Bull and Vogt, 1979; Ewert and

Nelson, 1991; Janzen and Paukstis, 1991; Valenzuela and Lance, 2004) and size (Doody,

1999; Booth 2000). In general, warm temperatures produce larger female turtles and cool

temperatures produce smaller males (Packard, 1991). Gutzke et al. (1987) and Packard

and Packard (1988) have shown that temperature in combination with hydric conditions

significantly affect hatchling body size. Consequently, a central paradigm of nesting

ecology is that environmental variation among nests can result from variation in maternal

nest site choice (Overall, 1994; Resetarits, 1996; Roosenburg, 1996; Warner and

Andrews, 2002). Thus, variation in offspring phenotype is influenced by more than the

combination of maternal genetic material and maternal resources allocated to each

offspring (Wade, 1998).

In reptiles, egg/clutch size generally correlates positively with female size

(Carpenter, 1960; Dunham and Miles, 1985; Wilbur and Morin, 1988; Gibbons and

Greene, 1990). Therefore, variable maternal effects such as egg size (Elgar and Heaphy,

1989; Roosenburg and Niewiarowski, 1998) must be removed when evaluating the

influence of environmental variation on offspring phenotype. For example, when

investigating seasonal changes in egg size, it is necessary to control for variation in

female size as larger females may nest at different times of the nesting season. Larger

females also tend to nest at a greater frequency than smaller females, thus they have a

greater capacity to produce multiple offspring/clutches per year (Horne et al., 2004).

Additionally, larger females may have longer reproductive periods per season as they

have greater potential to store and obtain obligatory reproductive resources (Kuchling,

1999; Doody et al., 2003; Moll and Moll, 2004).

Although maternal influences are well described in temperate oviparous

organisms that have restricted start time and duration of development (Kempthorne,

1969; Wright, 1969; Falconer, 1989; reviewed in Wade, 1998), there is little knowledge

of how species with facultative developmental timing are impacted by maternal

influences. Mechanisms for dissociating development from oviposition include cold

torpor (e.g., synchronization of hatching in bird siblings (Ewert, 1985)) and numerous

forms of early developmental diapause in fish (Murphy and Collier, 1997), mammals,

(Yamaguchi et al., 2006) and turtles (Booth, 2002). These mechanisms enable

developing embryos to conserve parental investment in care (PIC) (Congdon, 1989;

Congdon and Gibbons, 1990). Previous studies of chelonian species exhibiting

prolonged incubation periods have involved hatchling over-wintering (Congdon and

Gibbons, 1990). Species that can arrest metabolism early in development rather than

later should be able to conserve PIC, suggesting a weaker association between female

size and egg size, because less energy is needed during these prolonged developmental

periods. However, there is an additional constraint. There should be a tighter

relationship between female size and egg size when the hatchlings emerge into a

resource-poor environment (Nagle et al, 2003). Nagle et al. (2003) demonstrated that the

smooth softshell turtle (Apalone mutica) required greater amounts of PIC due to its lotic

post-hatching environment that was poor in nutrient resources. Females may provision

eggs with varying amounts of yolk (i.e., energy) to meet different post-hatching situations

(Congdon and Gibbons, 1985).

In turtles, embryonic diapause (ED) is a physiological mechanism that arrests

embryogenesis in the late gastrula stage. The induction of this arrest occurs well before

conditions adverse to embryogenesis arise, and active development begins after a

prolonged exposure to an environmental cue (e.g., cool temperatures). ED expression

can last for many months, thereby enabling oviposition well before environmental

conditions suitable for embryogenesis begin. ED arrests metabolism and reduces cellular

maintenance costs (i.e., catabolism of yolk; Ewert 1985) thereby reducing energy

expenditures (i.e., propagule size) when prolonged incubation is essential to offspring

survival. If there is an increase in energy expenditures due to postponed morphogenesis

then females should increase the energy allocation per egg at the cost of additional egg

production (Shine, 1980; Lloyd, 1988). It is assumed that increased energy allocation

will be represented by increases in egg size. However, during ED very little energy is

used to prolong incubation so additional resources are potentially available to post-

paritive lecithotrophy. Hatchlings can more quickly reach a positive energy balance due

to these energy reserves so they can grow more rapidly as young juveniles. Thus, the

advantages of larger egg size may be more related to post-emergence survival and

growth.

I studied the relationship between female size, offspring phenotype, and hatching

success in the white-lipped mud turtle (Kinosternon leucostomum), a turtle that

facultatively express ED (Ewert, 1991). Ranging from southern Mexico to western

Ecuador (Iverson, 1992) K. leucostomum utilizes a wide range of habitats, from large

permanent lakes and rivers to small ephemeral ponds (Berry and Iverson, 2001), and lays

multiple small clutches (1-6 eggs; Moll and Legler, 1971; Berry and Iverson, 2001; see

below) during an extended nesting season (Vogt, 1997).

Methods

I collected 143 eggs from 64 K. leucostomum females between October 12, 2002

and April 24, 2003 within the Los Tuxtlas region of southern Veracruz, Mexico. Females

were trapped using fyke-nets and funnel traps baited with canned tuna and maximum

straight-line carapace length and width were measured with digital calipers (Mitutoyo,

model: CD-12” CP) to the nearest 0.01 mm. I injected oxytocin, a hormone that triggers

smooth muscle contractions in the oviducts, into the inguinal region of gravid females to

induce oviposition (Ewert and Legler, 1978). Because oxytocin injection can fail to

induce complete clutch oviposition (Ewert and Legler, 1978), I used female carapace

length in analyzing maternal effects instead of female post-parturition wet mass. All

females were palpated before and after oviposition, females retaining eggs were noted

and excluded from clutch size analysis. Due to the small size of the females, I could

detect any remaining eggs. Immediately after oviposition, maximum egg length, and

maximum egg width were measured to the nearest 0.01 mm using digital calipers, and

egg wet mass was recorded on an electronic balance (Denver Instrument, XP-3000) to the

nearest 0.1 of a gram. All eggs were blotted dry with a paper towel before recording

mass. The formula V = (∏ /6000) * L* W2) calculated elliptical egg volume (Iverson

and Ewert 1991). I incubated eggs as described in chapter 3.

All statistical tests were performed on SAS (2001) and SYSTAT (Version 10)

software. When testing for relationships between female size and egg size, I used

multiple regressions with egg size parameters as the response variables and multiple t-

tests for determining significance of individual variables. To investigate female size and

hatching success, a multiple logistic regression with a one-way model was used to test for

differences in egg size parameters. Simple linear regression tested for seasonal effects on

egg mass and female size. I analyzed the relationships between female size and egg size,

and female size and clutch size using multiple regressions with maximum R-squared

improvements, simple correlations, and t-tests with Bonferroni adjusted probabilities.

Because female mass may include additional developing follicles and corpora lutea, I

used female maximum straight-line carapace length as a covariate to test for seasonal

differences in egg size. Non-transformed data was used to create the best-fit linear

regression lines; otherwise, data was log10 transformed. Square-root conversion

normalized clutch size data and hatching success (number of hatchlings per female). To

avoid clutch effects, I averaged sibling parameters when multiple individuals originated

from the same clutch. P-values were set at 0.05, and means were expressed + the

standard error (SE).

Results

Hatching success rate was 51.0%; 19 clutches failed, and 45 clutches produced 73

hatchlings. Female size, egg size, clutch size, hatchlings per clutch, and the size of

successful females are reported in Table 4.1. Larger females were not more successful in

2 producing hatchlings (r = 0.06, F1,41 = 2.57, P = 0.12; Figure 4.1). Egg size parameters

of embryos that successfully hatched (N = 73) are reported in Table 4.2. There were no

multivariate relationships between mean egg parameters and hatching success (Table

4.3). When the difference between egg length and hatching success was re-tested

independently, there was still no relationship (χ2 = 1.34, P = 0.25). The addition of

sibling relationships into the full model, more accurately predicted (r2 = 0.59, df = 68) the

relationship between egg sizes and hatching success; however, this result was invalidated

due to low sample size.

There was no seasonal effect on egg mass when the residuals of clutch size and

2 mean clutch egg mass were regressed against date of oviposition (r = 0.01, F1,63 = 0.19,

P = 0.67). Additionally, there were no seasonal changes in the size of gravid females (r2

< 0.001, F1,63 = 0.04, P = 0.84).

Egg size differed among clutches for all four metrics: egg wet mass (F61,71 = 3.13,

P < 0.001), egg volume (F61,71 = 3.37, P < 0.001), egg width (F61,71 = 2.97, P < 0.001),

and egg length (F61,71 = 3.36, P < 0.001). Egg length, width, volume, and mass increased

with female size (N = 46; F = 4,41 = 3.76, P < 0.001; Figure 4.2). However, egg mass was

the only significant variable in the model (t = 3.76, P < 0.001). Egg size parameters are

summarized in Table 4.2. Interestingly, clutch size also increased with female carapace

2 length (r = 0.17, F1,41 = 12.72, P < 0.001; Figure 4.1). Since egg mass explained the

most egg-size variation in relation to female size, it was used to investigate the effects of

female size and clutch size.

Linear regression of the residuals of mean egg mass and clutch size with female

2 carapace length was positive (r = 0.31, F1,63 = 27.94, P < 0.001) indicating that larger

females are still making larger eggs and clutches when corrected for the positive effect of

clutch size on egg size. This result is contrary to the typical trade off between egg size

and clutch size. Mean egg mass increased with each additional egg per clutch (r2 = 0.09,

F1,63 = 6.41, P = 0.01; Figure 4.3). Furthermore, an average increase of 8.5 mm in female

carapace length resulted in an additional egg per clutch, and increased individual egg

mass relates to only 4.04 % of the total increase in clutch mass.

Discussion

A positive correlation between egg size and clutch size was found in contrast to

the more typical negative relationship observed in most turtles. Thus, K. leucostomum

shows no evidence of a reproductive trade-off between egg and clutch size. A similar

positive relationship between egg size and female size occurs when egg size is limited by

the females pelvic opening, for example in the painted turtle, Chrysemys picta (Congdon

and Tinkle, 1982), and the slider turtle, Trachemys scripta (Congdon and Gibbons, 1983).

Yet, it is still regarded as an unusual reproductive pattern (reviewed in Congdon and

Gibbons, 1990), because there a negative correlation is more common (Congdon and

Gibbons, 1985). The small clutch size (1 - 5 eggs) in K. leucostomum suggests that there

may be two constraints affecting egg size and number, the pelvic aperture affecting egg

size and volumetric constraint due to the inflexibility of the carapace and plastron. No

small females were found with large clutches (3+ eggs). When controlling for body size,

larger females still produced more eggs.

Larger turtles in this study all had signs of advanced age, including heavily

melanistic shells and external soft tissues (Lovich et al., 1990), worn growth annuli or

excessive accumulation of growth annuli at the scute seems of the plastron (Moll and

Legler, 1971), and outward “bowing” of costal bones (Peter Pritchard, personal

communication). Although there was a wide range of female size, these observations

suggest that some of the largest turtles may be some of the oldest. It is not always true

that the largest females in the population are necessarily the oldest (Frazer et al. 1990).

Increased growth rates at an early age can overshadow the more gradual growth after

sexual maturity (Dunham and Gibbons, 1990; Congdon et al., 2003).

The positive correlation between egg size and female size was expected and not

surprisingly, egg length changed more rapidly than egg width. In addition, egg mass

increased at a greater rate with length rather than width suggesting female size constrains

egg width. The pelvic opening influences egg width, more readily than egg length and

can be a morphological constraint (Congdon and Gibbons, 1987). Egg length can be

variable due to yearly fluctuations in energy reserves devoted to reproduction (Congdon

and Gibbons, 1990); egg width should increase incrementally as female body size

increases (Congdon and Gibbons, 1987).

Excluding depredation and death due to increased energetic utilization during

prolonged development, death in otherwise healthy embryos is presumably related to

lethal mutations during embryogenesis and/or environmentally induced hardships. Turtle

embryos are susceptible to desiccation and excessive water uptake (Packard and Packard,

1988; Ackerman and Lott, 2004), but Kinosternid turtles have desiccation-resistant eggs

(Packard et al., 1984) due to thicker shells. Because of the thicker eggshell, they are less

able to absorb water after oviposition (Packard et al., 1982) and are thus highly dependent

on the initial egg water content. Larger eggs, with their corresponding higher water

content, and lower surface area to volume ratio (Packard, 1991), may have a selective

advantage during prolonged incubations if physiologically available water becomes a

limiting factor in development. Yet, no difference in survivorship among egg sizes was

observed, suggesting that the cause of embryonic death in this study was independent of

egg size – possibly cellular mutation or bacterial/fungal infections. Therefore, the

advantages of larger egg size may have a greater impact on post-emergence survival and

growth.

The clade Kinosternidae is comprised of approximately 22 species (Bonin et al.,

2006); all with very similar adult morphology, clutch size to female size relationships,

and egg size to female size relationships. Iverson and Ewert (1991) reported the egg

volume for K. leucostomum of unknown northern Central American origin (Mike Ewert,

personal communication) as 7.59 cm3 (N = 7), which falls midway in the range of the egg

volume found in this study. In addition, their reported mean egg length (37.17 mm) and

mean egg width (19.03 mm) were also within the range of egg sizes found in this study.

Similarly, clutch size reported here is not different from what Morales-Verdeja and Vogt

(1997) documented, nor did it vary greatly from the smaller non-ED expresser

Kinosternon subrubrum of the eastern deciduous US (mean clutch size = 3.0, range 1-5;

Gibbons et al., 1982). However, the clutch size of Kinosternon leucostomum found in

this study was less than the similarly sized Kinosternon sonoriense of the deserts of

southwestern United States and northwestern mainland Mexico (range 1-11; Ernst et al.,

1994). This species is also a known expresser of ED, with incubation periods of almost a

full year (Ewert, 1991). Hulse (1982) reports a positive correlation with female size and

egg size for K. sonoriense, however Gibbons et al. (1982) was not able to describe a

pattern between female size and clutch size in K. subrubrum. Thus, K. leucostomum is

more similar to K. sonoriense in regards to developmental timing and the relationship of

female size to egg size but more similar to K. subrubrum in clutch size.

Reproductive patterns of turtles fit into two broad types: those with few large

clutches of relatively small eggs, which are laid during a distinct nesting season (Type I)

and those with several small clutches of relatively large eggs, which are laid more or less

continuously (Type II; Moll, 1979; Moll and Moll, 2004). Type II reproduction is

predominantly observed in tropical turtles (Moll, 1979) while Type I nesters are

distributed globally. Tropical turtles (e.g., certain species of genera Acanthochelys,

Kinosternon, Phrynops, and Rhinoclemmys) known to be long-term historical residents

have derived a Type II reproductive strategy of “small clutches of large, brittle-shelled

eggs with long incubation periods” (Moll and Moll, 2004). Whereas recent radiation of

Trachemys into Meso-America from its temperate origins retain the Type II reproductive

characteristics; reviewed in Legler, 1990; see also Vogt, 1990. Trachemys scripta

venusta (Type I) is a medium-sized turtle (30 cm mean female carapace length; Moll and

Legler, 1971), which is sympatric with the much smaller K. leucostomum (Table 4.1;

Type II) in southern Mexico. Trachemys s. venusta nests within a two-month period

(clutch sizes ranging 5 – 22 eggs; ratio of egg length to female carapace length is 0.16

(3.81 cm/23.6 cm; Vogt, 1990)). In addition, T. s. venusta has the typical negative

correlation between egg size and female size (Moll and Legler, 1971) unlike the K.

leucostomum in this study. My results suggest that K. leucostomum has a Type II

reproductive strategy producing small clutches ranging 1 – 5 eggs with large eggs (mean

egg length 3.41 cm) relative to female size (mean female carapace 14.0 cm) and they

have a protracted 10 month nesting season (Morales-Verdeja and Vogt 1997).

Until now, no physiological mechanism has been proposed for the prolonged nesting

periods in Type II species. However, a compromise between the energetic needs of the

embryo and the hatchling may provide some insight. Egg yolk is composed of high-

energy lipids that are converted into new tissues throughout embryogenesis (Ewert, 1979;

Congdon et al., 1983b). Eggs must contain enough yolk for both development and

maintenance of all embryonic tissues (Ar et al., 2004). Although, turtles generally do not

exhibit parental care beyond nest construction, females allocate more yolk per egg than

necessary for embryonic development (Nagle et al., 1998). Yolk not used before

hatching is retained within the hatchling as residual energy reserves and can be used for

post-paritive lecithotrophy. Thus, increases in egg size and the subsequent increases in

PIC are not similar to what Nagle et al. (2003) documented in Apalone mutica, which

emerge into a nutrient-poor system. Furthermore, Nagle et al. (1998) reported that

hatchling emergence strategies in addition to developmental factors (e.g., temperature,

date of oviposition, and metabolic rates) affect Kinosternid parental investment. The

relationship between egg size and clutch size in K. leucostomum is weakened by the

embryos ability to arrest development during extended incubation periods by expressing

ED, thereby conserving PIC. I suggest that ED is an energy conservation mechanism in

Type II turtles that reduces energy expenditure for development (Congdon, 1989;

Congdon and Gibbons, 1990), resulting in more energy for PIC. The extended nesting

season facilitated by ED allows for more reproductive bouts compared to Type I

congeners perhaps resulting in similar annual reproductive output between the strategies.

Future research should exam female age, clutch frequency, and annual individual

variation in egg/clutch size amongst reproductive events to understand the advantages of

reproducing at smaller body sizes relative to delaying maturity to reach a larger female

body size (Gibbons et al., 1982; Congdon and Gibbons, 1990). Conceivably the

reproductive biology of K. leucostomum will be considered typical when we develop a

greater understanding of the life histories of turtles with Type II reproductive strategies.

Literature Cited Ackerman, R. A. 1991. Physical factors affecting the water exchange of buried reptile eggs. In D. C. Deeming, and M. W. J. Ferguson (ed.), Egg Incubation: Its Effects on Embryonic Development in Birds and Reptiles, pp. 193-211. Cambridge University Press: Cambridge, United Kingdom.

Ackerman, R. A., and D. B. Lott. 2004. Thermal, hydric, and respiratory climate of nests. In D. C. Deeming (ed.), Reptilian Incubation: Environment, Evolution, and Behavior, pp. 15-45. Nottingham University Press, United Kingdom of Great Britain..

Ackerman, R. A., R. Dmi’el, and A. Ar. 1985. Energy and water vapor exchange by parchment-shelled reptile eggs. Physiological Zoology 58:129-137.

Ades, G., C. B. Banks, K. A. Buhlmann, B. Chan, H. Chang, T. Chen, P. Crow, H. Haupt, R. Kan, J. Lai, M. Lau, H. Lin, S. Haitao, P. P. van Dijk, B. L. Stuart, and A. G. J. 2000. Turtle trade in Northeast Asia: Regional summary (China, Hong Kong, and Taiwan). Chelonian Research Monographs 2:52-54.

Ahsam, F., and A. Saeed. 1992. Some aspects of the breeding biology of the black softshell turtle, Aspideretes nigricans. Hamadryad 17:28-31.

Alvarez del Toro, M. 1960. Los Reptiles de Chiapas. Tuxtla Gutierrez, Chiapas, Mexico.

Anderson, N. J., and B. D. Horne. (in press, 2007). Observations on the Nesting Ecology of the Mississippi Mud Turtle, Kinosternon subrubrum hippocrepis Lacepède. Southeastern Naturalist.

Andrews, R. M., and S. Donoghue, 2004. Effects of temperature and moisture on embryonic diapause of the veiled chameleon (Chamaeleo calyptratus). Journal of Experimental Zoology 301A (8):629-635.

Ar, A., A. Belinsky, R. Dmi’el, and R. A. Ackerman. 2004. Energy provision and utilization. In D. C. Deeming (ed.), Reptilian Incubation: Environment, Evolution, and Behavior, pp. 144-186. Nottingham University Press, United Kingdom.

Artner, H. 1995. Welt-Erstnachzucht der Fujian-Sumpfschildkröte, Mauremys iversoni. EMYS, Vereinszeitschrift der Schildkrötenfreunde Österreich 2(4/2):5.

Artner, H. 2006a. Keeping and breeding of Pan’s box turtle Cuora pani pani Song, 1984 and Cuora pani aurocapitata Luo and Zong, 1988. In H. Artner, B. Balazas, and V. Loehr (eds), Turtles, pp. 52-68. Proceedings: International Turtle and Tortoise Symposium Vienna 2002.

Artner, 2006b. The Antilean sliders of the genus Trachemys Agassiz, 1857. In H. Artner, B. Balazas, and V. Loehr (eds.), Turtles, pp. 298-327. Proceedings: International Turtle and Tortoise Symposium Vienna 2002.

Baard, E. 1994. Cape Tortoises; Their Identification and Care. Cape Nature Conservation, Cape Town, South Africa.

Baker, J. R. 1938. The evolution of breeding seasons. In G. R. de Beer (ed.), Evolution:Essays on Aspects of Evolutionary Biology, pp. 161-177. Oxford University Press, London, United Kingdom.

Berry, J. F., and J. B. Iverson. 2001. Kinosternon leucostomum (Dumeril and Bibron) White Lipped Mud Turtle. Catalogue of American Amphibians and Reptiles 724.1-724.8.

Berry, J. F., M. E. Seidel, and J. B. Iverson. 1997. A new species of mud turtle (genus Kinosternon) from Jalisco and Colima, Mexico, with notes on its natural history. Chelonian Conservation Biology 2:329-337.

Birchard, G. F., 2004. Effects of incubation temperature. In D. C. Deeming (ed.), Reptilian Incubation: Environment, Evolution, and Behavior, pp. 103-124. Nottingham University Press, United Kingdom.

Blanc, F. 1974. Table de développement de Chamaeleo lateralis Gray, 1831. Annalesd’Embryologie et de Morphogénèse 7:99-115.

Bonin, F., D. Bernard, and A. Dupre. 2006. Turtles of the World. Translated by P. C. H. Pritchard. John Hopkins University Press, Baltimore, Maryland, United States of America.

Booth, D. T. 1999. Incubation temperature and growth of the Brisbane river turtle (Emydura signata) hatchlings. Proceedings of the Linnaean Society of New South Wales 121:45-52.

Booth, D. T. 2000. Incubation of eggs of the Australian broad-shelled turtle, Chelodina expansa (Testudinata: Chelidae), at different temperatures: effects on pattern of oxygen consumption and hatchling morphology. Australian Journal of Zoology 48(4):369-378.

Booth, D. T. 2002. The breaking of diapause in embryonic broad-shelled turtle, Chelodina expansa (Testudinata: Chelidae), at different temperatures: effects on pattern of oxygen consumption and hatchling morphology. Australian Journal of Zoology 48:369-378.

Booth, D. T. 2003. Composition and density of eggs from two species of freshwater turtles with twofold ranges in egg size. Comparative Biochemistry and Physiology Part A.134:129-137.

Bour, R. 1983. Trois populations endémiques du genre Pelusios (Reptilia, Chelonii, Pelomedusidae) aux îles Seychelles; relations avec les espèces africaines et malgaches. Bulletion of the National Natural History Museum.Paris, France (4)5: 343-382.

Bourret, R. 1941. Les Tortues de l'Indochine. Institute of Oceanography l'Indochine.

Boycott, R. C. 1989. Homopus signatus. Namaqualand speckled padloper, Peer's padloper (for southern race) (English); Gifskilpadjie, Klipskilpadjie, Namaqualandklip-skilpadjie (Afrikaans). In I. R. Swingland, and M. W. Klemens (eds.), The Conservation Biology of Tortoises, pp. 82-84. Occasional Papers. IUCN/SSC 5, Tortoise and Freshwater Specialists Group. IUCN, Gland, Switzerland.

Branch, W. R. 1988. Bill Branch's Field Guide to the Snakes and Other Reptiles of Southern Africa. New Holland Publishers, London, England.

Branch, W. R. 1989. Homopus femoralis. Greater padloper, Karoo tortoise (English); Vlakskilpad, Bergskilpadjie, Groter padloper (Afrikaans). In I. R. Swingland, and M. W. Klemens (eds.), The Conservation Biology of Tortoises, pp. 80-81. Occasional Papers. IUCN/SSC 5, Tortoise and Freshwater Specialists Group. IUCN, Gland, Switzerland.

Boycott, R. C., and O. Bourquin. 2000. The Southern African Tortoise Book: A guide to Southern African Tortoises, Terrapins, and Turtles. Privately published by O. Bourquin. KwaZulu-Natal, South Africa.

Braudeau E., M. Sene, and R. H. Mohtar. 2005. Hydrostructural characteristics of two African tropical soils. European Journal of Soil Science 56(3):375-388.

Broadley, D. G. 1981. A review of the genus Pelusios Wagler in southern Africa (Pleurodira: Pelomedusidae). Occasional Papers National Museum of Rhodesia, B, Natural Science 6:633-686.

Broadley, D. G. 1989. Kinixys natalensis. Natal hinged tortoise. In I. R. Swingland, and M. W. Klemens (eds.), The Conservation Biology of Tortoises, pp. 60-61. Occasional Papers. IUCN/SSC 5, Tortoise and Freshwater Specialists Group. IUCN, Gland, Switzerland.

Broadley, D. G. 1993. A review of southern African species of Kinixys Bell (Reptilia: Testudinidae). Annual of the Transvaal Museum 36:41-52.

Brown, W. S. 1974. Ecology of the aquatic box turtle, Terrapene coahuila (Chelonia, Emydidae) in northern Mexico. Bulletin of the Florida State Museum of Biological Sciences 19:1-67.

Bruin, R. de. 2006. Breeding the Indochinese box turtle Cuora galbinifrons galbinifrons. In H. Artner, B. Balazas, and V. Loehr (eds.), Turtles, pp. 69-78. Proceedings: International Turtle and Tortoise Symposium Vienna 2002.

Buhlmann, K. A. 1998. Ecology, terrestrial habitat use, and conservation of a freshwater turtle assemblage inhabiting a seasonally fluctuating wetland with emphasis on the life history of Deirochelys reticularia. Ph.D. Diss. University of Georgia.

Bull, J. J., and R. C. Vogt. 1979. Temperature-dependent sex determination in turtles. Science 206:1186-1188.

Burger, J. 1977. Determinants of hatching success in diamondback terrapin, Malaclemys terrapin. American Midland Naturalist 97:444-464.

Burke, V. J., J. W. Gibbons, and J. L. Greene. 1994. Prolonged nesting forays by common mud turtles (Kinosternon subrubrum). American Midland Naturalist 131(1):190-195.

Cabrera, M. R. 1998. las Tortugas Continentales de Sudameerica Austral. Privately printed, Córdoba, Argentina, 108, 6 plates.

Cairncross, B. L., and J. C. Greig. 1977. Note on variable incubation period within a clutch of eggs of the leopard tortoise (Geochelonia paradalis) (Chelonia: Cryptodira: Testudinidae). Zoologica Africana 12:255-256.

Cagle, F. R. 1952. The status of the turtles Graptemys pulchra Baur and Graptemys barbouri Carr and Marchand, with notes on their natural history. Copeia 1952: 223-234.

Caldwell, J. P., and J. T. Collins. 1981. Turtles in Kansas. AMS Publishers, Lawrence, Kansas.

Cameron, M. 2004. Evolutionary Significance of Embryonic Diapause in Turtles. Unpublished Thesis. University of Guelph. Guelph, Ontario, Canada.

Campbell, G. S., 1985. Soil Physics with BASIC: Transport Models for Soil Plant Systems. Elsevier, Amsterdam, Netherlands.

Cann, J. 1998. Australian Freshwater Turtles. Beaumont Publishing. Singapore, China.

Cansdale, G. 1955. Reptiles of West Africa. Penguin Books, London, England.

Carpenter, G. C. 1960. Reproduction in Oklahoma Sceloporus and Cnemidophorus. Herpetologica 16:175-182.

Carr, J. L., and R. B. Mast. 1988. Natural history observations of Kinosternon herrerai (Testudines: Kinosternidae). Trianea (Acta Ciencia Tecnologia Inderena) 1:87- 97.

Castano-Mora, O.V. 1986. Contribution to the study of the reproduction of Podocnemis lewyana Dumeril Reptilia Chelonia Pelomedusidae. Caladasia 15:665-668.

Cathey, H. M. 1990. USDA plant hardiness zone map. United States Department of agriculture. Miscellaneous Publication (1475). United States National Arboretum, Agriculture Research Service, Washington, DC.

Christiansen, J. L., and A. E. Dunham. 1972. Reproduction of the yellow mud turtle (Kinosternon flavescens flavescens) in New Mexico. Herpetologica 28:130-137.

Cintra, R., and C. Yamashita, 1989. Notes on the nesting ecology of Platemys macrocephala in the Brazilian Pantanal. Herpetological Review 20(3):65-66.

Congdon, J. D. 1989. Proximate and evolutionary constraints on energy relations of reptiles. Physiological Zoology 62(2):356-373.

Congdon, J. D., and J. W. Gibbons. 1983. Relationship of reproductive characteristics to body size in Pseudemys scripta. Herpetologica 39:147-151.

Congdon, J. D., and J. W. Gibbons, 1985. Egg components and reproductive characteristics of turtles: relationship to body size. Herpetologica 4(2):194-205.

Congdon, J. D., and J.W. Gibbons. 1987. Morphological constraint on egg size: A challenge to optimal egg size theory? Proceedings of the National Academy of Sciences of the United States of America. 84:4145-4147.

Congdon, J. D., and J. W. Gibbons. 1990. Turtle eggs: their ecology and evolution. In J.W. Gibbons (ed.), Life History and Ecology of the Slider Turtle, pp. 109-123. Smithsonian Institution Press, Washington, D. C.

Congdon, J.D., and D. W. Tinkle. 1982. Reproductive energetics of the painted turtle(Chrysemys picta). Herpetologica 38:228-237.

Congdon, J. D., J. W. Gibbons, and J. L. Greene. 1983a. Parental investment in the chicken turtle (Deirochelys reticularia). Ecology 64:419-425.

Congdon, J. D., D. W. Tinkle, and P. C. Rosen. 1983b. Egg components and utilization during development in aquatic turtles. Copeia 1:264-268.

Congdon, J. D., R. D. Nagle, A. E. Dunham, C. W. Beck, O. M. Kinney, and R. S. Yeomans. 1999. The relationship of body size to survivorship of hatchling snapping turtles (Chelydra serpentina): An evaluation of the “bigger is better” hypothesis. Oecologia 121(1):224-235.

Congdon, J. D., R. D. Nagle, O. M. Kinney, R. C. Van Loben Sels, T. Quinter, and D. W. Tinkle. 2003. Testing hypotheses of aging in long-lived painted turtles (Chrysemys picta). Experimental Gerontology 38(7):765-772.

Das, I. 1995. Turtles and Tortoises of India. Oxford University Press, Bombay, India.

Davidowitz, G. 2002. Does precipitation variability increase from mesic to xericbiomes? Global Ecology and Biogeography 11(2):143-154.

Deeming, D. C., and M. W. J. Ferguson. 1991. Physiological effects of incubation temperature on embryonic development in reptiles and birds. In D. C. Deeming and M. W. J. Ferguson (eds.), Egg Incubation: Its Effects on Embryonic Development in Birds and Reptiles, pp. 147-171. Cambridge University Press, Cambridge, United Kingdom.

De La Fuente, M. S. 2003. Two new Pleurodiran turtles from the Portezuelo Formation (Upper Cretaceous) of Northern Patagonia, Argentina. Journal of Paleontology 77:559-575.

Delaney, R. 1997. Reproductive ecology of the allied rock-wallaby, Petrogale assimilis. Australian Mammalogy 19(2):209-218.

Denlinger, D. L. 1985. Hormonal control of diapause. In G. A. Kekurt and L. I. Gilbert (eds.), Comprehensive Insect Physiology, Biochemistry, and Pharmacology, Vol 8, pp. 353-412. Pergamon Press, Oxford, United Kingdom.

Denlinger, D. L. 1998. Maternal control of fly diapause. In T. A. Mousseau and C. W. Fox (eds.), Maternal Effects as Adaptations, pp. 275-287. Oxford University Press, New York.

Dhruvajyoti, B. 1998. Female reproductive cycle in Hardella thurjii Gray from Northern India. Hamadryad. 22(2):95-106

Dobie, J. L. 1971. Reproduction and growth in the alligator snapping turtle, Macroclemys temminckii (Troost). Copeia 1971:645-658.

Dodd, C. K., Jr. 1988. Synopsis of the biological data on the loggerhead sea turtle Caretta caretta (Linnaeus 1758). U. S. Fish and Wildlife Service. Biology Report 88(14).

Doody, J. S. 1999. A test of the comparative influences of constant and fluctuating incubation temperatures on the phenotypes of hatchling turtles. Chelonian Conservation and Biology 3:529-531.

Doody, J. S., A. Georges, and J. E. Young. 2003. Twice every second year: reproduction in the pig-nosed turtle, Carretochelys insculpta, in the wet-dry tropics of Australia. Journal of Zoology (London) 259(2):179-188.

Doody, J. S., A. Georges, J. Young, M. Pauza, A. Pepper, R. Alderman, L. Rachael, and M. Welsh. 2001. Embryonic aestivation and emergence behaviour in the pig- nosed turtle, Carettochelys insculpta. Canadian Journal of Zoology 79(6):1062- 1072.

Doody, J. S., A. Georges, and J. E. Young. 2003. Twice every second year: reproduction in the pig-nosed turtle, Carettochelys insculpta, in the wet-dry tropics of Australia. Journal of Zoology (London) 259(2):179-188.

Dunham, A. E., and J. W. Gibbons. 1990. Growth of the slider turtle. In J. W. Gibbons (ed.), Life History and Ecology of the Slider Turtle, pp. 135-145. Smithsonian Institution Press, Washington, D.C.

Dunham, A. E., and D. M. Miles. 1985. Patterns of covariation in life history traits of squamate reptiles: the effects of size and phylogeny reconsidered. American Naturalist 126:231-257.

Eglis, A. 1962. Tortoise behavior: a taxonomic adjunct. Herpetologica 18:1-8.

Einem, G. E. 1956. Certain aspects of the natural history of the mud turtle, Kinosternon baurii. Copeia 2:186-188.

Elgar, M. A., and L. J. Heaphy. 1989. Covariation between clutch size, egg weight and shape evidence for chelonians. Journal of Zoology 219:137-152.

Elmslie, L. J. 2001. Strain differences in Helix pomatia L. (Gastropoda: Pulmonata): Diapause, digging, growth rate, and shell fill. Journal of Molluscan Studies 67(1):121-124.

Ernst, C. H. 1992. Trachemys gaigeae. Catalog of American Amphibians and Reptiles 538:1-4.

Ernst, C. H., and R. W. Barbour. 1989. Turtles of the World. Smithsonian Institution Press. Washington, D. C.

Ernst, C. H., J. E. Lovich, and R. W. Barbour. 1994. Turtles of the United States and Canada. Smithsonian Institution Press, Washington, D.C.

Ernst, C.H., R. G. M. Altenburg, R.W. Barbour. 1997. Turtles of the World (CD-ROM). Springer Publishing: 1st edition.

Evans, M. E. K., and J. J. Dennehy. 2005. Germ banking: bet-hedging and variable release from egg and seed dormancy. The Quarterly Review of Biology 80:431- 451.

Ewert, M. A. 1979. The embryo and its egg: development and natural history. In M. Harless and H. Morlock (eds.), Turtles: Perspectives and Research, pp. 333-413. Wiley, New York.

Ewert, M. A. 1985. Embryology of turtles. In: C. Gins, F. Billet, and P. F. A. Maderson (eds.), Biology of the Reptilia, Vol 14, pp 75-267. Wiley, New York.

Ewert, M. A. 1991. Cold torpor, diapause, delayed hatching and aestivation in reptiles and birds, In D. C. Deeming, and M. W. J. Ferguson (eds.), Egg Incubation: Its Effects on Embryonic Development in Birds and Reptiles, pp.173-191. Cambridge, United Kingdom.

Ewert, M. A., and J. M. Legler. 1978. Hormonal induction of oviposition in turtles. Herpetologica 34:314-318.

Ewert, M. A., and C. E. Nelson. 1991. Sex determination in turtles: diverse patterns and some possible adaptive values. Copeia 1991:50-69.

Ewert, M. A., and D. S. Wilson. 1996. Seasonal variation of embryonic diapause in the striped mud turtle (Kinosternon baurii) and general considerations for conservation planning. Chelonian Conservation Biology 2(1): 43-54.

Falconer, D. S. 1965. Maternal effects and selection response. In J. Geerts (ed.), Genetics Today, pp. 763-774. Pergamon Press, Oxford, United Kingdom.

Falconer, D. S. 1989. Introduction to Quantitative Genetics. Wiley, New York.

Farrell, R. F., and T. E. Graham. 1991. Ecological notes on the turtle, Clemmys insculpta in northwestern New Jersey. Journal of Herpetology 25:1-9.

Feldman, M. 1982. Notes on reproduction in Clemmys marmorata. Herpetological Review 13:10-11.

Flatt, T., R. Shine, P.A. Borges-Landaez, and S. J. Downes. 2001. Phenotypic variation in an oviparous montane lizard (Bassiana duperreyi): the effects of thermal and hydric incubation environments. Biological Journal of the Linnean Society 74(3):339-350.

Flores-Villela, O. A., and G. R. Zug. 1995. Reproductive biology of the chopontil, Claudius angustatus (Testudines: Kinosternidae), in Southern Veracruz, Mexico. Chelonian Conservation Biology 1:181-186.

Frazer, N. B. 1992. Sea turtle conservation and halfway technologies. Conservation Biology 6:179-184.

Frazer, N. B., J. W. Gibbons, and J. L. Greene. 1990. Life tables of a slider turtle population. In J. W. Gibbons (ed.), Life History and Ecology of the Slider Turtle, pp. 183-200. Smithsonian Institution Press, Washington, D.C.

Freeman, S., and J. C. Heron. 1998. Evolutionary Analysis. Prentice-Hall, Inc. New Jersey.

Freiberg, M. A. 1967. Tortugas de la Argentina. Cienc. Invest. 23: 351-363.

Freiberg, M. A. 1981. Turtles of South America. T. F. H. Publishers, Inc., Neptune, New Jersey.

Fuduka, S. 1952. Function of the pupal brain and subesophageal ganglion in the production of non-diapause and diapause eggs in the silkworm. Annotated Zoology 149-155. Japan.

Fukada, H. 1965. Breeding habits of some Japanese reptiles (critical review). Bull. Kyoto Gak University Service B 27:65-82.

Gause, G. F. 1932. Experimental studies on the struggle for existence. I. Mixed population of two species of yeast. Journal of Experimental Biology 9:389-402.

Georges, A., C. Limpus, and R. Stoutjesdijk. 1994. Hatchling sex in the marine turtle Caretta caretta is determined by proportion of development at a temperature, not daily duration of exposure. Journal of Experimental Zoology 270(5): 432-444.

Georges, A., K. Beggs, J. E. Young, and J. S. Doody. 2005. Modelling development of reptile embryos under fluctuating temperature regimes. Physiological and Biochemical Zoology 78(1):18-30.

Gibbons, J. W., and D. H. Nelson. 1978. The evolutionary significance of delayed emergence from the nest by hatchling turtles. Evolution 32(2):297-303.

Gibbons, J. W., and J. L. Greene, 1990. Reproduction in the slider and other species of turtles. In J. W. Gibbons (ed.). Life History and Ecology of the Slider Turtle, pp. 124-134. Smithsonian Institution Press, Washington. D.C.

Gibbons, J. W., J. L. Greene, and K. K. Patterson. 1982. Variation in reproductive characteristics of aquatic turtles. Copeia (4):776-784.

Gidis, M., and Y. Kaska. 2004. Population size, reproductive ecology and heavy metal levels in eggshells of the Nile soft-shell turtle (Trionyx triunguis) around thermal Lake Kuekuertlue (sulphurous), Mugla-Turkey. Fresenius Environmental Bulletin Vol. 13, no. 5 pp. 405-412.

Gilbert, S. F. 1998. Conceptual breakthroughs in developmental biology. Journal of Bioscience 23:169-176.

Gilbert, S. F. 2001. Ecological developmental biology: developmental biology meets the real world. Developmental Biology 233:1-12.

Gilbert, S. F., J. M. Opitz, and R. A. Raff. 1996. Resynthesizing evolutionary and developmental biology. Developmental Biology 173:357-372.

Giovanni, G. 2005. Elevage et reproduction de la Platamyde de l'Amazone, Batrachemys raniceps (Gray, 1855). Manouria 8 (27):27-32.

Godfray, H. C. J., and G. A. Parker. 1991. Clutch size, fecundity, and parent-offspring conflict. Philosophical Transmissions of the Royal Society B. 332:67-79.

Goff, C. C. and D. S. Goff, 1935. On the incubation of a clutch of eggs of Amyda ferox (Schneider). Copeia 1935:156.

Grime, J. P. 1977. Evidence for the existence of three primary strategies in plants and its relevance to ecological and evolutionary theory. American Naturalist 111:1169- 1194.

Grime, J. P. 1979. Plant Strategies and Vegetation Processes. Wiley, New York.

Grossmann, P., and M. Reimann. 1991. Beiträge zum Verhalten und Nachzucht von Phrynops tuberculatus (Lüderwaldt 1926) im Aqua-Terrarium. Sauria 13(2):3-6.

Gutzke, W. H. N., G. C. Packard, M. J. Packard, and T. J. Boardman. 1987. Influence of the hydric and thermal environment on eggs and hatchling of painted turtles (Chrysemys picta). Herpetologica 43:393-404.

Haagner, G. V. 1994. Chelonii, Pelomedusidae, Pelusios nanus African dwarf hinged terrapin. Reproduction. African Herpetological News 21:18.

Hairston, N. G., Jr., and W. R. Munns. 1984. Timing of copepod diapause as an evolutionary stable strategy. American Naturalist 123:733-751.

Hand, S. C. 2000. Induction of quiescence and diapause during life cycles of aquatic invertebrates: mechanisms and implications. American Zoologist 40(6): 1044- 1045.

Hand, S. C., and J. E. Pobrasky. 2000. Bioenergetics of diapause and quiescence in aquatic animals. Thermochimica acta 349:31-42.

Harvel, C. D. 1990. The ecology and evolution of inducible defenses. Quarterly Review of Biology 65(3):323-340.

Hasegawa, K. 1952. Studies on the voltinism of the silkworm, Bombyx mori L., with special reference to the organs controlling determination of voltinism. Journal of Facilitative Agricultural 1:83 -124. Tottori University, Japan.

Hofstra, J. 1995. Kweken met de moerasschildpad, Mauremys mutica. Lacerta 53(6): 187-191.

Honegger, R. E. 1986. Zur Pflege und langjährigen Nachzucht von Siebenrockiella crassicollis (Gray, 1831). (Testudines: Emydidae). Salamandra 22(1):1-10.

Horne, B. D. 1993. Courtship behavior and oviposition of captive Acanthochelys pallidipectoris Freiberg. Journal of Herpetology 24(1):25.

Horne, B. D., R. J. Brauman, M. J. C. Moore, and R. A. Seigel. 2004. Reproductive and nesting ecology of the yellow-blotched map turtle, Graptemys flavimaculata: implications for conservation and management. Copeia 4:729-738.

Howe, H. F. 1978. Initial investment, clutch size, and brood reduction in the common grackle (Quisculus quiscula L.) Ecology 59:1109-1112.

Huffaker, C. B. 1958. Experimental studies on predation: dispersion factors and predator- prey oscillations. Hilgardia 27:343-383.

Hulse, A. C. 1982. Reproduction and population structure in the turtle, Kinosternon sonoriense. Southwest Naturalist 27:447- 456.

Inchaustegui Miranda, S. J. 1973. Las tortugas Dominicanas de agua dulce Chrysemys decussata vicina y Chrysemys decorata (Testudinata, Emydidae). Thesis, University of Autonmous. Santo Domingo, Dominican Republic.

Ireland, L. C., and C. Gans. 1972. The adaptive significance of the flexible shell of the tortoise, Malacocherus tornieri. Animal Behavior 20(4);778-781.

Iverson, J. B. 1980. The reproductive biology of Gopherus polyphemus (Chelonia: Testudinidae). American Midland Naturalist 103:353-359.

Iverson, J. B. 1988. Distribution and status of Creaser's mud turtle, Kinosternon creaseri. Herpetological Journal 1(7):285-291.

Iverson, J. B. 1989. Natural history of the Alamos mud turtle, Kinosternon alamosae (Kinosternidae). Southwestern Naturalist 34:134-141.

Iverson, J. B. 1991. Phylogenetic hypotheses for the evolution of modern Kinosternine turtles. Herptologica Monograph 5:1-27.

Iverson, J. B. 1992. A Revised Checklist with Distribution Maps of the Turtles of the World. Privately printed. Richmond, Indiana.

Iverson, J. B., and M. A. Ewert, 1991. Physical characteristics of reptilian eggs and a comparison with avian eggs. In D. C. Deeming, and M. W. J. Ferguson (ed.). Egg Incubation: Its Effects on Embryonic Development in Birds and Reptiles. pp. 87- 100. Cambridge University Press. Cambridge, United Kingdom.

Iverson, J. B., E. L. Barthelmess, G. R. Smith, and C. E. DeRivera. 1991. Growth and reproduction in the mud turtle Kinosternon hirtipes in Chihuahua, Mexico. Journal of Herpetology 25:64-72.

Jackson, D. R. 1988. Reproductive strategies of sympatric freshwater emydid turtles in northern peninsular Florida. Bulletin of the Florida State Museum Biological Sciences 33(3):113-158.

Janzen, F. J. 1995. Experimental evidence for the evolutionary significance of temperature-dependent sex determination. Evolution 49(5):864-873.

Janzen, F. J., and G. L. Paukstis. 1991. A preliminary test of the adaptive significance of environmental sex determination in reptiles. Evolution 45: 435-440.

Janzen, F. J., J. K. Tucker, and G. L. Paukstis. 2000. Experimental analysis of an early life-history stage: Avian predation selects for larger body size of hatchling turtles. Journal of Evolutionary Biology 13(6):947-954.

Jones, R. L., T. C. Majure, and K. R. Macaro. 1991. Graptemys kohnii (Mississippi map turtle). Herpetological Review 22:24-25.

Judd, F. W., and J. C. McQueen. 1980. Incubation, hatching, and growth of the tortoise, Gopherus berlandieri. Journal of Herpetology 14:377-380.

Kempthorne, O. 1969. An Introduction to Genetics Statistics. Iowa State University Press, Ames.

Kennett, R., A. Georges, and M. Palmer-Allen. 1993. Early developmental arrest during immersion of eggs of a tropical freshwater turtle, Chelodina rugosa. (Testudinata: Chelidae), from Northern Australia. Australian Journal of Zoology 41:37-45.

Kingslover, J. G., and R. B. Huey. 1998. Evolutionary analysis of morphological and physiological plasticity in thermally variable environments. American Zoologist 38:545-560.

Kitimasak, W., K. Thirakhupt, and D. L. Moll. 2003. Eggshell Structure of the Siamese Narrow-headed Softshell Turtle Chitra chitra Nutphand, 1986 (Testudines: Trionychidae). ScienceAsia 29:95-98.

Klerks, M. 2006. Keeping and breeding the Chinese box turtle, Cuora flavomarginata (Gray, 1863). In: H. Artner, B. Balazas, and V. Loehr (Eds.): Turtles, pp.13-17. Proceedings: International Turtle and Tortoise Symposium Vienna 2002.

Kuchling, G. 1999. The Reproductive Biology of the Chelonia. Springer-Verlag Berlin Heidelberg. Germany.

Lacey, E. P. 1998. What is an adaptive environmentally induced parental effect? In T. A. Mousseau and C. W. Fox. (eds.), Maternal Effects as Adaptations, pp. 54-66. Oxford University Press, New York.

Lack, D. 1966. Population Studies of Birds. Oxford University Press, New York.

Lahanas, P. N. 1982. Aspects of the life history of the southern black-knobbed sawback, Graptemys nigrinoda delticola Folkerts and Mount. Master's thesis, Auburn University, Auburn, Alabama.

Le, M.D. 2006 Systematics, Biogeography, and Conservation Status of the Turtle Family Geoemydidae. Ph.D. Diss. Columbia University, New York.

Legler, J. M. 1966. Notes on the natural history of a rare Central American turtle, Kinosternon angustipons Legler. Herpetologica 22:118-122.

Legler, J. M. 1990. The genus Pseudemys in Mesoamerica: taxonomy, distribution, and origins. In: J. W. Gibbons (ed.), Life History and Ecology of the Slider Turtle, pp 82-105. Smithsonian Institution Press, Washington D.C.

Legler, J. M., and R. G. Webb. 1961. Remarks on a collection of Bolson tortoises, Gopherus flavomarginatus. Herpetologica 17:26-37.

Leshem, A., A. Ar, and R. A. Ackerman. 1991. Growth water and energy metabolism of the soft-shelled turtle Trionyx triunguis embryo effects of temperature. Physiological Zoology 64(2): 568-594.

Lloyd, D. G. 1988. A general principle for the allocation of limited resources. Evolutionary Ecology 2:175-187.

Long, D. R. 1986. Lipid content and delayed emergence of hatchling yellow mud turtles. Southwestern Naturalist 31:244-246.

Lotka, A. J. 1925. Elements of Physical Biology. Williams and Wilkins, Baltimore, Maryland.

Loveridge, A. and E. E. Williams. 1957. Revision of the African tortoises and turtles of the suborder Cryptodira. Bulletin of the Museum of Comparative Zoology. Harvard 115:163-557.

Lovich, J. E., C. J. McCoy, and W. R. Garstka. 1990. The development and significance of melanism in the slider turtle. In J. W. Gibbons (ed.), Life History and Ecology of the Slider Turtle, pp. 233-256. Smithsonian Institution Press, Washington, D.C.

MacArthur, R. H. 1958. Population ecology of some warblers of northeastern coniferous forests. Ecology 39:599-619.

Mahapatra, M. S., S. K. Mahata, and B. R. Maiti. 1986. Circadian rhythms in serotonin, norepinehrine, and epinephrine contents of the pineal-paraphyseal complex of the soft-shelled turtle (Lissemys punctata punctata). General Comparative Endocrinology 74:215-220.

Maran, J. 2006. Observations on Gabonese chelonians. In H. Artner, B. Balazas, and V. Loehr (eds.), Turtles, pp. 351-373. Proceedings: International Turtle and Tortoise Symposium Vienna 2002.

Márquez M., R. 1990. FAO species catalogue. Vol. 11: Sea turtles of the world. An annotated and illustrated catalogue of sea turtle species known to date. FAO Fisheries Synopsis. No 125, Vol. 11. Rome, FAO.

Masaki, S. 2002. Ecophysiological consequences of variability in diapause intensity. European Journal of Entomology 99(2):143-154.

McKeown, S., and R. G. Webb. 1982. Softshell turtles in Hawaii. Journal of Herpetology 16:107-111.

Medem, F. 1960. Datos zoo-geográficos y ecologícos sobre los Crocodylia y Testudinata de los rios Amazonas, Putumayo y Caqueta. Caldasia 8:341-351.

Medem, F. 1961. Contribuciónes al conocimiento sobre la morfología, ecología y distribución geográfica de la tortuga Kinosternon dunni K. P. Schmidt. Novedades Colombianas 1:446-476.

Medem, F. 1962. La distribución geográfica y ecología de los Crocodylia y Testudinata en el Departamento del Choco. Revista Academica de Colombia Ciencia Ex Fisiologia Natural. 11:279-303.

Medem, F. 1966. Contribuciónes al conocimiento sobre la ecología y distribución geográfica de Phrynops (Batrachemys) dahli; (Testudinata, Pleurodira, Chelidae). Caldasia 9:467-489.

Medem, F. 1983. Reproductive data on Platemys platycephala (Testudines: Chelidae) in Colombia. In: A. G. J. Rhodin and K. Miyata (eds.), Advances in Herpetology and Evolutionary Biology, pp. 429-434. Museum of Comparative Zoology. Harvard,

Menu, F., J. P. Roebuck, and M. Viala. 2000. Bet-hedging diapause strategies in stochastic environments. American Naturalist 155 (6):724-734.

Milstead, W. W., and D. W. Tinkle. 1967. Terrapene of western Mexico, with comments on the species groups in the genus. Copeia 1967:180-187.

Miroslaw, M., P. Dawidowicz, and E, Rygielska. 2005. Hide, rest, or die: a light- mediated diapause response in Daphnia magna to the threat of fish predation. Freshwater Biology 50(1):141-146.

Mittermeier, R. A., and R. A. Wilson. 1974. Redescription of Podocnemis erythrocephala (Spix, 1824), an Amazonian pelomedusid turtle. Papeis Avulusos de Zoologia, Sao Paulo, Brazil 28:147-162.

Moll, D., and E. O. Moll, 1990. The slider turtle in the neotropics: adaptation of a temperate species to a tropical environment. In J. W. Gibbons (ed.), Life History and Ecology of the Slider Turtle, pp. 152-161. Smithsonian Institution Press, Washington, D.C.

Moll, D., and E. O. Moll. 2004. The Ecology, Exploitation, and Conservation of River Turtles. Oxford University Press, New York.

Moll, E. O. 1979. Reproductive cycles and adaptations. In M. Harless and H. Morlock (eds.), Turtles: Perspectives and Research, pp. 305-332. Krieger Publishing Company, Florida.

Moll, E. O., and J. M. Legler. 1971. The life history of a neotropical slider turtle, Pseudemys scripta (Schoepff), in Panama. Bull of the Los Angles County Museum of Natural History Science. 11:1-102.

Morafka, D. J., K. E. Spangenberg, and V. A. Lance, 2000. Neonatology of reptiles. Herpetological Monographs 14:353-370.

Morales-Verdeja, S. A., and R. C. Vogt. 1997. Terrestrial movement and activity patterns in Kinosternon leucostomum. Copeia 1:123-130.

Morris, K. A., G. C. Packard, T. J. Boardman, G. L. Paukstis, and M. J. Packard. 1983. Effect of the hydric environment on growth of embryonic snapping turtles (Chelydra serpentina). Herpetologica 39:272-285.

Mount, R. H. 1975. The Reptiles and Amphibians of Alabama. Auburn University Agricultural Experimental Station, Auburn, Alabama.

Murphy, W. J., and G. E. Collier. 1997. A molecular phylogeny for aplocheiloid fishes (Atherinomorpha, Cyprinodontiformes): The role of vicariance and the origins of annualism. Molecular Biology and Evolution 14(8):790-799.

Mrosovsky, N. 1982. Sex ratio bias in hatchling sea turtles from artificially incubated eggs. Biological Conservation 23:309-314.

Mrosovsky, N., and C. L. Yntema, 1980. Temperature dependence of sexual differentiation in sea turtles: implications for conservation practices. Biological Conservation 18:271-280.

Nadzhafou, D. A., and T. M. Iskenderov. 1994. The peculiarities of reproduction of Levantine viper (Vipera lebetina obtusa). Zoologicheskii Zhurnal 73(6):79-84.

Nager, R. G., P. Monaghan, D. C. Houston. 2000. Within-clutch trade-offs between the number and quality of eggs: experimental manipulations in gulls. Ecology 81(5):1339-1350.

Nagle, R. D., V. J. Burke, and J. D. Congdon. 1998. Egg components and hatchling lipid reserves: Parental investment in Kinosternid turtles from the southeastern United States. Comparative Biochemistry and Physiology 120(1):145-152.

Nagle, R. D., M. V. Plummer, J. D. Congdon, and R. U. Fischer. 2003. Parental investment, embryo growth, and hatchling lipid reserves in softshell turtles (Apalone mutica) from Arkansas. Herpetologica 59(2):145-154.

Nelson, D. 1997. Alabama Endangered Wildlife Projects. Alabama Red-Belly Turtle Population Survey: Population Ecology of the Alabama Redbelly Turtle (Pseudemys alabamensis) Alabama Department of Conservation and Natural Resources; 1997 Period Covered: 1 October 1996 - 30 September 1997/Annual Performance Report Project Number: AL E-001/Studies 14.

Niekisch, M., B. Farkas, U. Fritz, and H. D. Duc. 1997. Rekordgrößen bei Weichschildkröten im Stadtzentrum von Hanoi, Vietnam. Herpetofauna 19 (107): 28-34.

Nolan, D. 2006. Using scanning electron microscopy to study tortoise and turtle eggshell structure in captivity. In H. Artner, B. Balazas, and V. Loehr (eds.), Turtles, pp.583-592. Proceedings: International Turtle and Tortoise Symposium Vienna 2002.

Nutphand, W. 1979. The Turtles of Thailand. Mitghadung Press. Bangkok, Thailand.

Overall, K. L. 1994. Lizard egg environments. In L.J. Vitt and E.R. Pianka (eds.), Lizard Ecology: Historical and Experimental Perspectives, pp 51-72. Princeton University Press, Princeton, New Jersey.

Packard, G. C. 1991. Physiological and ecological importance of water to embryos of oviparous reptiles. In D. C. Deeming and M. W. J. Ferguson (eds.), Egg Incubation, Its Effect on Embryonic Development in Birds and Reptiles, pp. 213- 228. Cambridge University Press, Cambridge, United Kingdom.

Packard, G. C. 1999. Water relations of chelonian eggs and embryos: is wetter better? American Zoologist 39(2):289-303.

Packard, G. C., and M. J. Packard. 1988. The physiological ecology of reptilian eggs and embryos. In C. Gans and R. B. Huey (eds.), Biology of the Reptilia, Vol 16, pp. 423-605. Alan R. Liss, Inc., New York.

Packard, G. C., and M. J. Packard. 2004. To freeze or not to freeze: adaptations for overwintering by hatchlings of the North American painted turtle. Journal of Experimental Biology 207(17):2897-2906.

Packard, G. C., M. J. Packard, T. J. Boardman, and M.D. Ashen. 1981. Possible adaptive value of water exchanges in flexible-shelled eggs of turtles. Science 213:471-473.

Packard, M. J., and G. C. Packard, 1986. The effect of water balance of eggs on growth and calcium metabolism of embryonic painted turtles (Chrysemys picta). Physiological Zoology 59:398-405.

Packard, M. J., G. C. Packard, and T. J. Boardman. 1982. Structure of eggshells and water relations of reptilian eggs. Herpetologica 38:136-155.

Packard, M. J., J. B. Iverson, and G. C. Packard. 1984. Morphology of shell formation in eggs of the turtle Kinosternon flavescens. Journal of Morphology 181:22-28.

Parker, G. A., and M. Begon. 1986. Optimal egg size and clutch size: effects on environment and maternal phenotype. American Naturalist 128:573 -592.

Parker, G., and D. W. Mock. 1987. Parent-offspring conflict over clutch size. Evolutionary Ecology 1:161-174.

Paton, P. 2002. Meteorological and Hydrological Summary for Barro Colorado Island, Smithsonian Tropical Institute. Unpublished Report. Meteorological and Hydrological Branch of the Panama Canal Authority.

Pearse, D. E., and J. C. Avise. 2001. Turtle mating systems: behavior, sperm storage, and genetic paternity. The Journal of Heredity 92(2):206-211.

Pearse, D. E., F. J. Janzen, and J. C. Avise. 2002. Multiple paternity, sperm storage, and reproductive success of female and male painted turtles (Chrysemys picta) in nature. Behavioral Ecology and Sociobiology 51:164-171.

Pianka, E. R. 1969. Sympatry of desert lizards (Ctenotus) in Western Australia. Ecology 50:1012-1030.

Pieau, C. 1971. Sur la proportion sexuelle chez les embryons de deax Chéloniens (Testudo graeca L. et Emys orbicularis L.) issus d'oeufs incubés artificiellement. CR. Academy of Sciences. Paris. France. 272:3071-3074.

Pieau, C. 1974. Sur la differentiation sexuelle chez des embryons d’ Emys orbicularis L. (Chélonien) issus d’oeufs incubés dans le sol au curs de l’été 1973. Bulletin de la Société Zoologique de France 99:363-376.

Polisar, J. 1996. Reproductive biology of a flood-season nesting freshwater turtle of the northern neotropics: Dermatemys mawii in Belize. Chelonian Conservation and Biology. 2:13-25.

Pritchard, P. C. H. 1979. Encyclopedia of Turtles. T. F. H. Publishers, Inc., Neptune, New Jersey.

Pritchard, P. C. H., and P. Trebbau. 1984. Turtles of Venezuela. Society for the Study of Amphibians and Reptiles. Venezuela, South America.

Resetarits, W. J. J. 1996. Oviposition site choice and life history evolution. American Zoologist 36:205-215.

Rhen, T., and J. W. Lang. 1995. Phenotypic plasticity for growth in the common snapping turtle: effects of incubation temperature, clutch, and their interaction. American Naturalist 146(5):726-747.

Roosenburg, W. M. 1996. Maternal condition and nest site choice: an alternative for the maintenance of environmental sex determination? American Zoologist 36:157- 168.

Roosenburg, W. M., and A. E. Dunham. 1997. Allocation of reproductive output: egg and clutch size variation in the diamondback terrapin. Copeia. 2:290-297.

Roosenburg, W. M., and P. Niewiarowski. 1998. Maternal effects and the maintenance of environmental sex determination. In T.A. Mousseau and C.W. Fox (eds.), Maternal Effects and Adaptations, pp. 307- 322. Oxford University Press, New York.

Sachsse, W. 1974. Zum Fortpflanzungsverhalten von Clemmys muhlenbergii bei weitgehender Nachmung der natürlichen Lebensbedingungen im Terrarium. Salamandra 10:1-14.

Sachsse, W., and A. A. Schmidt. 1976. Nachzucht in der zweiten Generation von Staurotypus salvinii mit weiteren Beobachtungen zum Fortpflanzungsverhalten (Testudines, Kinosternidae). Salamandra 12:5-16.

SAS Institute Inc. 2001. Release 8.02. Cary, North Carolina.

Savage, J. 2002. The Amphibians and Reptiles of Costa Rica: A Herpetofauna Between Two Continents, Between Two Seas. Chicago University Press, Chicago, Illinois.

Schilde, M., and E. Lehr. 2002. Care and breeding of Cyclemys tcheponensis (Bourret, 1939). Radiata.11(1):31-34.

Seigel, R. A., and K. C. Dodd, Jr. 2000. Manipulation of turtle populations for conservation: Halfway technologies or viable options? In M. Klemens (ed.), Turtle Conservation, pp. 218-238. Smithsonian Institution Press, Washington, D.C.

Seigel, R. A., and N. B. Ford. 2001. Phenotypic plasticity in reproductive traits: Geographical variation in plasticity in a viviparous snake. Functional Ecology 15 (1):36-42.

Senneke, D. 2006. Husbandry and breeding of Indotestudo elongata in a temperate environment. In H. Artner, B. Balazas, and V. Loehr (eds.), Turtles, pp. 96-101. Proceedings: International Turtle and Tortoise Symposium Vienna 2002.

Seymour, J. E., and R. E. Jones. 2000. Humidity terminated diapause in the tropical braconid Microplitis demolitor. Ecological Entomology 25:1-5.

Shaffer, H. B., P. Meylan, and M. L. McKnight. 1997. Tests of turtle phylogeny: molecular, morphological, and paleontological approaches. Systematic Biology. 46(2): 235-268.

Shealy, R. M. 1976. The natural history of the Alabama map turtle, Graptemys pulchra Baur, in Alabama. Bulletin of the Florida State Museum of Biological Sciences 21:47-111.

Shine, R. 1980. “Cost” of reproduction in reptiles. Oecologia 46:92-100.

Smith, M. A. 1923. On a collection reptiles and batrachians from the island of Hainan. Journal of Natural History Society of Siam 6:195-212.

Smith, M. A. 1931. The Fauna of British India, including Ceylon and Burma. Reptilia and Amphibia, vol. 1, Loricata and Testudines. Taylor and Francis, London, England.

Smith, C. C. and S. D. Fretwell. 1974. The optimal balance between size and number of offspring. American Naturalist 108:499-506.

Spawls, S., K. Howell, R. Drewes, and J. Ashe. 2002. A Field Guide to the Reptiles of East Africa. San Diego: Academic Press.

Stearns, S. C. 1992. The Evolution of Life Histories. Oxford University Press, Oxford, United Kingdom of Great Britain.

Stearns, S. C., and R. E. Crandall. 1981. Bet-hedging and persistence as adaptations of colonizers. In G. G. E. Scudder and J. L. Reveal (eds.), Evolution Today. Hunt Institute, Philadelphia, Pennsylvania.

Stein, R. 2006. Captive care and breeding of the Malagasy spider tortoise, Pyxis arachnoides arachnoides Bell, 1827. In H. Artner, B. Balazas, and V. Loehr (eds.), Turtles. pp 511- 517. Proceedings: International Turtle and Tortoise Symposium Vienna 2002.

Stuart, B. L., and J. F. Parham. 2006. Recent hybrid origins of three rare Chinese turtles. Conservation Genetics. 8(1): 169-175.

Stuart, J. N., and C. W. Painter. 1997. Trachemys gaigeae (Big Bend slider). Reproduction. Herpetological Review 28:149-150.

Sultan, S. E. 1987. Evolutionary implications of the phenotypic plasticity in plants. Evolutionary Biology 21:127-178.

Sultan, S. E. 2003. Commentary: the promise of ecological developmental biology. Journal of Experimental Zoology 296B:1-7.

Swingland, I. R., and M. J. Coe. 1979. The natural regulation of giant tortoise populations on Aldabra Atoll: movement, polymorphism, reproductive success and mortality. Journal of Animal Ecology 48:639-654.

Taskavak, E., and M. K. Atatür. 1998. Habitats and distribution of the Euphrates soft- shelled turtle, Rafetus euphraticus (Daudin, 1802) in southeastern Anatolia; some observations on its biology and factors endangering its survival. Chelonian Conservation Biology 3(1):20-30.

Tauber, M. J., C. A. Tauber, and S. Masaki. 1986. Seasonal Adaptations of Insects. Oxford University Press, Oxford, United Kingdom.

Thompson, M. B. 1985. Functional significance of the opaque white patch in eggs of Emydura macquarii. In G. Grigg, R. Shine, and H. Ehmann (eds.), Biology of Australian Frogs and Reptiles, pp. 387-395. Surrey Beatty and Sons. Chipping Norton, New South Wales, Australia.

Thompson, M. B. 1987. Water exchange in reptilian eggs. Physiological Zoology 60:1-8.

Tinkle, D. W. 1967. The life and demography of the side-blotched lizard, Uta stansburina. Miscellaneous Publications of the Museum of Zoology, University of Michigan. 132:1-182.

Trivers, R. L. 1972. Parental investment and sexual selection. In B. Campbell (ed.), Sexual Selection and Descent of Man, pp. 136-179. Aldine, Chicago, Illinois.

Trivers, R. L. 1974. Parent-offspring conflict. American Zoologist 14:249-264.

Trivers, R. J. 1985. Social Evolution. Cummings, Menlo Park, California.

Turtle Conservation Fund (TFC). 2003. “Top 25 Turtles on Death Row: The Worlds Top 25 Most Endangered Turtles.”

Valenzuela, N. 2000. Multiple paternity in side-neck turtles Podocnemis expansa: evidence from microsatellite DNA data. Molecular Ecology 9:99-105.

Valenzuela, N., and V. A. Lance. 2004. Temperature Dependent Sex Determination in Vertebrates. Smithsonian Institution Books, Washington, DC.

Van Dijk, P. P., B. L. Stuart, and A. G. J. Rhodin. 2000. Asian Turtle Trade: Proceedings of a Workshop on Conservation and Trade of Freshwater Turtles and Tortoises in Asia, Phnom Penh, Cambodia, 14 December 1999, p. 164. Chelonian Research Foundation.

Van Valen, L. M. 1976. Energy and evolution. Evolutionary Theory 1:179-229.

Vanzolini, P. E. 1977. A brief biometrical note on the reproductive biology of some South American Podocnemis (Testudines: Pelomedusiidae). Pap. Avul. Zool., Sao Paulo, Brazil 31:79-102.

Vermersch, T. 1992. Lizards and Turtles of South-central Texas. Eakin Press, Austin, Texas.

Vijaya, J. 1982. Rediscovery of the forest cane turtle (Heosemys silvatica) of Kerala. Hamadryad 7(3):2-3.

Vincent, M. 2005. Maintenance en captivité de l'Emyde lépreuse (Mauremys leprosa, Schweigger, 1812). Manouria 29:42-44.

Vogt, R. C. 1980. Natural history of the map turtles Graptemys pseudogeographica and G. ouachitensis in Wisconsin. Tulane Studies of Zoology and Botany 22:17-48.

Vogt, R. C. 1990. Reproductive parameters of Trachemys scripta venusta in Southern Mexico. In: J. W. Gibbons (ed.). Life History and Ecology of the Slider Turtle, pp, 162-170. Smithsonian Institution Press. Washington, D C.

Vogt, R. C. 1997. Las ranas de Laguna del Zacatal. In E. G. Soriano, R. Dirzo, and R. Vogt (eds.) Historia Natural de Los Tuxtlas, pp. 500-503. Universidad Nacional Autónoma de México, México.

Vogt, R. C., and O. Flores-Villela. 1992. Effects of incubation temperature on sex determination in a community of Neotropical freshwater turtles in southern Mexico. Herpetologica 48:265-270.

Volterra, V. 1926. Variations and fluctuations of numbers of individuals in animal species living together. Reprinted in 1931 in R. N. Chapman (ed.), Animal Ecology, McGraw-Hill, New York.

Vyas, R. 1996. Breeding data on Lissemys punctata from western India. Hamadryad 21: 45-46.

Wade, M. J. 1998. The evolutionary genetics of maternal effects. In T. A. Mousseau and C. W. Fox (eds.), Maternal Effects and Adaptations, pp 5-21. Oxford University Press, New York.

Warner, D. A., and R. M. Andrews. 2002. Laboratory and field experiments identify sources of variation in phenotypes and survival of hatchling lizards. Biological Journal of the Linnaean Society 76105-124.

Whitaker, Nikhil. 2000. Incubation of Aspideretes gangeticus eggs and long term sperm storage in females. Hamadryad 25(2):198-200.

Wibbels, T., F. C. Killebrew, and D. Crews. 1991. Sex determination in Cagle's map turtle: Implications for evolution, development, and conservation. Canadian Journal of Zoology 69: 2693-2696.

Wilbur, H. M., and P. J. Morin. 1988. Life history evolution in turtles. In C. Gans, and R. Huey (eds.), Biology of the Reptilia, Vol 16, pp. 387-440. Alan R. Liss, Inc., New York.

Win Ko Ko, Khin Myo Myo, and Kyaw Moe. 2006. Final report of freshwater turtle conservation to a Chindwin River segment between Htamanthi Village and Khamanthi. September 2005 – June 2006. Report to Wildlife Conservation Society, Yangon, Myanmar.

Wourms, J. P. 1972. The developmental biology of annual fishes III. Pre-embryonic and embryonic diapause of variable duration in the eggs of annual fishes. Journal of Experimental Zoology 182:389-414.

Wright, S. 1969. Evolution and the Genetics of Populations. Vol 2. University of Chicago Press, Chicago, Illinois.

Yamaguchi, N., H. L. Dugdale, and D. W. Macdonald. 2006. Female receptivity embryonic diapause, and superfetation in the European badger (Meles meles): Implications for the reproductive tactics of males and females. Quarterly Review of Biology 81(1):33-48.

Yamashita, C. 1990. Hydromedusa maximiliani. Ecology. Herpetological Review 21(1): 19.

Yasukawa, Y., T. Yabe, and H. Ota. (in press). Mauremys japonica (Japanese Pond Turtle). In P. C. H. Pritchard, and A. G. J. Rhodin (eds.), The Conservation Biology of Freshwater Turtles. IUCN/SSC Tortoise and Freshwater Turtle Specialist Group. Chelonian Research Foundation, Lunenburg, MA: Chelonian Research Monographs.

Zappalorti, R. T. 1976. The Amateur Zoologist's Guide to Turtles and Crocodilians. Stackpole Books, Harrisburg, Pennsylvania.

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Appendix A. ED categorical modeling database. Species list with range (within geographic bans, Y = Yes and N = No), egg type (B = Brittle and P = Pliable), phylogeny (close phylogenetic relationship to a species possessing ED, Y = Yes and N = No), and ED expression (documentation of the traits expression, Y = Yes, N = No, UK = Unknown, P = Probable ED expression, NP = No Probable ED expression, and HP = Highly probable ED expression).

In Range* Egg Type Phylogeny** ED Expression Acanthochelys macrocephala Y B 1 Y UK Acanthochelys pallidipectoris Y B 2 Y Y 2 Acanthochelys radiolata Y B 3 Y UK Acanthochelys spixii Y B 4 Y UK Actinemys marmorata Y B 5 N N 6 Amyda cartilaginea N B 7 N P Apalone ferox Y B 8 N N 8 Apalone mutica Y B 5 N N 3 Apalone spinifera Y B 3 N N 3 Aspideretes gangeticus Y B 9 Y Y 9 Aspideretes hurum Y B 10 Y P Aspideretes leithii Y B 10 Y P Aspideretes nigricans Y B 11 Y P Batagur baska Y P 12 N N 12 Callagur borneensis N P 12 N N 12 Caretta caretta Y P 13 N N 13 Carretochelys insculpta N B 14 N N 14 Chelodina cannii Y B 15 Y Y 15 Chelodina expansa Y B 15 Y Y 15 Chelodina longicollis Y B 15 Y P Chelodina mccordi Y B 15 Y P Chelodina novaequinae N B 15 Y N 15 Chelodina oblonga Y B 15 Y P Chelodina parkeri N B 15 Y HP Chelodina pritchardi Y B 15 Y P Chelodina reimanni N B 15 Y N 15 Chelodina rugosa Y B 16 Y Y †, 16

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