MORPHOLOGICAL SIGNATURES OF HYBRIDIZATION BETWEEN THE
ENDEMIC ENDANGERED PECOS GAMBUSIA (GAMBUSIA NOBILIS)
AND INVASIVE GAMBUSIA SPP. AT BITTER LAKE NATIONAL
WILDLIFE REFUGE, NEW MEXICO
by
Garrett Gill
A Thesis Submitted in Partial Fulfillment
Of the Requirements for the Degree
MASTERS OF SCIENCE
Major Subject: Biology
West Texas A&M University
Canyon, Texas
December 2020
ABSTRACT
Fishes of the southwestern United States are among the most imperiled fishes in the world. Of the risks that these fishes face, extinction through hybridization is potent and is often facilitated by human activities. For example, native fish species may hybridize with closely related, introduced congeners. Moreover, these hybridization events are often difficult to detect and even more so among small, cryptic species. I attempt to identify and quantify the sites and extent of hybridization between the native
Gambusia nobilis and introduced Gambusia affinis or Gambusia geiseri at Bitter Lake
National Wildlife Refuge in Chaves Co., New Mexico, U.S.A., as well as identifying any possible barriers to fish movement on the property. This was done through studying the range of variability of morphological characteristics within populations and salinity throughout different wetlands at Bitter Lake National Wildlife Refuge. Morphology suggestive of invasive or hybrid Gambusia populations were found at sites in the southern portion of the property. The morphology of fish sampled from bitter creek and sinkholes were more indicative of G. nobilis and showed less variability in morphology relative to southern sampling sites. Bitter Lake, with a salinity 2.3 times that of seawater, may be halting the progress of invasive Gambusia spp. northward through the property.
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ACKNOWLEDGEMENTS
First and foremost, I would like to thank my adviser, Dr. James Bradley Johnson, and members of my thesis committee, Dr. David Sissom, Dr. Richard Kazmaier, and Dr.
Erik Crosman, for their guidance and help. I would also like to thank the Kilgore graduate student research grant program at West Texas A&M University for providing funding for this project. I would like to thank my family and my girlfriend Kaitlyn for all of the encouragement and love that they have given me, and for keeping me sane at times. I would like to thank U.S Fish and Wildlife Service for access to Bitter Lake
National Wildlife Refuge and for material support for my research. In particular, I would like to thank Wildlife Biologist Jeff Beauchamp for his assistance.
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Approved:
______
James B. Johnson, PhD Date Co-Chairman, Thesis Committee
______
W. David Sissom, PhD Date Co-Chairman, Thesis Committee
______
Richard T. Kazmaier, PhD Date Member, Thesis Committee
______
Eric T. Crosman, PhD Date Member, Thesis Committee
______
Head, Life, Earth and Date Environmental Sciences
______
Dean, College of Agriculture Date and Natural Sciences
______
Dean, Graduate School Date iv
TABLE OF CONTENTS
Introduction……………………………………………………………….………….……1
Morphological Variation in Gambusia spp. at BLNWR……………….…………...….6
Variation in Salinity among Wetlands at BLNWR…………………………………….6
Morphology and Variation in Salinity…………………………………….……………6
Methods………………………………………………………………………..…………..6
Study Site and Sampling……………………………………………….………………6
Character Analysis……………………………………………………………………..8
Statistical Analysis………………………………………………………………...….10
Morphological variation in Gambusia spp. at BLNWR…………………………….10
Variation in salinity among wetlands at BLNWR…………………..……………11
Morphology and variation in salinity…………………………………...……….11
Results……………………………………………………………………………………12
Morphological Variation in Gambusia spp. at BLNWR……………………………...12
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Variation in Salinity among Wetlands at BLNWR…………...………………………13
Morphology and Variation in Salinity………………….……………………………..14
Discussion……………………………………………….……………………………….14
Issues with Maintaining Protected Status………………..……………………………18
Additional Threats to Consider………………………………….……………………19
LIST OF TABLES
Table
1. Character coding for Gambusia spp. Characteristics measured at Bitter Lake
National Wildlife refuge. Columns represent the assigned grade given for a
character. Dashes represent the lack of numeral use for the character…………. 30
2. Morphological character loadings for CAP 1 and CAP 2 axis of the db-RDA.…31
LIST OF FIGURES
Figure
1. Locations of the four extant populations of endangered Pecos gambusia within
Texas and New Mexico, USA. ……………..…………………………….……..32
2. Sampling sites and wetlands at Bitter Lake National Wildlife Refuge, New
Mexico. Sites were sampled throughout 2018 and 2019…………………...……33
3. Morphological characters used to evaluate Gambusia spp. at Bitter Lake National
Wildlife Refuge ………………….………………..………………………..……34
4. Ordination of the first to axis of the db-RDA (CAP 1 and CAP 2). Sampling sites
are shown as colored points with 95% confidence ellipses. Loadings for the
morphological characters are shown in black text.………………...…………….35
5. Salinity measurements for different wetland types at Bitter Lake National Wildlife
Refuge, New Mexico. Observations (log transformed) are shown as black points.
Means (±SE) are shown in red. Maximum salinity tolerance for G. affinis is
shown by the dotted line…...………………….…………………………………36
6. Log mean salinity for wetlands at Bitter Lake National Wildlife Refuge, Chaves
Co., NM. Data was collected across 2018-2019…………………………………37
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7. Visualization of the relationship between the major axis of morphological
variation yielded from the db-RDA (CAP 1) and the coefficient of variation of
salinity …………………………………………………………………………...38
CHAPTER 1
MORPHOLOGICAL SIGNATURES OF HYBRIDIZATION BETWEEN THE
ENDEMIC ENDANGERED PECOS GAMBUSIA (GAMBUSIA NOBILIS)
AND INVASIVE GAMBUSIA SPP. AT BITTER LAKE NATIONAL
WILDLIFE REFUGE, NEW MEXICO
INTRODUCTION
Aquatic habitats of the southwestern United States are imperiled (Miller et al.
1989) because of anthropogenic activities (Fagan 2002, Dias et al. 2017). Modification of the natural flow regime of streams can cause physical changes to channel structure, sediment load, and thermal regime, which often leads to biological changes in species composition, trophic structure, and community composition (Marchetti et al. 2001).
Many of New Mexico’s rivers suffer from high levels of siltation, loss of riparian habitat, and bank destabilization (Propst 1999). Loss of spring flow poses a threat to fishes dwelling in spring-fed waters, desert streams and cienegas, as it can reduce the amount of suitable habitat available (Echelle et al. 1972).
Human consumption of water has continued to rise, increasing the demand on desert groundwater (Shepard 1993). This demand has caused a lowering of the water table (McGuire et al. 2003, Robson and Banta 1995, Gutentag et al. 1984). This lowering 1 of the water table has caused habitat fragmentation as well as habitat loss, which are the largest threats to biodiversity worldwide (Vitousek et al. 1997). In addition to induced changes in hydrology, humans have also introduced species into novel habitats in the
Southwest which have negatively impacted native species through competition for resources and space, direct predation, and hybridization (Hoagstrom et al. 2010, Rhymer and Simberloff 1996). In a study by Hoagstrom et al. (2010), the Rio Grande silvery minnow (Hybognathus amarus) was found to have been nearly extirpated by the plains minnow (Hybognathus placitus) in a few years post-contact in a stretch of the Pecos
River between the Fort Sumner Dam and Brantley Reservoir, New Mexico, USA.
The species at the highest risk of extinction through hybridization are rare species that are hybridizing with an introduced, closely related species that is abundant (Rhymer and Simberloff 1996). This risk of extinction through hybridization can be caused by the compromising of the native species through outbreeding depression, genetic introgression, and a decrease in fertility (Arnold 1997, Rhymer and Simberloff 1996).
This risk of hybridization is further enhanced by anthropological activities (Lamont et al.
2003, Byers 2002). Possible reasons for this are the introduction of historically separated species and impairment of natural coexisting taxa to distinguish between conspecifics and heterospecifics (Schwarz and McPheron 2007, Grabenstein and Taylor 2005, Rubidge and Taylor 2001, Grosholz 2002). Increased hybridization rates following anthropogenic disturbances have been documented in North American coyotes (Canis latrans) and gray wolves (Canis lupus) as well as bluehead (Catastomus discobulus) and Utah suckers
(Catastomus arden; Bangs et al. 2017, Kyel et al. 2006, Leonard et al. 2005). This is of
2 particular concern between species that have weak pre- and post-zygotic isolating mechanisms (Hubbs 1955).
The Pecos gambusia (Gambusia nobilis) is a small fish (<45 mm) (Rosen and
Bailey 1963) that is a surface feeder (Bednarz 1979). Its diet consists of aquatic insects and larvae, but it is generally an opportunistic feeder that does not specialize on a food source (Bednarz 1979). Pecos gambusia are active throughout the day, but highest activity levels correlate with insect activity at night (Bednarz 1979). Pecos gambusia were observed to use submerged cliffs, overhangs, and aquatic vegetation as cover
(Bednarz 1979). Pecos gambusia was historically found in spring systems throughout the
Lower Pecos River drainage (Echelle et al. 1989). However, extant Pecos gambusia populations are currently found in only four locations in the Pecos drainage (Figure 1,
Echelle et al. 1989, Echelle and Echelle 1980). This reduction in range is largely because of loss of spring habitats as a result of groundwater pumping (Hubbs et al. 1983). Despite this reduction in range, Pecos gambusia are numerous where found with studies at Bitter
Lake National Wildlife Refuge estimating the Pecos gambusia populations there to total over 30,000 individuals (Bednarz 1979). These populations are located in spring fed streams, cienegas, and sinkholes found on the property. Bednarz (1979) also estimated
Blue Spring to contain approximately 900,000 Pecos gambusia. Pecos gambusia, being adapted to spring systems, is extremely intolerant of variation in water physiochemistry, such as salinity, pH, temperature, and water hardness (Hubbs et al. 1983).
Additionally, two other Gambusia species, the mosquitofish (Gambusia affinis) and the Largespring gambusia (Gambusia geiseri), have been introduced into the Pecos 3
River drainage for mosquito control (Sanchez et al. 2013, Lee and Burgess 1980, Hubbs et al. 1995). The Largespring gambusia is native to the San Marcos and Comal Springs of
Central Texas but is now invasive in spring systems throughout western Texas and New
Mexico (Nico and Fuller 2018, Sanchez et al. 2013, Hubbs and Springer 1957).
Mosquitofish are a global invasive and have been found in waters ranging from streams and ponds to drainage ditches and sewage drains (Hubbs 2000, Arthington and Lloyd
1989, Cherry et al. 1976, Moyle and Nichols 1973). In addition to this, Mosquitofish have a much wider range of salinity levels that it can tolerate, being able to survive in waters with salinity as high as 41 ppt (Hubbs 2000).
While these species are closely related, there are morphological traits that can be used to distinguish between the Gambusia species. One method for this is to examine characteristics of the gonopodium, the male intromittent organ. This elongated anal fin ray has hooks, rays and scale patterns along the distal end. Species have unique combinations of these features that have been useful for identification (Rivas 1963,
Greenfield 1983). These species also differ in body morphology as well. Pecos gambusia are associated with having deep bodies, heads, and caudal peduncles (Rodriguez 2017,
Page and Burr 2011). They are tan in coloration and have a streak of pigment extending from the eye towards the ventral side of the head called an eye streak (Page and Burr
2011). Mosquitofish have a more streamlined body composition than Pecos gambusia, have body spots, and are associated with eye streaks as well (Page and Burr 2011).
Largespring gambusia have a streamlined body and spots on the body and fins
(Rodriguez 2017, Page and Burr 2011). Variation in these characteristics does occur
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(Stoopes et al. 2013, Pyke 2005, Echelle and Echelle 1986). Additionally, allometry, the scaling of traits with body size, effects characters in some poeciliid fishes (Horth et al.
2010)
There is evidence of hybridization between the invasive Gambusia spp. and the endangered Pecos gambusia (Hubbs et al. 2002) but the severity of introgression between these species appears to be variable. For example, Swenton (2011) evaluated populations at Bitter Lake National Wildlife Refuge in New Mexico and suggested that hybridization occurs but is not commonplace. However, a more recent study done at Balmorhea State
Park in Texas found that hybridization was extensive (Rodriguez 2017). These findings raise concerns as to whether hybridization is as widespread in other locations as well given the timespan between these studies. Hybridization is a process that can change rapidly over time and space (Engebretsen et al. 2016) particularly for taxa such as
Gambusia who have relatively short lifespans and mature and reproduce relatively quickly. For example Pecos gambusia have an interbrood interval of only 52 days (Hubbs et al. 2002). Because of the restricted range and the threat of hybridization, it is important to regularly assess the prevalence of hybridization in Pecos gambusia.
I evaluated the prospect of hybridization by assessing the morphology of
Gambusia spp. and water salinity at Bitter Lake National Wildlife Refuge. The objectives for this project were as follows:
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Morphological Variation in Gambusia spp. at BLNWR
I evaluate hybridization between Pecos gambusia and invasive Mosquitofish and
Largespring gambusia by assessing morphological variation of Gambusia at Bitter
Lake National Wildlife Refuge.
Variation in Salinity among Wetlands at BLNWR
I explore the complex wetland structure in relation to salinity, which may limit the
movement of Gambusia spp. at Bitter Lake National Wildlife Refuge.
Morphology and Variation in Salinity:
Finally, I evaluate the association between morphological variation and salinity to
determine if invasive/hybrid Gambusia are associated with more environmentally
variable habitats.
METHODS
Study Site and Sampling
My research was conducted at Bitter Lake National Wildlife Refuge (BLNWR), a property managed by the U.S. Fish and Wildlife Service located approximately 10 km east of Roswell, New Mexico (Lat: N33.47426, Lon: -W104.41370). The mission of this property was initially to provide wintering habitat for migratory birds (“About the
Refuge”, URL: https://www.fws.gov/refuge/bitter_lake/). In the past, research at Bitter
Lake National Wildlife Refuge has focused primarily on the threats to and conservation of its many threatened or endangered species, such as the Interior Least Tern (Sternula
6 antillarum athalassos), Noel’s amphipod (Gammarus desperatus), and the Pecos sunflower (Helianthus paradoxus).
The northern portion of the property is comprised of sinkholes, a spring-fed creek
(Bitter Creek), and Bitter Lake (Figure 2). The southern portion of the property is managed wetlands and their connecting canals (Figure 2). The Pecos River and its oxbows comprise the eastern edge (Figure 2). Several sinkholes on this property are thought to contain natural populations of Pecos gambusia (Sinkhole 7, Sinkhole 27, Sago
Sinkhole, and Dragonfly Spring Sinkhole, Hubbs et al. 1983). Populations were also established in six other sinkholes during the 1970s (Hubbs et al. 1983).
In order to determine the presence and extent of hybridization between populations of Gambusia at Bitter Lake National Wildlife Refuge, eight sites were sampled during the fall and spring of 2018 and 2019. Gambusia spp. were found in seven of the sites. Three sites were isolated in sinkholes, three were in interconnected canals, one was located on Bitter Creek, and one was located on the southern property known as the Farm (Figure 2). Fish were sampled using 23 cm x 45 cm minnow traps with either
0.6 cm or 1.3 cm mesh (Gee’s Minnow Napper, Tackle Factory, Fillmore, New York,
USA). Minnow traps are non-invasive, creating little substrate disturbance which was intended to protect the numerous species of endangered invertebrates found at Bitter Lake
National Wildlife Refuge. Traps were set each evening near sunset and checked the following morning. Traps were set without bait so as not to introduce nutrients to these sensitive aquatic systems.
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Once traps were collected, all fish were placed in 19-l buckets with roughly six l of water from the wetland to minimize differences in water quality, chemistry, temperature, and oxygen levels. In addition, a thermometer was placed in each bucket for continuous temperature monitoring. After fish from each site were processed, each minnow trap and bucket were sanitized with a 1% solution of Virkon-S to reduce the possibility of pathogen transfer between sampling locations.
To understand the possible distribution of fish at Bitter Lake National Wildlife
Refuge, I analyzed salinity (ppt) at 28 sites at Bitter Lake National Wildlife Refuge during the timeframe of the morphometric data collection (2018 and 2019, Figure 2). Not all of the sites sampled had fish. The salinity measurements were provided by the United
States Fish and Wildlife Service at Bitter Lake National Wildlife Refuge. Salinity data were collected using a digital water probe that measured salinity to within 1% of actual salinity levels (YSI Professional Plus Multiparameter Instrument, YSI Incorporation,
Yellow Springs, Ohio, USA).
Character Analysis
Morphometric measurements of 76 individuals were collected, all of which were female. Only females were used in the analysis for three reasons. First, males were found to be extremely sensitive to the stress of being handled and measured, particularly those from spring-fed localities such as Bitter Creek. Second, the distribution of male
Gambusia was also extremely disjunct in space and time, with many locations lacking males for many months out of the year (Swenton and Kodric-Brown 2012). Third,
Gambusia are extremely sexually dimorphic, which would require analysis to be
8 performed separately for males and females, however the rarity of males in the sampling rendered this impossible.
After collection, individual females analyzed in this study were transferred to a polyacrylic tank (29.7 cm x 20.3 cm x 19.5 cm). The tank was then placed on a flat platform with the tank at one end and a digital camera (Nikon d5700, Nikon Corporation,
Tokyo Japan) at the other. Each fish was then gently corralled next to the glass and an image of the fish was captured at a resolution of 6000 by 4000 pixels. A ruler was included in the frame of each image to provide scale. This approach resulted in minimal handling of the fish. Fish were then observed for 30 min to ensure recovery before being released into the sampling location from which they were collected.
From each image, standard length (mm) and body depth (mm) were collected using ImageJ software (Schneider et al. 2012). Qualitative characters for post-anal streak, mouth pigment, eye streak, later band, anal pigment, dorsal fin spots, caudal fin spots, and body spots were assessed from the images (Figure 3) and graded on a scale of 0 to either 1, 3 or 6, depending on the trait (Table 1). The post-anal streak is a line of pigment extending from the anus of the fish distally. Mouth pigment is a blotch of pigment found around the upper snout of the fish, and the eye streak is a narrow blotch of pigment starting at the bottom of the eye and extending ventrally. The lateral band is a loose band of pigmentation that runs along the side of the fish. Anal pigment is a blotch of pigment surrounding the anus. This is particularly apparent in females ready to mate.
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Statistical Analysis
Morphological variation in Gambusia spp. at BLNWR.— Permutational multivariate analysis of variance (PERMANOVA) was used to compare multivariate morphological variation among sampling localities. PERMANOVA is similar to least-squares multivariate analysis of variance (MANOVA) in that multiple independent variables can be used to predict multiple dependent variables. However, unlike MANOVA,
PERMANOVA does not require the model to conform to a particular error distribution
(Anderson 2005). Data such as the morphological data collected in this study (mixed discrete/continuous data) would violate the assumptions of least-square MANOVA, in particular the assumption of multivariate normality.
PERMANOVA is a distance (dissimilarity) based approach (Anderson 2001).
Gower dissimilarity was chosen to measure the dissimilarity between data points because it is appropriate for mixed discrete and continuous data (Gower 1971). PERMANOVA compares the sample centroid in Gower’s space for each level of the independent variable
(i.e. sampling site) to test the null hypothesis that the sampling site centroids are equidistant (Anderson 2001). The dependent variables were the 10 morphological traits
(Table 1) and the independent variables were sampling site and standard length (to account for allometric variation). The model was performed using 999 permutations. To visualize differences among sampling site centroids a distance-based redundancy analysis was used (db-RDA hereafter; Legendre and Legendre 2012). PERMANOVA and db-
RDA were performed using the package VEGAN (Oksanen et al. 2019) in the R statistical programming platform (v3.6.3, R core team 2017).
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Variation in salinity among wetlands at BLNWR. — To compare salinity among wetland types (Figure 2), I applied a generalized linear mixed model (GLMM) with wetland type (Bitter Creek, Bitter Lake, managed wetlands, rivers and oxbows, and sinkholes) as a fixed independent variable. To account for repeated measurements at each site, I included sample number as a random factor. Log salinity (ppt) was the dependent variable. GLMM was performed in the lme4 package (v1.23, Bates et al. 2015) in the R statistical programming platform (v3.6.3, R core team 2017). Alpha values were set at
0.05 for this project.
Morphology and variation in salinity. — I compared the site means for the first axis of the db-RDA (CAP 1, see below) and coefficient of variation in salinity for that site to look for a relationship between variation in salinity and changes in morphology.
To accomplish this I reduced the salinity data to sites that were also fish sampling sites. I calculated the coefficient of variation for salinity at each site as follows:
푠 퐶푉 = × 100 푥̅
Where s is the standard deviation for salinity and 푥̅ is the mean salinity. The coefficient of variation provides a measure of dispersion around the mean, expressed as a percentage. Salinity measurements were log transformed prior to the calculation of means and standard deviation for each site to approximate normality. The coefficient of variation was then used as the dependent variable in a linear regression with the site centroids of the primary morphological axis from the db-RDA (CAP 1, Figure 7) as the
11 independent variable. This model predicts variation in salinity as a function of morphology. Alpha values for this project were set to 0.05.
RESULTS
Morphological variation in Gambusia spp. at BLNWR
The PERMANOVA model suggested that the relationship between standard length and morphological variation was statistically significant; however, standard length
2 had little predictive power (F1,60 =18.628, P = 0.001, R = 0.111). This suggests that the species diagnostic features used in this study (Table 1) are weakly associated with body size, and thus the interpretation of morphological variation is unlikely to be solely attributable to allometric variation among populations.
PERMANOVA also suggested that sampling site was significantly associated
2 with morphological variation (F6,60 = 14.937, P = 0.001, R = 0.533). Interpretation of morphological variation among sites was accomplished by plotting site centroids against the primary axis from the db-RDA (Figure 4). The primary axis of variation (CAP 1) accounted for 66.8% of the variation found among sites. CAP 1 summarized variation in dorsal spots, caudal spots, post-anal streak, anal pigment, mouth pigment, eye streak, color, lateral band, body depth, and body spots (Table 2). The secondary axis of variation
(CAP 2) accounted for 16.2% of the variation in morphology and summarized variation in color, body spots, caudal spots, dorsal spots, mouth pigment, eye streak, post-anal streak, lateral banding, body depth, and anal pigment (Table 2). 12
Sinkholes and Lost River Flume samples clustered together and were associated with tan coloration, mouth pigment and eye streak (Figure 4, Table 2). Fish sampled from the Visitor Center were similar in respect to CAP 1 but were separated somewhat on CAP
2 being associated with lateral banding, eye streak, and body depth (Figure 4, Table 2).
Centroids for the sampling sites The Farm and Spring 2 were separated on CAP 1 from other sites and were somewhat separated from one another on CAP 2 (Figure 4). The
Farm and Spring 2 were associated with caudal spots, post-anal streak and anal pigment
(Figure 4, Table 2). Fish sampled from sinkholes Lost River Flume and the Visitor Center showed similar variance in morphology (95% confidence ellipses) which was considerably less variance than demonstrated in fish collected from The Farm and Spring
2 (Figure 4).
Variation in salinity among wetlands at BLNWR
GLMM indicated that significant variation in salinity existed among wetland types (X2 = 302.26, df = 4, P = <0.001). Salinity at Bitter Creek was on average similar to managed wetlands and rivers and oxbows (Figure 5). However, Bitter Creek had very little variation in salinity compared to these habitats (Figure 5). Salinity in sinkholes was greater than other habitats with the exception of Bitter Lake (Figure 5). Bitter Lake was on average 2.3 times saltier than seawater. In addition, spatial visualization suggests
Bitter Lake to be a significant barrier to the movement of fish into and out of Bitter Creek
(Figure 6).
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Morphology and variation in salinity
The regression model was not statistically significant (β = 0.809, SE = 1.508, t =
0.537, df = 5, P = 0.615, R2 = 0.05). These data do not suggest a trend with greater variance in salinity being associated with the CAP 1 axis (Figure 7). I did not detect morphological variation being higher in sites with greater variation in salinity.
DISCUSSION
This study suggests that populations of Gambusia at Bitter Lake National Wildlife
Refuge differ considerably in morphology among sites. In addition, the observed morphological variation at some sampling sites is constant with Pecos gambusia, with caveats. Rodriquez (2017) measured morphology and used micro-satellites to identify
Pecos gambusia at Balmorhea State Park, Texas. The author found that deep bodies, eye streaks, tan coloration, and lateral banding was associated with Pecos gambusia. These traits are seen in fish sampled from the sinkholes, Lost River Flume (located on Bitter
Creek), and the Visitor Center at Bitter Lake National Wildlife Refuge. These sites were specifically described as Pecos gambusia populations in the species recovery plan (Hubbs et al. 1983). However, fish from sinkholes Lost River Flume and the Visitor Center also had body spots and mouth pigment, traits which Rodriquez (2017) associated with
Largespring gambusia or putative hybrids. In Page and Burr (2011), body spots were associated with Largespring gambusia.
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The Farm and Spring 2 separated distinctly and were associated with caudal spots, dorsal spots, post-anal streak, and anal pigment. Rodriquez (2017) found that anal pigment was associated with Pecos gambusia. However, caudal fin spots and dorsal fin spots are associated with Mosquitofish and Largespring gambusia (Page and Burr 2011).
In addition, the Farm and Spring 2 showed a great deal of variation in morphology (95%
CI). Increased phenotypic variation is commonly seen in hybrid populations (Grant and
Grant 2019, Tobler and Carson 2010). In the late 1970s, Pecos gambusia X Mosquitofish hybrids were collected from this region of the refuge (Echelle and Echelle 1980). The characteristics associated with different sites suggests that Pecos gambusia can be found primarily in locations such as sinkholes, Bitter Creek and possibly near the Visitor
Center. Fish from the Farm and Spring 2 may be Mosquitofish or Mosquitofish-like hybrids. However, in absence of a molecular analysis it is not possible to say for certain.
As mentioned above, characteristics were observed in possible Pecos gambusia sites that did not coincide with all of the previously described characteristics for this species. The morphological differences between these species are extremely subtle and vary considerably among populations within a species to such a degree that measurements of the male intromittent organ, the gonopodium, is required for certain species identification (Hubbs et al. 1983, Stoops et al. 2013). In addition, Pecos gambusia is likely somewhat divergent between extant populations because of isolation, both as a result of modern anthropogenic factors but also drying since the end of the Pleistocene
(Reeves 1973). This divergence is a major consideration mentioned in the species recovery plan (Hubbs et al. 1983). Also, Echelle and Echelle (1986) measured
15 characteristics different from those presented here at all extant populations except Bitter
Lake National Wildlife Refuge and found significant variation between the four populations studied. Thus, the lack of congruence between morphological variability described in this study and variation described elsewhere could simply reflect long-term population divergence.
I documented significant variation in salinity in the wetlands at Bitter Lake
National Wildlife Refuge. While variable salinity is generally within the tolerances of invasive Mosquitofish and thus unlikely to prohibit its movement through most wetlands at Bitter Lake National Wildlife Refuge, Chervinski (1982) suggested that with prior exposure and conditioning, Mosquitofish were able to survive in 100% seawater (35 ppt).
Hubbs (2000) described a population of Mosquitofish which survived for a week at 41 ppt. Sinkholes were generally found to be intermediate in salinity with a large variance.
This contrasts with the wetlands harboring Pecos gambusia populations found at Bitter
Creek, which are relatively less saline and very limited variability because of being spring fed. Several sinkholes at Bitter Lake National Wildlife Refuge are thought to have natural populations of Pecos gambusia (Sinkhole 7, Sinkhole 20, Sinkhole 27, Sago
Sinkhole and Dragonfly Spring Sinkhole; Hubbs et al. 1983). In 1973 supplemental stocking of Pecos gambusia occurred in Sinkhole 20 (Hubbs et al. 1983). During the
1970s populations of Pecos gambusia were established in six other sinkholes at Bitter
Lake National Wildlife Refuge (Hubbs et al. 1983). Both the natural and established sinkholes at Bitter Lake National Wildlife Refuge have continued to reinforce the
16 population of Pecos gambusia, as they provide a robust barrier to fish movement on the property.
Likewise Bitter Lake presents a barrier to fish movement because of its high salinity. The hyper-salinity of Bitter Lake is caused by the salt deposits present in the soil and their concentration in the water by evaporation, which occurs in the arid climate of
Southeastern New Mexico. The inflow into Bitter Lake is Bitter Creek. Two possible out flows exist, one southeast and the other northeast of Bitter Lake. Bitter Lake experiences large changes in water level over the course of the year, and thus the only opportunity for a connection to be made with the managed wetlands would be during later summer to mid-fall as a result of monsoonal rains. This connection during most years is minimal. In addition, at its most diluted, Bitter Lake is still quite saline. This means that in order for
Mosquitofish to reach Bitter Creek, they would have to be able to withstand elevated salinity that is far beyond what has been demonstrated as their thresholds. This would provide serious challenge for 2.5 cm fish to cross Bitter Lake (distance of 1.25 km minimum). This project supports the hypothesis that Bitter Lake acts as a physical barrier to invasive gambusia spreading into Bitter Creek.
I hypothesized that morphology would correlate with variation in salinity. This is because of Pecos gambusia being a spring adapted species. This hypothesis was not supported by the data. Spring systems support numerous endemic species in the Western
United States (Davis et al. 2017, Minckley 1991). This endemism is thought to have arisen through aridification at the end of the last ice age, leaving previously continuous aquatic communities separated into disjunctive systems (Thompson and Anderson 2000, 17
Van Devender and Spaulding 1979). These isolated spring populations persisted and evolved into distinct populations (Davis et al. 2017). Spring systems can be very stable systems. In fact, the recovery plan for Pecos gambusia postulates that Mosquitofish may have an advantage over Pecos gambusia in unstable habitats (“downstream waters removed from spring influence”; Hubbs et al. 1983). Pecos gambusia may reverse this dynamic in the stability of spring habitats (Hubbs et al. 1983). It could be assumed that
Pecos gambusia, being found primarily in spring systems, would generate a pattern of
Pecos gambusia morphotypes being more prevalent in sites with limited variation in salinity. However, this prediction was not supported. This could be for several reasons.
First, the morphological traits measured here may be an imperfect predictor of parental or hybrid identity. For example Rodriquez (2017) found hybrid Pecos gambusia X
Largespring gambusia to be more similar to Largespring gambusia morphologically.
Secondly, sinkholes are much more variable with respect to salinity than spring habitats.
Clearly, Pecos gambusia do quite well in sinkhole habitats.
Issues with Maintaining Protected Status
One area of concern is that of maintaining protected status for Pecos gambusia.
Endangered species hybrids are not explicitly covered under the Endangered Species Act
(ESA). Pecos gambusia is currently listed as an endangered species. This listing provides additional protection that would not be provided otherwise. This is crucial for the continued management for Pecos gambusia. While there are hybrids that have received protection under the ESA, this has been controversial (Erwin 2017, O’Brien and Mayr
1991).
18
Additional Threats to Consider
In the 1983 recovery plan (Hubbs et al. 1983), there were factors that were given credit as the reasons for the decline of Pecos gambusia throughout its range. The two factors that were mentioned were loss of habitat and the introduction of exogenous fishes
(Hubbs et al. 1983). The two species that most directly impact Pecos gambusia are
Mosquitofish and Largespring gambusia. These two species have been introduced throughout the range of Pecos gambusia and are listed as impacting Pecos gambusia primarily through direct competition and hybridization. Hybridization, particularly with
Mosquitofish was listed as one of the factors affecting abundance and distribution, among others. However, at the time, hybridization was not thought to be an immediate threat to most of the populations. Because of the findings of this study, and those presented by
Rodriguez (2017), there is evidence that this statement may need to be further evaluated.
While there is much to be concerned about regarding habitat loss and competition for invasive fishes, evidence points to the need to include hybridization as well.
To close, the data gathered in this study supports the hypothesis that there is hybridization occurring on the property between Pecos gambusia and Mosquitofish.
There is also evidence that there are physical barriers that have prevented Mosquitofish from spreading further throughout the property, particularly the high levels of salinity found in Bitter Lake. This is particularly important as it does not seem possible for
Mosquitofish to reach the northern portion of the property without being transported by humans. The results from this study coupled with the findings of Rodriquez (2017) highlight the need for continual monitoring of the status of Pecos gambusia. These results
19 also suggest that the Pecos gambusia population found at Bitter Creek (e.g. Lost River flume) are an excellent candidate for a more in-depth genomics study.
20
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Table 1. Character coding for Gambusia spp. characteristics measured at Bitter
Lake National Wildlife Refuge 2018-2019. Columns represent the assigned
grade given for a character. Dashes represent the lack of numeral use for the
character. Characters 0 1 2 3 4 5 6
Lateral None Light, Dusky, Dark, Light, Dusky, Dark, Band Thin Thin Thin Thick Thick Thick Anal None Small, Small, Small, Large, Large, Large, pigment Light Dusky Dark Light Dusky Dark Post-anal None Light Dusky Dark −− −− −− streak Mouth None Light Dusky Dark −− −− −− pigment Eye streak None Light Dusky Dark −− −− −− Dorsal Spots Absent Present −− −− −− −− −−
Caudal spots Absent Present −− −− −− −− −−
Body spots Absent Present −− −− −− −− −−
Color −− Grey Taupe Tan −− −− −−
30
Table 2. Morphological character loadings for CAP 1
and CAP 2 axis of the db-RDA used to evaluate
morphological variation in Gambusia at Bitter Lake
National Wildlife Refuge, 2018-2019 Characteristic CAP 1 CAP 2 Body Depth (mm) -0.28576 -0.56038
Lateral Band -0.10477 -0.52011 Post Anal Streak 1.106567 -0.36178 Anal Pigment 0.729309 -0.7362
Mouth Pigment 0.372929 -0.03722 Eye Streak 0.222321 -0.31213 Dorsal Spots 1.407811 0.06513
Caudal Spots 1.277782 0.086369 Color 0.112009 0.224526 Body Spots -0.6075 0.220332
31
Figure 1. Locations of the four extant populations of endangered Pecos gambusia within Texas and New Mexico, USA. Larger markers indicate
Pecos gambusia locations.
32
Figure 2. Sampling sites and wetlands at Bitter Lake National Wildlife
Refuge, New Mexico used to evaluate morphological variation in
Gambusia, 2018-2019.
33
analstreak
streak -
Dorsal fin spots fin Dorsal Lateral band Lateral Pigment Anal Post pigment Mouth Eye spots fin Caudal spots Body
------
6 2 3 4 5 7 8 1
spp. at Bitter Lake National Wildlife Wildlife National Lake at spp. Bitter
Gambusia
characteristics used to evaluate evaluate to used characteristics
Morphological
Figure 3. 3. Figure Refuge.
34
. Sampling .
2019
-
used to evaluate evaluate to used
lipses.
RDA (CAP 1 and CAP 2) CAP and 1 (CAP RDA
-
at Bitter Lake National Wildlife Refuge, 2018 Wildlife Bitter Refuge, National at Lake
Gambusia
Figure 4. Ordination of the first to axis Ordination 4. first the db the of of Figure variation in morphological el as shown points colored confidence with 95% are sites
35
is shown by the dotted line. line. dotted the shown is by
G. affinis G.
r r
. Observations (log transformed) are shown as black points. shown shown are (±SE) black transformed) Means (log red. in as are Observations .
2019
-
, 2018 ,
Figure 5. Salinity measurements for different wetland types at Bitter Lake National Wildlife Refuge, New New Wildlife Refuge, National Bitter at wetland types measurements different Lake Salinity 5. for Figure Mexico tolerance fo Maximumsalinity
36
Lost River Flume
Figure 6. Log mean salinity (log(ppt)) for wetlands at Bitter Lake National
Wildlife Refuge, Chaves Co., NM, 2018-2019
37
Figure 7. Visualization of the relationship between the major axis of morphological variation yielded from the db-RDA (CAP 1) and the coefficient of variation of salinity (Salnity CV). Morphometric and salinity data collect at Bitter Lake National Wildlife Refuge during
2018 and 2019.
38