Body Shape Diversification of Pecos Pupfish (Cyprinodon Pecosensis)

Western Kentucky University TopSCHOLAR®

Masters Theses & Specialist Projects Graduate School

Spring 2017 Body Shape Diversification of Pecos Pupfish (Cyprinodon Pecosensis) on Varying Habitats as Evaluated by Geometric Morphometrics Qianna Xu Western Kentucky University, [email protected]

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Recommended Citation Xu, Qianna, "Body Shape Diversification of Pecos Pupfish (Cyprinodon Pecosensis) on Varying Habitats as Evaluated by Geometric Morphometrics" (2017). Masters Theses & Specialist Projects. Paper 2017. http://digitalcommons.wku.edu/theses/2017

This Thesis is brought to you for free and open access by TopSCHOLAR®. It has been accepted for inclusion in Masters Theses & Specialist Projects by an authorized administrator of TopSCHOLAR®. For more information, please contact [email protected]. BODY SHAPE DIVERSIFICATION OF PECOS PUPFISH (CYPRINODON PECOSENSIS) IN VARYING HABITATS AS EVALUATED BY GEOMETRIC MORPHOMETRICS

A Thesis Presented to The Faculty of the Department of Biology Western Kentucky University Bowling Green, Kentucky

In Partial Fulfillment Of the Requirement for the Degree Master of Science

By Qianna Xu

May 2017 BODY SHAPE DIVERSIFICATION OF PECOS PUPFISH (CYPRINODON PECOSENSIS) IN VARYING HABITATS AS EVALUATED BY GEOMETRIC MORPHOMETRICS

I I ~/ ]..tf /17 Date DEDICATION

This work is dedicated to my parents, who always love me and support every one of my life decisions. ACKNOWLEDGEMENTS

I would like to thank Dr. Michael Collyer for advising me throughout my two years at WKU. I am grateful for his guidance, patience, and support, most importantly for introducing me to the field of evolutionary biology and biometry. I also thank Dr. Philip

Lienesch and Dr. Jarrett Johnson for their helpful comments and suggestions as my

Graduate Advisory Committee members. Thank you to Alexandra Snyder and Dr.

Thomas Turner, Museum of Southwestern Biology at UNM, for their generous hospitality to allow us access to the museum specimens, supplies, and laboratory space for our fish photo booth.

Thank you to Mr. Chaise Gilbert for being a collaborative and supportive colleague, and for his companionship on our endless driving trips to New Mexico.

Hannah Chaney and Haley D. Austin provided invaluable assistance with digitizing the fish photos in the lab. I appreciate all the help from Jessica Dunnegan, Melanie Redden, and the rest of the biology office staff at WKU.

I would also like to thank my mom and dad for their invaluable understanding, support, and love during my eight years of studying in the United States. I want to thank all my friends for their company and giving me a home away from home.

This work was supported through funds from the WKU Research and Creative

Activities Program (RCAP) to Dr. Michael Collyer, the Graduate Student Research Grant from the WKU Graduate School, and the WKU Department of Biology.

iv TABLE OF CONTENTS

Introduction ...... 1

Ecology of the Pecos pupfish in the Pecos River System ...... 2

Geometric Morphometrics and Ecomorphology of Pecos Pupfish ...... 4

Conservation of Pecos Pupfish ...... 6

Objectives and Hypotheses ...... 7

Methods and Materials ...... 9

Morphological Data Collection and Processing ...... 10

Statistical Analyses and Logic for Initial Model Building ...... 12

Results ...... 16

Shape Allometry ...... 16

Sexual Dimorphism in Body Shape ...... 16

Spatial Effects on Body Shape Variation ...... 17

Temporal Effects on Body Shape Variation ...... 21

Discussion ...... 22

Bibliography ...... 30

Appendix ...... 50

v LIST OF FIGURES Figure 1. Map of the Pecos River (highlighted in red) based on a map from Handbook of Texas Online, Delmar J. Hayter, “Pecos River”...... 44 Figure 2. General sampling areas in Chaves County, New Mexico within: (a) Bitter Lake National Wildlife Refuge, and (b) Bottomless Lake State Park. Sampling sites are indicated as red dots. Habitat types were categorized as following: WF = waterfowl impoundments; SH = deep sinkholes; BLM = Bitter Lake outflow marsh; BL = Bitter Lake. Map data: Google, DigitalGlobe...... 45 Figure 3. Anatomical landmark definition for geometric morphometric analyses of Pecos pupfish. The landmarks colored in red are “fixed” landmarks, including: most anterior point of maxilla; insertion points for dorsal, caudal, anal, and pectoral fins; intersection of operculum and ventral; dorsal margin of the operculum; and center of the eye. The landmarks colored in blue are the sliding semi-landmarks for the fish body curvature estimation...... 46 Figure 4. Visualization of allometric effects on the predicted shapes of the sex-habitat groups as illustrated by the first principal component (PC 1) of the predicted shape against the log of centroid size. Sex-habitat groups are denoted by colors as: yellow = Male BL, black = Male BLM, grey = Male SH, green = Male WF, orange = Female BL, red = Female WF, purple = Female SH, blue = Female BLM...... 47 Figure 5. Principal component (PC) plot indicating shape variation of male Pecos pupfish among 27 localities, and among habitat types, including upper Bitter Lake (BL), Waterfowl impoundments (WF), deep sinkholes (SH), and BLM outflow marsh (BLM). Thin-plate-spline deformation grids depicting the most extreme body shapes are included to facilitate visual interpretation...... 48 Figure 6. Principal component (PC) plot indicating shape variation of female Pecos pupfish among 27 localities, and among habitat types, including upper Bitter Lake (BL), Waterfowl impoundments (WF), deep sinkholes (SH), and BLM outflow marsh (BLM). Thin-plate spline deformation grids depicting the most extreme body shapes are included to facilitate visual interpretation...... 49

vi LIST OF TABLES

Table 1. Results of non-parametric multivariate analysis of variance (np-MANOVA) for the analysis of shape variation and its association with size (i.e. shape allometry) in Pecos pupfish. (α = 0.05) ...... 40 Table 2. Results of non-parametric multivariate analysis of variance (np-ANOVA) for assessing the effects of habitat complex designation and sampling period on lateral body shape, as well as the effect of appropriate interactions. (α = 0.05) ...... 41 Table 3. Results of non-parametric multivariate analysis of variance (np-MANOVA) for assessing the effects of locality and habitat complex designation on lateral body shape, as well as the effect of specific localities within habitat complexes. (α = 0.05) ...... 42 Table 4. Results of non-parametric multivariate analysis of variance (np-MANOVA) for assessing the effects of locality and sampling periods, as well as the effect of sampling periods within specific localities on lateral body shape. (α = 0.05) ...... 43

vii BODY SHAPE DIVERSIFICATION OF PECOS PUPFISH (CYPRINODON PECOSENSIS) IN VARYING HABITATS AS EVALUATED BY GEOMETRIC MORPHOMETRICS Qianna Xu May 2017 53 Pages Directed by: Michael Collyer, Philip Lienesch, and Jarrett Johson Department of Biology Western Kentucky University During the 19th and 20th centuries, alterations to the Pecos River in New Mexico and Texas, USA due to anthropogenic activities, including damning and river channelization, vast water extraction for irrigation, as well as pollution of associated habitats, have greatly impacted the fish fauna within the drainage. One of the endemic fish species, the Pecos pupfish (Cyprinodon pecosensis), might be the most affected.

Historically abundant and widespread large populations have been disrupted and became a series of small isolated subpopulations that persist at a few highly fragmented habitats restricted to a small area in southern New Mexico. The connectivity among these habitats is extremely low, and can potentially prevent any gene flow among subpopulations, which might eventually result in morphological divergence among subpopulations in face of different ecological conditions. Here I utilized landmark-based Geometric

Morphometrics to evaluate body shape variation of the Pecos pupfish at 26 different localities categorized into four general habitat types that each differ greatly in ecological properties.

Results from this study suggest that, despite significant sexual dimorphism, body shape morphology of Pecos pupfish varied in response to spatial heterogeneity and it was most intensely influenced by specific localities within habitat types. There were overlaps of the convex hull regions of morphospace among the four habitat types, implying that ecomorphological dynamics of the Pecos pupfish were rather site-specific. Moreover,

viii temporal variation of body morphology was statistically significant but not comparable to body shape variation among different localities. The empirical data collected from this study provides preliminary evidence for phenotypic diversity of Pecos pupfish in varied ecological conditions, which has important implications for the future conservation management of Pecos pupfish diversity and viability. Such implications could be extended to other endemic desert fishes in disrupted habitats.

ix INTRODUCTION

The southwestern United States is susceptible to radical changes in environmental conditions (Miller 1961). In addition to climatic fluctuations, anthropogenic disturbance has greatly affected this area. During the 19th and 20th centuries, both physical and chemical alterations including damming, river channelization, extensive groundwater depletion, as well as oil field pollution of associated habitats, have caused drastic changes in the arid deserts, and have threatened the spatial distributions and assemblages of the fish fauna endemic to this area (Williams et al. 1985; Hoagstrom and Brooks 1999; Fagan et al. 2005). North American desert fishes are typically vulnerable to environmental disturbance because of their restricted distribution. According to Williams (1981), 39 desert fish species have been listed as endangered or threatened by the Department of the

Interior of United States.

The Pecos pupfish, Cyprinodon pecosensis (Cyprinodontiformes:

Cyprinodontidae), might be one of the most affected small-bodied desert fish species.

The species was once the most abundant and widespread fish in the Pecos River basin in

New Mexico and Northern Texas (Echelle and Echelle 1978; Echelle and Corner 1989;

Wilde and Echelle 1992). However, the large meta-population has been disrupted during the past two centuries due to habitat degradation, as well as hybridization with introduced species. The species has greatly declined in abundance, and the remaining genetically pure populations now persist in small numbers and at a few isolated sites only, in scattered sinkholes, lakes, streams, and saline springs in southern New Mexico. As of

2013, the Pecos pupfish is listed as a vulnerable species on the IUCN Red List

(NatureServe 2013), and it is classified as a threatened species of the State of New

Mexico in 1988 (Propst 1999; Garrett et al. 2002).

As habitat degradation increases because of the dewatering of habitats, so does the frequency of exotic fish introduction into the Pecos River, leading to intensified competition over depletable resources. All could result in elimination of the endemic desert fishes in this ecosystem. Therefore, if natural habitats and fish communities cannot be restored, understanding the evolutionary trajectories of the remaining sub-populations of C. pecosensis in the disrupted habitats is one of the priorities for its conservation.

Ecology of the Pecos pupfish in the Pecos River System

The pupfishes, genus Cyprinodon, occupy diverse aquatic habitats in the deserts of

Southwestern United States. They have a notable tolerance of a wide range of harsh environmental conditions, especially of salinity levels from brackish to very saline, and temperatures from near freezing to as high as 45 C (Miller 1981; Moyle and Cech 2000;

Hoagstrom and Brooks 1999). They can also be found in water bodies of low dissolved oxygen content (Collyer 2003). Historically, the Pecos pupfish was widely distributed throughout the Pecos River valley, which extended 926 miles from Mora County, north- central New Mexico, through the eastern portion of New Mexico and south to its confluence with the Rio Grande in southwestern Texas (Figure 1; Echelle and Echelle

1978; Childs et al. 1996). However, since the 1980s, the introduction of the sheepshead minnow, Cyprinodon variegatus, into the Pecos River system has extirpated the endemic pure Pecos pupfish populations via hybridization and rapid genetic introgression throughout the Texas portion of the Pecos River (Echelle and Conner 1989). Yet, up until mid-1990s, there had been no sign of hybridization in the New Mexico portion of the

Pecos pupfish habitats, probably because the upward migration of the fish is thwarted damming of the river (Echelle et al. 1997). Remnant genetically pure Pecos pupfish populations primarily occur in more saline portions of upper Pecos River near Roswell basin, Chaves Co., New Mexico, and are confined to nearby isolated springs and gypsum sinkholes (Williams et al. 1985). Furthermore, excessive groundwater extraction for agricultural use and construction of five major reservoirs in the past few decades had significantly lowered the Pecos River water table and consequently caused loss of much historic wetland habitat and intensified habitat fragmentation for the Pecos pupfish in this area.

The remaining populations are presently restricted along approximately 70km of the upper Pecos River and occur most abundantly in specific localities at the Bitter Lake

National Wildlife Refuge (BLNWR) and Bottomless Lakes State Park (BLSP) near

Roswell, New Mexico (Hoagstrom and Brooks 1999). Due to the loss of flood flow, the connectivity among off-channel habitats is often very low or presumably non-existent, resulting in declined genetic integrity and potentially altered local adaptions within and among populations. In the 1990s, federal and state agencies constructed man-made impounded marshes and creeks within the BLSP, which continue to help maintain some degree of gene flow among some remnant Pecos pupfish populations (Hoagstrom and

Brooks 1999), but habitat depletion remains a threat.

The Pecos pupfish generally has a body shape visibly similar to other pupfishes in

North America, which is laterally compressed, deep bodied, with typically a supra- terminal to terminal mouth and protruding lower jaw, suggesting adaptations to a mid- water and surface-feeding lifestyle (Eddy and Underhill 1978; Soltz and Naiman 1978;

Sublette et al. 1990). They are opportunistic omnivores and feed mainly on a variety of algae and detritus (Davis 1981). The fish generally can reach reproductive maturity in less than one year at a body size of approximately 20mm (Garrett et al. 2002), but adults can grow to a maximum of 60mm in total body length (Kodric-Brown 1977; Page and

Burr 1991). Pupfishes have a relatively short lifespan with only a few adults surviving past one year (Kodric-Brown 1977; Garrett 1981). Pecos pupfish are typically sexually dimorphic with reproductive males exhibiting several morphological traits including larger size and a bright breeding coloration uniformly distributed over a breeding male’s body (Kodric-Brown 1990; Collyer 2003; Kodric-Brown 2004). As breeding behaviors of

Pecos pupfish have not been well studied, it is unclear how body shape varies within and between sexes, although sexual dimorphism has been observed in some habitats (Collyer et al. 2015b). Pupfish reproductive effort and morphology can be greatly affected by physical conditions, as well as co-existence of other fish species within habitats.

However, because of Pecos pupfish’s notable tolerance to high salinity, co-occurrence of other fishes is rare in highly saline waters. In the BLNWR and BLSP habitat complexes,

Pecos pupfish only co-occurs with plain killifish (Fundulus zebrinus) and the endangered

Pecos gambusia (Gambusia nobilis) at a few gypsum sinkholes of low to medium salinity

(3–23ppt) (Hoagstrom and Brooks 1999).

Geometric Morphometrics and Ecomorphology of Pecos Pupfish

The field of geometric morphometrics (GM) aims to describe organismal forms and their variation within and among organisms, based on the spatial relationship of anatomical features. Because morphometrics studies usually investigate multiple traits, GM also integrates the application of multivariate statistical methods to study shape covariation to

4 other variables (Rohlf and Marcus 1993; Bookstein 1998). The application of the GM approach has long been used in many biological disciplines, including systematics, evolutionary biology, increasingly in ecology, and developmental biology. Traditionally, morphology was described qualitatively and heavily addressed anatomical properties.

The emergence of GM allowed rigorous quantification of shape data by capturing the organisms’ geometry (Rohlf and Marcus 1993). In the late 20th century, GM shifted from emphasizing linear distance measurements to using 2-dimensional or 3-dimensional landmark coordinates, or outline analysis, for quantifying and analyzing shape variation

(Brookstein and Rohlf 1990; Adams et al. 2004). The most commonly employed approach in modern geometric morphometric studies is the landmark-based geometric morphometric technique, which has been widely used to assess shape variations in plants and animals, including fishes (Adams et al. 2004; Bookstein 1997; Adams and Collyer

2009). Landmark-based geometric morphometric (GM) methods include mathematical decomposition of “size” and “shape” in landmark configurations, plus multivariate analyses of these attributes. GM methods have been shown to be capable of describing much more morphological variation richness within and among organisms and are equipped with powerful statistical analysis and comprehensive visual representation of results (Collyer et al. 2015a).

Cyprinodon is known for great morphological diversity within and among species, even with little genetic basis for differentiation in some cases (e.g., Collyer et al.

2005). Within-species morphological variation often reflects spatial and temporal environmental heterogeneity (e.g., Collyer et al. 2015b). Abiotic features such as temperature, salinity, turbidity, and dissolved oxygen content are greatly associated with

5 fish abundance and viability, which could have the potential to influence morphological diversification directly through thermal or osmotic factors (Langerhans et al. 2003;

Collyer 2003). Constantz (1981) described pupfishes inhabiting springs of low velocity as more deep-bodied and robust in terms of overall body shape compared to stream pupfishes, a difference which may have resulted from the difference in drag or turbidity between the two types of habitat (Collyer 2003). The White Sand pupfish (Cyprinodon tularosa) exhibited significant body shape divergence when introduced into new habitats, and their body shape corresponds to habitat conditions, for example, higher salinity is associated with more streamlined body shape (Collyer et al. 2005). Besides, food availability and community structure also have the potential to affect the fish’s morphological features (phenotypic plasticity) indirectly via intra- and interspecific interactions, which may shape the path of morphological divergence in sympatry and allopatry (Brown and Wilson 1956). With Pecos pupfish sub-populations subject to diverse ecological conditions, I expected that adaptive morphological modifications would also be induced in Pecos pupfish in dissimilar habitats.

Conservation of Pecos Pupfish

There is a clear need to understand how environmental heterogeneity correlates with local adaptation and evolutionary trajectories of the Pecos pupfish to make informed conservation decisions (Storfer 1999). Previous studies have mainly focused on species assemblage and fish abundance within habitat complexes, as well as life history traits of the Pecos pupfish (Hoagstrom and Brooks 1998, Swaim and Boeing 2008). For example,

Swaim and Boeing (2008) showed Pecos pupfish had lower fitness and negatively affected body conditions with coexistence of other fishes. In a series of studies, Kodric-

Brown explicitly described the breeding pattern (1977, 1988), social status determination

(1993), and sexual selection (1990, 1997) of the Pecos pupfish in response to ecological features of habitats. Nevertheless, understanding how abiotic and biotic factors affect body shape divergence in allopatric and sympatric Pecos pupfish sub-populations remains an unaccomplished objective. An assessment of body morphology divergence would provide evidence for phenotypic diversity within and among different ecological conditions (Watters et al. 2003). However, empirical studies that reveal such complex interactions involving abiotic and biotic factors are rare, simply because replication of natural populations that would allow such complex analysis is also infrequent. The occurrence of multiple habitats within the BLNWR and the BLSP provides natural replications of varied multi-species communities (Swaim and Boeing 2008) that permit sympatric and allopatric studies, which would help reveal the relationship between fish body morphology and interacting ecological factors. This study will grant further insights of environmental variation (especially via habitat fragmentation) on the potentially distinct evolutionary trajectories for small isolated Pecos pupfish sub-populations in terms of morphological divergence, which will contribute information for better conservation management of Pecos pupfish diversity and sustainability.

Objectives and Hypotheses This study examined the diversity of lateral body morphology, as evaluated by geometric morphometrics, in natural Pecos pupfish (C. pecosensis) populations occurring at

BNWLR and BLSP habitat complexes:(1) between sexes (i.e. sexual dimorphism) among isolated sub-populations; (2) associated with spatial heterogeneity subject to different habitat type and locality; (3) in response to temporal changes. Since temporal effects

7 could potentially prompt fluctuated environmental gradients, as well as interspecific interaction due to food availability in different seasons, the populations could potentially adapt in response to temporal changes.

I attempted to test the following null hypotheses for body shape divergence among Pecos pupfish populations: (1) no difference in shape between mature males and females (sexual dimorphism); (2) no difference in shape among populations that inhabit different habitat types; (3) no spatial heterogeneity effect on body shape among populations occurring at different localities; (4) no temporal heterogeneity effect on body shape among populations.

MATERIALS AND METHODS

A total of 1188 Pecos pupfish specimens from museum collections, including 62 samples at 26 localities within the Bitter Lake National Wildlife Refuge (BLNWR) and

Bottomless Lake State Park (BLSP) near Roswell, New Mexico (Figure 2), were used in this study for investigating lateral body shape variations. All specimens were obtained at the Museum of Southwestern Biology, University of New Mexico (see Appendix 1 for

Museum of Southwestern Biology catalog numbers). Samples used were obtained from repeated sampling between 1940 and 2003 at isolated habitat complexes within BLNWR and BLSP. The 26 localities could be grouped into 4 habitat complexes based on their physical properties, habitat conditions, and habitat isolation: deep sinkholes (SH), Bitter

Lake region (BL), Waterfowl impoundments (WF), and the Bitter Lake outflow marsh

(BLM). Among the four habitat types, the sinkhole (SH) habitats are the smallest and most isolated and vary greatly in surface area, depth, and temporally variable salinity

(Hoagstrom and Brooks 1999; Collyer et al. 2015b). The Bitter Lake (BL) habitat complex includes 5 localities that are roughly located northwest of Bitter Lake and flows directly into it: Sago Spring, Sinkhole 31, Sinkhole 32, and two divisions of Bitter Creek.

(Although Sinkhole 31 and Sinkhole 32 are named for their geophysical depressions, they are comparatively shallow than other isolated sinkholes and connected to other BL habitats via Bitter Creek. These habitats are physically marsh-like compared to the deep sinkholes, which have highly incised banks and little littoral vegetation.) The waterfowl impoundments (WF) are located in the BLNWR and consist of a number of individual units that vary in ecological properties (e.g. surface area, salinity, community structure;

Hoagstrom and Brooks 1999). The last habitat complex, BLM, is an extensive marshland

9 that flows out from Lea Lake. Collections from only two localities were incorporated in this study.

I also included samples collected during different months (April, May, June, July,

September, October, November, and December) if there was repeated sampling within a year, to consider temporal effects. During the process, it was brought to my attention that one sample collected in 1988 at Figure Eight Lakes comprised fish caught and kept in aquarium over several months before preservation. The sample, therefore, was removed from the analysis. Moreover, some juvenile specimens exhibited underdeveloped crania compared to adults. Hence, juvenile specimens were also removed from the analysis to avoid morphological variation due to ontogeny. Thus the sample size for analysis consisted of 1117 specimens (555 females and 562 males).

Morphological Data Collection and Processing

Adult Pecos pupfish were used in this study to minimize ontogenetic effect on morphological divergence. Mature males possess relatively darker and larger dorsal and anal fins, with a black terminal band on the caudal fin. Females have dark ocelli near the posterior base of the dorsal fin (Echelle and Echelle 1978). Based on these characteristics, fish were separated into male and female groups with visual identification assisted by using a Nikon ShuttlePix P-400R digital microscope. Each individual was then given a specific identification number.

Up to 10 male and 10 female mature individuals ranging from large to small were haphazardly selected from each sample so that the association between size and shape

(i.e. allometry) could be analyzed (Mosimann 1970; Collyer et al. 2015a). All individuals from a collection were included if the sample contained less than 20 specimens.

Digital images of each specimen were captured using a Nikon D90 camera mounted approximately 10 centimeters above the specimen on a portable copy stand

(Collyer et al. 2005; Langerhans et al. 2003). The left lateral side of each individual was photographed with flash lighting. Each specimen was patted dry to limit optical distortion and placed on a piece of grid index card, with a 1-mm incremental scale bar.

Landmark configurations were then generated: 11 “fixed” anatomical landmarks and 45 “sliding” semi-landmarks were digitized on the photographs of left lateral side of fish (Figure 3), using tpsDig2 software, version 2.27 (Rohlf 2012). The “fixed” landmarks were chosen to reflect easily identifiable and homologous points among all specimens: (1) most anterior point of maxilla, (2) anterior insertion of dorsal fin, (3) dorsal insertion of caudal fin, (4) ventral insertion of caudal fin, (5) posterior insertion of anal fin, (6) anterior insertion of anal fin, (7) superior insertion of pectoral fin, (8) insertion of pectoral fin, (9) dorsal origin of operculum, (10) intersection of operculum and ventral, and (11) center of the eye (Langerhans et al., 2004; Collyer et al. 2015a). The semi-landmarks were placed evenly in-between “fixed” landmarks. The semi-landmarks are free to slide along tangent directions on curves (Collyer et al. 2015a), and therefore, characterizing more realistic body shape by obtaining morphological information of curves (see below).

To eliminate any non-shape components of variation, including size, position and orientation, geometric shape information was extracted with a Generalized Procrustes

Analysis (GPA; Rohlf and Slice 1990; Viscosi and Cardini 2011). GPA rescaled, shifted,

11 and rotated the landmark configurations, therefore aligning the configurations to a standard size, position, and orientation, respectively, through Procrustes superimposition

(Rohlf and Slice 1990; Adams et al. 2004; Rohlf and Marcus 1993). During GPA, an algorithm was used to minimize the “Procurstes distances” between semi-landmarks. The only variation after this process was exclusively shape variation. Raw landmark coordinates were translated into Procrustes residuals during superimposition, which could then be used for subsequent statistical analyses. The Procrustes fit of the raw landmark coordinates and GPA were performed using R, version 3.2.3 (R Core Team 2015), utilizing the “geomorph” package, version 3.0.1 (Adams et al. 2016).

Statistical Analyses and Logic for Initial Model Building

Fishes grow continuously as they age throughout their lifetime. Samples collected from the field usually contain a mix of ages and sizes, which could lead to potential confounding effects between shape differences and heterogeneity in the sizes of the samples. Even though shape and size are quantitatively independent measures via GPA, shape and size can still be correlated. It is generally advised that all comparative shape analyses involving organisms take into account size allometry: the covariation between shape and size (Klingenberg 1998). Therefore, it is logical to consider shape-size allometry as the first covariate in our analysis (Collyer et al. 2015b). I performed a

Principal Component Analysis (PCA) and a multivariate analysis of variance

(MANOVA) to initiate the morphometric analysis. In all models, alpha was set to 0.05 for significance testing. A PCA using covariance matrices of the aligned X-Y coordinates was used to visualize the overall shape variation. Specimens were categorized into 8 groups based on sex and habitat type: Female-BL, Male-BL, Female-SH, Male-SH,

Female-WF, Male-WF, Female-BLM, and Male-BLM. Then the body size (logCsize) of each group was plotted against the first principal component scores (PC 1) of predicted values. Body size was quantified as log-transformed centroid size, where centroid size was determined as the square root of the sum of the square distances of all landmarks from their centroid (Bookstein 1991). The centroid size measures the amount of dispersion of each landmark around the center of the landmark configuration. I then performed a Procrustes analysis of variance (Procrustes ANOVA) with a randomized residual permutation procedure (i.e. RRPP; 10,000 iterations) to assess shape covariation with fish body size (i.e. allometry; Adams et al. 2004; Collyer et al. 2015a). Then a

Procrustes ANOVA with RRPP (10,000 iterations) was performed to determine the patterns of shape variation associated with group.

For analysis of morphological divergence among groups, I was interested in the relative amount of phenotypic change associated with size attributable to multiple factors, including sex, habitat type, specific locality, month, and year. I performed a non- parametric multivariate analysis of variance (np-MANOVA) with RRPP (1,000 iterations) on various linear models that included body size, sex, habitat type, locality, month, and year collected, plus interactions: Body Shape ~ log(Centroid size) + Group

+ log(Centroid size):Group, where group represents sex, habitat, locality, month, and year. The ANOVA results for this model are represented in Table 1. A factor was considered biologically interpretable or meaningful if it explained more than 5% of the total shape variation (R2 > 0.05). Based on the complete linear model, sex was a strong indicator of morphological divergence, thus, I separate sex groups and analyze males and females separately in subsequent analyses in order to make meaningful inferences.

For each sex group, a non-parametric MANOVA was performed with several models to assess shape variation attributable to habitat type, specific locality, month, and year. The models were designed to test the nested random effects of one factor within a specific level of another factor. The nested effects of interactions between habitat type and month; month and year; habitat type and locality; locality and year; and locality and month were analyzed with Nested MANOVA (RRPP = 1,000 iterations). For each sex group, the model was structured as following: Body Shape ~ log(Centroid size)*Habitat + Habitat/Month + Month/Year + Month:Year:Habitat. The ANOVA results of this model are represented in Table 2. Because shape data are multidimensional, typical model selection criteria (e.g., Akaike’s information criterion) are prone to favor over-fitting models (sensu Bedrick and Tsai 1997; Davis et al. 2016).

As a method of model selection, I simply chose the final model as one that included meaningful effects and excluded those effects with small effect size (R2 < 0.05). If significant distinctions were identified (R2 > 0.05) with the Nested MANOVA, we then performed a Procrustes ANOVA with pairwise comparisons (via the advanced.procD.lm function in geomorph) using a complex linear model to measure the amount of shape variation explained by each variable among groups.

The morphological types, represented by the sample mean of each population, were plotted on a Principal Component (PC) plot with convex hulls to help visualize the most extreme values (Collyer et al. 2015a). Deformation grids were generated to display the transformation of the mean shape of specimen associated with each locality in tangent space along the principal axes (PC 1 and PC 2). All GM analyses were performed in R,

14 version 3.2.3 (R Core Team 2015), utilizing the “geomorph” package, version 3.0.1

(Adams et al. 2016).

RESULTS

Shape Allometry

The body shapes from a total of 1117 adult specimens from 63 samples of Pecos pupfish natural populations from BLNWR and BLSP were quantified for analyses. Shape allometry was significant and significantly different among Pecos pupfish populations

(Table 1; Figure 4). The overall trend showed that larger individuals exhibited more lateral body shape variation. The np-MANOVA with RRPP (1,000 iterations) also indicated a strong influence of size (measured as logarithm of centroid size) on body shape variation (i.e. shape allometry; Table 1), as size was responsible for approximately

15.5% of shape variation. The interactions between size and other factors including habitat complex, locality, and year were all significant (P < 0.05) with one exception of sampling period (month). The various significant interactions between size and other factors also indicated that shape allometries varied among different sub-populations.

Therefore, it was rational to consider the influence of static body shape allometry among other variables and appropriate interactions between shape allometry and other variables.

Nevertheless, there was no evidence of size variation among different habitat complexes

(R2 = 0.009). Month-to-month and year-to-year variation were also not influential factors associated with body shape (R2 = 0.006 and 0.005 for Month and Year, respectively).

Sexual Dimorphism in Body Shape

Geometric morphometric analyses revealed significant sexual dimorphism of body shape and body shape allometry in Pecos pupfish. Initial evaluation of linear models suggested that either sex (R2 = 0.064) or a sex by habitat complex interaction (R2 = 0.126) was

16 significant and explained a sufficient amount of shape variation in body morphology (R2

>0.05). Moreover, when plotting the GPA aligned shapes (y-axis) versus log of centroid size (x-axis), there was a substantial divergence in the male and female allometric patterns (Figure 4). Within each of the four habitat complexes, the slope of the allometric regression line was greater for males than females, indicating that larger male individuals exhibited more body shape variation than female individuals of similar sizes. The multivariate analysis of variance (MANOVA) also confirmed that Pecos pupfish were sexually dimorphic in terms of body shape as sex explained a relatively large amount of shape variation (R2 = 0.073, P = 0.001; Table 1). When the mean shape configurations of male and female Pecos pupfish were compared, males generally exhibit more deep- bodied shapes with larger caudal peduncle regions in comparison to female fish.

Spatial Effects on Body Shape Variation

Multiple models were used to assess the effects of several variables that had potentially influenced morphology. I considered size (log of centroid size), habitat complex type, specific localities, month, year, and appropriate interactions of these variables as potential sources of body shape variation in Pecos pupfish populations (Body Shape ~ log(Centroid size)*Habitat + Habitat/Month + Month/Year + Month:Year:Habitat).

Males and females were analyzed separately as subsets to examine sources of shape variation aside from the contribution of sexual dimorphism. The np-MANOVA analyses performed with RRPP (1,000 iterations) suggested that shape allometry was still significant upon separating males and females (Table 2). Regardless of sex, body shape was significantly different among specific localities (P = 0.001). Specific locality alone explained 7.22% of shape variation in Pecos pupfish (Table 1). Habitat complex type

17 could be an important source of shape variation but not as much as locality. Although the effect of habitat was significant (P = 0.001), for both sexes, habitat type was only associated with slightly more than 5% of variation. Females and male shape allometries were significantly different among different habitat complexes (evidenced by significant size-habitat interactions), thus, the effect of habitat complex needed to be further investigated.

For females, the np-MANOVA with RRPP (1,000 iterations), performed to assess the shape change in relation to the effect of habitat type with nested random effect of other factors, suggested statistical significance of shape variation among different habitat complexes (P = 0.001; Table 2). However, interestingly, such effect of habitat complex was not significant in males (P = 0.056). Meanwhile, habitat type may be of some statistical significance in female fish, but it was not a meaningful (i.e. biologically interpretable) source of shape variation as it only explained little shape variation (R2 =

0.052). Further, shape variation among habitat within month was significant in both sexes but it poorly explained shape change in either of the sex groups (R2 = 0.028 and R2 =

0.031 for females and males, respectively). The interaction of monthly changes within years within habitat complexes was significant for females (P = 0.001) and for males (P =

0.038), but it also failed to explain a sufficient amount of shape variation (R2 = 0.008 and

R2 =0.004 for females and males, respectively). I only consider a factor as biologically interpretable or meaningful if it explained more than 5% of the total shape variation (R2 >

0.05). Therefore, a reduced model was employed to focus on the effect of specific locality while controlling the effects of allometry and the resulting model was Body

Shape ~ log (Centroid size) + Locality + Habitat/Locality. Model selection suggested that

18 locality and locality within the fixed effect of habitat complex were significant and meaningful (Table 3) — locality nested within habitat complex was a stronger factor in determining shape variation than habitat alone. Moreover, for both males and females, locality alone was associated with approximately the same amount of shape variation

2 2 (R male = 0.155; R female = 0.185) as the amount explained by habitat plus locality nested within habitat, which again confirmed that habitat was not a strong and meaningful factor of shape variation compared to specific locality. This suggests that ecomorphological dynamics are locality- or site-specific. Thus, I subsequently focused on shape variation among different localities while account for a common shape allometry.

Visual evidence indicated shape variation among specific locations, among and within habitat types for males and females (Figure 5 and Figure 6, respectively). The first and second principal components (PCs) of body shape variation were plotted on a 2- dimensional plot with thin plate spline transformation grids to facilitate interpretation of the most extreme body shapes. Convex hulls were shown to provide an overview of shape variation among the four habitat complexes even though shape variation was not quite consistent within each habitat complex. The principal component analyses revealed that, cumulatively, PC1 and PC2 accounted for 42.74% of the total variation in females and 59.52% in males. For males, thin-plate-spline transformation grids illustrated that

PC1, the axis of greatest shape variation, is associated with streamlining in lateral body shape. Comparatively, males with more positive PC1 scores appeared to have streamlined body shape, whereas negative PC1 scores corresponded to deep-bodied shapes. Positive

PC2 scores appeared to correspond to a more surface orientated, pelagic body shape in males. This can be seen with the more superior snouts and deeper caudal peduncles, for

19 fish of similar body depth—traits typically associated with mid-water and surface feeding

(Keast and Webb 1966; Gatz Jr. 1979). For females, transformation grids showed that

PC2 is associated with body streamlining, where individuals with more positive PC2 scores had more elongated lateral body shapes, and more negative PC2 scores corresponded to more deep-bodied and robust female individuals. The greatest shape variation, PC1, appeared to be associated with head shape and caudal peduncle area in females. Females with more positive PC1 scores had more terminal snouts and narrower caudal peduncle regions, whereas females with more negative PC1 scores had more pelagic body shapes (superior-oriented snouts and larger caudal peduncle regions).

For both sexes, the resulting convex hulls were not distinct but rather had broad regions of overlap between two or more point clusters of habitat complexes, suggesting low probability of distinct shape variation among different habitat complexes. The BL and WF hulls were large and mostly overlapping for both sexes. Exceptionally, the deep sinkhole (SH) males were clustered and overlapped the least with any other habitat complexes. The SH males were also more distinct along PC2 with mostly negative PC2 scores in comparison with males from other habitat complexes. The SH female lateral body shapes were more disperse when compared to SH males but not as diverse as the

BL and BLM females. Intriguingly, the BL sample means were similar among males and females, with the exception of the sample from Sinkhole 31. The two BLM samples were divergent but fell within the overlapping BL and WF convex hulls. Nevertheless, males tended to have a wider range of PC1 scores in comparison with females, but vice versa for PC2 scores. This suggested that males had more body shape variation associated with

20 the first principal component, while females tended to have more shape variation associated with PC2.

Temporal Effects on Body Shape Variation

To investigate the potential temporal effects of shape variation among localities, I applied an np-MANOVA with RRPP procedure (1,000 iterations) with the initial linear model

(Body Shape ~ log(Centroid size)*Habitat + Habitat/Month + Month/Year +

Month:Year:Habitat; Table 2). The result suggested that monthly change in body shape was significant regardless of sex (P = 0.001), which explained 5.56% of variation in females and 7.06% of variation in males. Monthly changes within years were also significant (P = 0.001), but the interaction only accounted for little shape variation in

Pecos pupfish (R2 < 0.05). However, neither monthly nor yearly changes in body shape were greater than shape variation among different localities in either males or females, which means that sampling periods within years were meaningful sources of shape variation, but not compared to locality variation. The data revealed no clear evidence of morphological divergence due to the interaction between size and month or year, either.

Furthermore, a Procrustes ANOVA (RRPP = 1,000) performed with a reduced model focusing on the nested effect of month or year after accounting for the effect of locality

(Body Shape ~ log(Centroid size) + Locality/Year + Year + Locality/Month + Month;

Table 4) also confirmed that although shape variation was significant during different sampling periods (month and year), neither month nor year explained sufficient amount shape variation in both sexes. Therefore, the effects of month and year on the Pecos pupfish morphological divergence were subsequently considered not biologically meaningful.

DISCUSSION

Pecos pupfish in the upper Pecos River basin provides an exceptionally valuable opportunity to investigate phenotypic diversification in response to differential ecological conditions in desert aquatic ecosystems. In this study, geometric morphometric and statistical analyses revealed distinctive morphological divergence of body shape among

Pecos pupfish sub-populations at the Bitter Lake Wildlife Refuge and Bottomless Lake

State Park in New Mexico. Besides ontogenetic effects on shape change and sexual dimorphism, Pecos pupfish’s lateral body morphology was significantly divergent at various localities. The divergence could be a product of the fish being exposed to varying selective pressures imposed by ecological heterogeneity associated with dissimilar localities within the area. Although temporal effects on the fish lateral body shape were statistically significant, such seasonal and yearly changes were minor sources of shape variation compared to variation among localities. Nevertheless, whether the morphological divergence in Pecos pupfish lateral body shape occurred due to local adaptation or non-genetic phenotypic plasticity remained vague. No genetic assessment has yet been performed in reference to the putative sub-populations in this system.

Moreover, comprehensive data of ecological parameters (salinity, dissolved oxygen, pH, sinkhole area, and predation pressure, etc.) among all sampling sites is sparse due to the fact that the samples were collected by different researchers over several decades. Yet, this initial assessment of morphological divergence among discrete sub-populations of

Pecos pupfish, combined with possible future investigations on population genetic

22 structure, could have important implications for the conservation of this species of concern, and other fish species highly endemic to the arid desert ecosystem.

Shape change occurs as an organism grows larger in size and when it goes through different developmental phases. It was not surprising that shape allometry, the covariation between fish size and lateral body shape, was apparent in the Pecos pupfish regardless of sex. Many studies show extensive shape changes occurs during larval and juvenile stages of fishes while adult phase is usually characterized by developmental stability (for example, Koumoundouros et al. 1999; Loy et al. 1998; Strauss and Fuiman

1985). In this study, although juvenile specimens were removed from analyses, I found that shape change associated with growth was still remarkable in adult Pecos pupfish for the fact that size (defined as centroid size) explained the most shape variations among all other factors considered in my models. By regressing shape variation on the logarithm of centroid size, there was a clear pattern that, in general, larger individuals showed more diversifications in lateral body shape. Meanwhile, this trend was not as notable for the

BLM subpopulations as for the other subpopulations presumably because of the small sample size of the BLM subpopulations included in the analyses. The waterfowl impoundment (WF) subpopulations exhibited the most shape variation for a given body size conceivably because the waterfowl impoundments within BLNWR and BLSP varies greatly in environmental conditions such as size, depth, surface area, etc., which hypothetically led to greater body shape diversification in Pecos pupfish sub-populations that resides at different WF locations. Besides spatial difference, shape allometry differed between male and female Pecos pupfish.

Sexual dimorphism in body shape could potentially reflect effects of sexual selection and different response to ecological heterogeneity between sexes (Fairbairn et al. 2007; Hassell et al. 2012). It has been well documented in previous studies that Pecos pupfish exhibit many distinct morphological characteristics between males and females, including size, coloration pattern, shape of ventro-lateral markings and breeding coloration (Echelle and Echelle 1978; Kodric-Brown and Nicoletto 1993). My findings confirmed that the Pecos pupfish is sexually dimorphic in term of body shape, apart from environmental features of which the fish experienced. The primary differences between male and female mean shapes were in body depth, relative head size and area of the caudal peduncle region. In comparison to males, females tended to be slenderer with elongated and narrower caudal peduncles. Males, by contrast, have larger anterior body depth. Furthermore, there was a clear disparity of the ontogenetic body shape variation between males and females. Among all habitat complexes, male Pecos pupfish body depth had a stronger positive correlation with increased size compared to females. The difference in the ontogenetic body shape variation between males and females is consistent with a study on the Blacktail Shiner (Cyprinella venusta) which is a small- bodied freshwater fish occurs in southern United States (Hood and Heins 2000). In this study, body shape of a single sample of Blacktail shiner was assessed with geometric morphometrics and it was found that males and females exhibited different ontogeny of body shape where males showed more changes in head shape as the fish grow larger in size, whereas females appeared to show more changes in abdomen shape. Such ontogenetic difference in shape change was thought to be associated with reproductive status of the fish. Similarly, the distinctive ontogenetic body shape variation of adult

Pecos pupfish could also be found as a function of female choice (i.e. sexual selection) in reproduction.

Many aquatic ecosystems contain habitats that differ in environmental features as well as community structure. Seasonal variation could also yield many differences in environmental gradients such as temperature, precipitation and dissolved oxygen content.

Thus, it is often considered as a major source of ecological heterogeneity in aquatic ecosystems (Allen et al. 2012). As a result, many fish species exhibit distinct morphologies among different habitats (Langerhans et al. 2003). Collyer et al. (2015b) observed distinct Pecos pupfish morphotypes associated with isolated habitat complexes within the Bitter Lake National Wildlife Refuge and Bottomless Lake State Park. This study greatly expanded the breadth of temporal and spatial distribution of sampled sites.

My results were consistent with prior findings that Pecos pupfish body shape variation had a significant ecological association with the types of habitat where fish resided.

Additionally, specific locality appeared to be a more important factor than habitat type, as locality explained more shape variation than habitat after accounted for shape allometry.

The ranges of body shape divergence overlap greatly for both sexes between BL and WF habitat complexes, both could be categorized as marshland habitat type (Hoagstrom and

Brooks 1999). The BLM marsh samples collected in BLSP, with presumably higher degree of habitat connectivity, produced intermediate body shapes that fell within the range of shape variation among marshland habitats. The overlap of body shape between habitat types was expected because of random assignment of samples to different habitat types without considering the fluctuating flow regimes and physio-chemical characteristics at different localities. Nonetheless, among various localities, Pecos pupfish

25 appeared to show corresponding changes in morphological characteristics, including but not limited to body depth, length of caudal peduncle and relative head size, which may have resulted from different flow regimes and salinity levels (Miller et al. 2005). Within the WF and BL habitat complexes, Pecos pupfish exhibited drastic shape variations among localities, especially along the PC1 axis for males where positive values corresponded to more streamlining in body profile. Females also exhibited more distinct variation in streamlining which was associated with PC 2 (positive values corresponded to more streamlined body profile). Among all localities, Unit 15 Waterfowl Lake and

Sinkhole 31 had the most robust shapes: deep-bodied with deep caudle peduncle and smaller heads. A series of studies on the White sand pupfish (Cyprinodon tularosa;

Collyer 2005; 2007; 2011) also provided sufficient evidence that the Cyprinodon is phenotypically plastic in response to varied salinity levels. Hence, it is reasonable to assume varying salinities among different localities could induce similar morphological changes in Pecos pupfish. In addition, food availability and distribution could also shift morphological features relevant to feeding mechanisms, such as head size and mouth positions (Hendary et al. 2002). Although physical parameters and environmental gradients of each locality could not be measured due to technical restrictions, clear hypotheses could be made from the information regarding environmental gradients in previous work on fish eco-morphology.

Contrary to marshland sub-populations, the deep sinkhole (SH) populations exhibited a comparatively obvious and consistent pattern of body shape divergence and the samples means were clustered in the PC space. The SH sub-populations possessed mean lateral body shape that was slender and had a slightly elongated caudle peduncle

26 with anal fin shifted anteriorly. Mouth position of the SH sub-populations was terminal to sub-terminal. This body type is presumably an adaptation for rover predation in deep pools with steep littoral area, and at the same time, sub-terminally positioned mouth would allow more benthic feeding to avoid competition with the surface-feeding Pecos gambusia (Gambusia nobilis) that often co-exist in some sinkholes of non-extreme salinities (Collyer 2015; Bednarz 1979). Compared to marshes or artificial waterfowl impoundments that are subject to fluctuating water levels, dissolved oxygen contents and flow, the gypsum sinkholes are relatively stable (Hoagstrom and Brooks 1999). Also, compared to the relatively higher connectivity and presumably more frequent gene flow in marshlands and waterfowl impoundments, sinkholes are typically isolated from nearby habitats; phenotypic diversity in the sinkhole habitats might be constrained due to the lack of gene flow. Yet, further investigation is required to gain insights to the evolutionary basis of these morphological divergences.

Implications to Conservation

The Pecos Pupfish Conservation Agreement in 1999 initiated a series of conservation plans to manage the Pecos pupfish populations within the Bitter Lake National Wildlife

Refuge and Bottomless Lake State Park (USFWS, 2000). The conservation actions administered by New Mexico state agencies and the Bureau of Land Management includes developing barriers to protect Pecos pupfish habitats from invasion of the

Sheepshead minnow (Cyprinodon variegatus) to prevent genetic introgression, regulating the use of Sheephead minnow as bait fish in New Mexico and Texas, and re-establishing pure populations of Pecos pupfish into restored historic habitats on lands managed by

New Mexico Fish and Wildlife Services (Edwards et al. 2004). Additionally, private

27 landowners were encouraged by the Landowner Incentive Programs to stock Pecos pupfish in their private ponds modified to imitate natural habitats with the aid of federal funding (Garrett 2002). The conservation efforts under this agreement have successfully reduced immediate threats to the species. In 2000, the U.S. Fish and Wildlife Services withdrew the proposal of listing Pecos pupfish as a federal endangered species, which was initially submitted in 1998. Creating artificial wetlands and waterfowl impoundments is a major part of Pecos pupfish conservation. Therefore, informed decisions of creating suitable habitats are important in order to reintroduce and maintain self-sustainability and variability of Pecos pupfish populations. Such a goal will not be accomplished without understanding the evolutionary basis of local adaptation in the established populations.

Phenotypic diversification is one of the fundamental evolutionary mechanisms, which often matches ecological conditions in which the organism occurs. Additionally, body shape often affects fishes’ fitness and survival (Webb 1984). Therefore, understanding which abiotic and biotic factors drive phenotypic diversification in body shape becomes a fundamental procedure in understanding the evolutionary trajectories, as well as maintaining variability of fishes in natural ecosystems. Meanwhile, further studies are necessary to determine how various combinations of environmental parameters at dissimilar localities affect phenotypic divergence, and future explicit tests of the environmental gradients are required to reveal the environmental basis of phenotypic divergence at different localities. Detailed experimental approaches could help elucidate which ecological factors are most important in driving evolutionary divergence, or to what extent phenotypic plasticity allows populations to persist in spite of environmental changes. This study provided an initial step in understanding the morphological

28 diversification of Pecos pupfish in ecologically divergent habitats. Most notably, marshland habitats (larger surface area, more aquatic vegetation, and more species rich communities) and sinkhole (small surface area compared to volume, higher salinity, less vegetation, and more depauperate communities) appear to provide a basis for substantial evolutionary divergence. Prior to Pecos River modification, including water extraction, such habitats were intermittingly connected, with sinkholes serving as sources for expansive marshlands, which were also inundated by river flooding (Hoagstrom and

Brooks 1999). Thus, loss of gene flow more so than habitat variation might augment evolutionary divergence. Efforts to restore natural habitats might have to also consider connectivity, lest the management of this species seeks to preserve distinct morphotypes in specific habitats.

Finally, this study contributes valuable information for future research and conservation decisions for Pecos pupfish, but implications could be extended to other endemic desert fish species. Unlike Pecos pupfish, many desert fish species are highly endemic with small geographic distributions. It is rare to have the opportunity to study replicated instances of population isolation in ecologically diverse habitats. The Pecos pupfish can be seen as a flagship species for desert fishes’ conservational biology.

Realizing that habitat fragmentation or environmental change can spur evolutionary divergence in small populations is an important implication for the conservation of any desert fish species.

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Table 1. Results of non-parametric multivariate analysis of variance (np-MANOVA) for the analysis of shape variation and its association with size (i.e. shape allometry) in Pecos pupfish. (α = 0.05)

Source of Variation df R2 Z P

Log(Csize) 1 0.155 30.865 0.001

Sex 1 0.073 29.447 0.001

Habitat 3 0.045 16.875 0.001

Locality 26 0.072 4.512 0.001

Month 7 0.044 9.449 0.001

Year 8 0.022 4.490 0.001

Log(Csize)*Sex 1 0.025 24.101 0.001

Log(Csize)*Habitat 3 0.009 4.742 0.001

Log(Csize)*Locality 26 0.026 2.027 0.002

Log(Csize)*Month 7 0.006 1.583 0.052

Log(Csize)*Year 8 0.005 1.223 0.154

Table 2. Results of non-parametric multivariate analysis of variance (np-ANOVA) for assessing the effects of habitat complex designation and sampling period on lateral body shape, as well as the effect of appropriate interactions. (α = 0.05)

Source of Variation df Females Males

R2 Z P R2 Z P

log(Csize) 1 0.108 25.362 0.001 0.214 29.979 0.001

Habitat 3 0.052 9.881 0.001 0.052 1.590 0.056

Month 7 0.056 5.078 0.001 0.071 2.564 0.001

log(Csize)*Habitat 3 0.070 13.112 0.001 0.012 3.204 0.001

Habitat* Month 4 0.028 5.199 0.001 0.031 6.116 0.001

Month* Year 10 0.041 3.278 0.001 0.031 2.717 0.001

Habitat* Month* 2 0.008 3.126 0.001 0.004 1.768 0.038

Year

Table 3. Results of non-parametric multivariate analysis of variance (np-MANOVA) for assessing the effects of locality and habitat complex designation on lateral body shape, as well as the effect of specific localities within habitat complexes. (α = 0.05)

Source of Variation df Females Males

R2 Z P R2 Z P

log(Csize) 1 0.108 25.885 0.001 0.229 30.182 0.001

Habitat 3 0.047 8.835 0.001 0.052 10.774 0.001

Locality 26 0.185 4.355 0.001 0.155 4.104 0.001

Habitat* Locality 23 0.138 3.898 0.001 0.103 3.337 0.001

Table 4. Results of non-parametric multivariate analysis of variance (np-MANOVA) for assessing the effects of locality and sampling periods, as well as the effect of sampling periods within specific localities on lateral body shape. (α = 0.05)

Source of Variation df Females Males

R2 Z P R2 Z P

log(Csize) 1 0.108 25.885 0.001 0.229 30.182 0.001

Locality 23 0.185 3.898 0.001 0.152 3.337 0.001

Month 7 0.021 3.196 0.001 0.028 5.445 0.001

Year 8 0.050 4.080 0.001 0.048 3.751 0.001

Locality*Year 3 0.041 13.949 0.001 0.037 3.935 0.001

Locality*Month 23 0.006 3.510 0.001 0.009 1.670 0.001

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Figure 1. Map of the Pecos River (highlighted in red) based on a map from Handbook of Texas Online, Delmar J. Hayter, “Pecos River”.

Bitter Lake National Wildlife Refuge

Imagery ©2017 Google, Map data ©2017 Google 2000 ft Figure 2. General sampling areas in Chaves County, New Mexico within: (a) Bitter Lake National Wildlife Refuge, and (b) Bottomless Lake State Park. Sampling sites are indicated as red dots. Habitat types were categorized as following: WF = waterfowl impoundments; SH = deep sinkholes; BLM = Bitter Lake outflow marsh; BL = Bitter Lake. Map data: Google, DigitalGlobe.

Figure 3. Anatomical landmark definition for geometric morphometric analyses of Pecos pupfish. The landmarks colored in red are “fixed” landmarks, including: most anterior point of maxilla; insertion points for dorsal, caudal, anal, and pectoral fins; intersection of operculum and ventral; dorsal margin of the operculum; and center of the eye. The landmarks colored in blue are the sliding semi-landmarks for the fish body curvature estimation.

Figure 4. Visualization of allometric effects on the predicted shapes of the sex-habitat groups as illustrated by the first principal component (PC 1) of the predicted shape against the log of centroid size. Sex-habitat groups are denoted by colors as: yellow = Male BL, black = Male BLM, grey = Male SH, green = Male WF, orange = Female BL, red = Female WF, purple = Female SH, blue = Female BLM.

Male: WF Male: SH Male: BLM Male: BL Female: WF Female: SH Female: BLM Female: BL

SH BL WF BLM

Figure 5. Principal component (PC) plot indicating shape variation of male Pecos pupfish among 27 localities, and among habitat types, including upper Bitter Lake (BL), Waterfowl impoundments (WF), deep sinkholes (SH), and BLM outflow marsh (BLM). Thin-plate-spline deformation grids depicting the most extreme body shapes are included to facilitate visual interpretation.

SH BL WF BLM

Figure 6. Principal component (PC) plot indicating shape variation of female Pecos pupfish among 27 localities, and among habitat types, including upper Bitter Lake (BL), Waterfowl impoundments (WF), deep sinkholes (SH), and BLM outflow marsh (BLM). Thin-plate spline deformation grids depicting the most extreme body shapes are included to facilitate visual interpretation.

Appendix 1. Collection information for samples used in this study: Museum of Southwestern Biology (MSB, University of New Mexico) catalog numbers, sampling locations, date of sampling, number of specimens used in this study, salinity level (low = 3-12 ppt; medium = 10-23 ppt; high = 22-86 ppt), number of co-occurring species are listed. Note: although named “Sinkhole 31” and “Sinkhole 32”, these localities are shallow depressions forming marshes connected by Bitter Creek, and are physically quite different than other sinkholes in the study, which are isolated deeper depressions with incised banks and little littoral vegetation (Hoagstrom and Brooks 1999).

General MSB Locality Habitat Month/Year No. Salinity Number Complex Specimen of Co- Location Catalog occurring # species BLNWR 44652 Bitter BL 11/1999 20 Low 5 Creek, confluence BLNWR 46961 Bitter BL 11/2000 7 Low 5 Creek, confluence BLNWR 43668 Bitter BL 05/1999 20 Low 5 Creek, weir. BLNWR 44649 Bitter BL 11/1999 20 Low 5 Creek, weir. BLNWR 46821 Bitter BL 05/2000 16 Low 5 Creek, weir. BLNWR 46958 Bitter BL 11/2000 20 Low 5 Creek, weir. BLNWR 5177 Sago BL 05/1987 13 Low 4 Spring BLNWR 44656 Sago BL 11/1999 20 Low 4 Spring BLNWR 5165 Sinkhole BL 05/1987 17 Low 1 31 BLNWR 44659 Sinkhole BL 11/1999 20 Low 4 32 BLNWR 46831 Sinkhole BL 05/2000 22 Low 4 32 BLNWR 46964 Sinkhole BL 11/2000 20 Low 4 32 BLSP 5179 Marsh BLM 05/1987 20 Unknown 3 Outflow, weir.

BLSP 49238 Lea Lake BLM 04/2002 20 Low 4 Outflow BLNWR 948 Figure SH 07/1940 15 Medium 0 Eight Lakes BLNWR 30006 Figure SH 06/1988 18 Medium Unknown Eight Lakes BLNWR 85034 Figure SH 12/1994 19 Medium 0 Eight Lakes, lower BLNWR 62539 Figure SH 07/1994 20 Medium 1 Eight Lakes, upper BLNWR 62643 Figure SH 12/1994 20 Medium 1 Eight Lakes, upper BLNWR 79496 Figure SH 10/1998 20 Medium 1 Eight Lakes, upper BLNWR 49725 Figure SH 06/2003 20 Medium 1 Eight Lakes, upper BLNWR 79493 Mirror SH 10/1998 17 Medium 3 Lake, sinkhole BLNWR 79482 Sinkhole SH 10/1998 18 High 0 16 BLNWR 79479 Sinkhole SH 10/1998 20 High 0 19 BLNWR 5221 Sinkhole SH 05/1987 20 Low 3 20 BLNWR 79478 Sinkhole SH 10/1998 20 Low 3 20 BLNWR 43659 Sinkhole SH 05/1999 20 Low 3 20 BLNWR 46795 Sinkhole SH 04/2000 20 Low 3 20 BLNWR 46815 Sinkhole SH 05/2000 20 Low 3 20 BLNWR 46836 Sinkhole SH 06/2000 20 Low 3 20 BLNWR 46859 Sinkhole SH 07/2000 20 Low 3 20 BLNWR 46904 Sinkhole SH 09/2000 20 Low 3

20 BLNWR 46928 Sinkhole SH 10/2000 20 Low 3 20 BLNWR 46951 Sinkhole SH 11/2000 10 Low 3 20 BLNWR 5166 Sinkhole SH 05/1987 20 Low 2 37 BLNWR 78596 Sinkhole SH 05/1987 20 Low 2 37 BLNWR 43661 Sinkhole SH 05/1999 20 Low 2 37 BLNWR 46816 Sinkhole SH 05/2000 20 Low 2 37 BLNWR 46930 Sinkhole SH 10/2000 20 Low 2 37 BLNWR 43664 Sinkhole 7 SH 05/1999 20 Low 2 BLNWR 46818 Sinkhole 7 SH 05/2000 20 Low 2 BLNWR 46933 Sinkhole 7 SH 10/2000 20 Low 2 BLNWR 46954 Sinkhole 7 SH 11/2000 18 Low 2 BLNWR 55305 Oxbow 1, WF 10/1999 20 Unknown 0 eat of Unit 3&5 BLNWR 56962 Oxbow 1, WF 10/2001 20 Medium 4 Unit 3 BLNWR 5189 Unit 15 WF 05/1987 13 Low 7 Waterfowl Lake BLNWR 3132 Unit WF 09/1944 20 Unknown Unknown 15&16 Waterfowl Lakes BLNWR 5173 Unit 16 WF 05/1987 24 High 3 Waterfowl Lake BLNWR 55294 Unit 16 WF 10/1999 19 Low 3 Waterfowl Lake BLNWR 5141 Unit 17 WF 05/1987 18 Low 3 Waterfowl Lake BLNWR 55269 Unit 2 WF 10/1999 20 Unknown 0 Waterfowl Lake BLNWR 5163 Unit 3 WF 05/1987 20 Medium 4 Waterfowl Lake BLNWR 55270 Unit 3 WF 10/1999 20 Medium 4

Waterfowl Lake BLNWR 56958 Unit 3 WF 10/2001 20 Medium 4 Waterfowl Lake BLNWR 62527 Unit 3 WF 10/2002 20 Low 4 Waterfowl Lake BLNWR 5185 Unit 5 WF 05/1986 20 Medium 5 Waterfowl Lake BLNWR 55273 Unit 5 WF 10/1999 16 Medium 5 Waterfowl Lake BLNWR 5206 Unit 6 WF 05/1987 5 Low 4 Waterfowl Lake BLNWR 55281 Unit 6 WF 10/1999 18 Medium 4 Waterfowl Lake BLNWR 56956 Unit 6 WF 10/2001 20 Low 4 Waterfowl Lake BLNWR 55288 Unit 7 WF 10/1999 20 Low 7 Waterfowl Lake BLNWR 56954 Unit 7 WF 10/2001 20 Low 7 Waterfowl Lake BLNWR 56946 Unit 7 WF 10/2001 20 Low 7 Waterfowl Lake