Population Genetics of the Endangered Giant Kangaroo Rat,
Dipodomys ingens, in the Southern San Joaquin Valley
Nicole C. Blackhawk, B.S.
A Thesis Submitted to the Department of Biology California State University, Bakersfield In Partial Fulfillment for the Degree of Masters of Science
Summer 2013
Copyright
By
Nicole Cherri Blackhawk
2013
Summer 2013
Population Genetics of the Endangered Giant Kangaroo Rat,
Dipodomys ingens, in the Southern San Joaquin Valley
Nicole C. Blackhawk
This thesis has been accepted on behalf of the Department of Biology by their supervisory committee:
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Population Genetics of the Endangered Giant Kangaroo Rat, Dipodomys ingens, in the
Southern San Joaquin Valley
Nicole C. Blackhawk
Department of Biology, California State University, Bakersfield
Abstract
The Giant Kangaroo Rat (Dipodomys ingens) is a federally and state-listed endangered
species, endemic to the San Joaquin Valley, Carrizo and Elkhorn Plains, and the Cuyama Valley.
Populations of the endangered Giant Kangaroo Rat (Dipodomys ingens) have decreased over the
past 100 years because of habitat fragmentation and isolation. Changes in the population
structure that can occur due to habitat fragmentation can significantly affect the population size
and the dispersal of these animals. Dr. David Germano and I collected small ear clippings from
male and female Giant Kangaroo Rats from six sites along the southern San Joaquin Valley to
determine the genetic population structure of this species in this part of their range. We
predicted that geographic distance and isolation of populations would decrease genetic
relatedness compared to populations closer together. Having a better understanding of the
genetic structure in this species will help with conservation actions, such as translocating
individuals within the range of the species. These data were compared to published estimates of
genetic diversity of Giant Kangaroo Rats in the Carrizo Plain to the west and the Panoche area to the north, the other large population centers of this species.
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Table of Contents
Page List of Tables………………………………………………………………………….. 4
List of Figures………………………………………………………………...……….. 5
CHAPTER 1: Species Background and Purpose of the Study……………...…. … 7
Literature Cited………………………………………………………………………... 21
CHAPTER 2………………………………………………………………………….. 24
Population Genetics of the Endangered Giant Kangaroo Rat, Dipodomys ingens, in the Southern San Joaquin Valley
Introduction……………………………………………………………………………. 25
Methods and Materials………………………...……………………………………… 27
Results…………………………………….…………………………………………… 30
Discussion……………………………….…………………………………………….. 33
Acknowledgements……………………………………………………………………. 39
Literature Cited………………………………………………………………………... 39
List of Figures…………………………………………………………………………. 48
CHAPTER 3: Conservation Actions and the Reassessment of Giant Kangaroo 54 Rat Habitat Occupancy…..…………………………………………………………..
Genetic Rescue……………………….……………………………………………….. 57
Translocation as a Conservation Tool………………………………………………… 59
Effects of Habitat Fragmentation……………………………………………………… 63
Reassessment of Habitat Occupancy………………………………………………….. 65
Conclusions…………………………………………………..……………………….. 67
Acknowledgements………………………………………………………………….... 69
Literature Cited………………………………………………………………………... 70
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List of Tables
CHAPTER 2
TABLE 1………………………………………………………………………..……. 42
Observed allele frequencies by locus and population of Giant Kangaroo Rat (Dipodomys ingens) from the southern San Joaquin Valley, California
TABLE 2…………………………………………………………………..…………. 44
Descriptive statistics for six populations across seven loci, including the mean sample size (n), proportion of polymorhphic loci (P), number of alleles (NA), number of effective alleles (Ne), information index (I), observed heterozygosity (Ho), expected heterozygosity (HE), the mean number of private alleles, and the fixation (F) indices of Giant Kangaroo Rat (Dipodomys ingens) from the southern San Joaquin Valley, California
TABLE 3…………………………………………………………………..…………. 45
Population comparisons of six populations of Giant Kangaroo Rat (Dipodomys ingens) in the southern San Joaquin Valley using an analysis of molecular variance (AMOVA)
TABLE 4…………………………………………………………………..…………. 46
Estimates of F-statistics across six populations of Giant Kangaroo Rat (Dipodomys ingens) in the southern San Joaquin Valley by locus FIS: standard genetic variation within populations; FST: standard genetic variation between populations; FIT: to partition genetic variation
TABLE 5……………………………………………………………..………………. 47
Pairwise values of FST, ΦPT, and Nei’s genetic distance for six populations of Giant Kangaroo Rat (Dipodomys ingens) in the southern San Joaquin Valley, California
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List of Figures
CHAPTER 1
FIGURE 1……………………………………………………………………. 8
The Giant Kangaroo Rat (Dipodomys ingens). This individual was caught at a study site (North Lokern) west of Buttonwillow, Kern County, California, in the southern San Joaquin Valley. (Photographed by Nicole Blackhawk).
FIGURE 2……………………………………………………………………. 13
The pictures show the differences in appearance of a normal seed cache (A) of the Giant Kangaroo Rat (Dipodomys ingens) compared to a population that has been experiencing food shortage (B). (Photographed by Nicole Blackhawk).
FIGURE 3……………………………………………………………………. 15
Locations of Giant Kangaroo Rat (Dipodomys ingens) populations sampled for this project (orange triangles). The green triangles indicate the populations sampled previously by Good et al. (1997) and Loew et al. (2005). The grey shading indicates the species’ historic range (IUCN 2012).
FIGURE 4……………………………………………………………………. 17
Three sites in the Lokern area of the southern San Joaquin Valley where I collected genetic samples of Giant Kangaroo Rats (Dipodomys ingens). A: North Lokern study site. B: Study site south of the North Lokern site. C: Study site south of Buttonwillow, California on the southeast portion of the Lokern. (A and C photographed by David J. Germano; B photographed by Nicole Blackhawk).
FIGURE 5……………………………………………………………………. 19
Two sites in the southern San Joaquin Valley where I collected genetic samples of Giant Kangaroo Rats (Dipodomys ingens). A: Midway study site. B: Study site northwest of Midway site and southwest of the Lokern sites. (Photographed by David J. Germano).
FIGURE 6……………………………………………………………………. 20
The northern most site (Northwest Belridge study site) where I collected genetic samples from Giant Kangaroo Rats (Dipodomys ingens) in the southern San Joaquin Valley. (Photographed by David J. Germano).
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CHAPTER 2
FIGURE 1……………………………………………………………………. 49
Locations of Giant Kangaroo Rat (Dipodomys ingens) populations sampled for this project (orange triangles). The green triangles indicate the populations sampled previously by Loew et al. (2005). The grey shading indicates the species’ historic range (IUCN 2012)
FIGURE 2……………………………………………………………………. 50
Locations of Giant Kangaroo Rat (Dipodomys ingens) populations sampled for this project (orange triangles). The grey shading indicates the species’ historic range (IUCN 2012)
FIGURE 3……………………………………………………………………. 51
Visualization of Mantel test for geographic distance (GGD) vs. pairwise population ΦPT (PhiPTP) for six populations of Giant Kangaroo Rat (Dipodomys ingens) in the southern San Joaquin Valley, California
FIGURE 4……………………………………………………………………. 52
Visualization of the percentage variation explained by the first three axes of the Principal Coordinate Analysis of six populations of Giant Kangaroo Rat (Dipodomys ingens) in the southern San Joaquin Valley, California
FIGURE 5……………………………………………………………………. 53
UPGMA tree based on a pairwise population matrix of Nei’s genetic distances for six populations of Giant Kangaroo Rat (Dipodomys ingens) in the southern San Joaquin Valley, California
CHAPTER 3
FIGURE 1…………………………………………………………………….. 66
The historic range of the Giant Kangaroo Rat (Dipodomys ingens). The red triangles represent the places that I traveled to that did not have Giant Kangaroo Rats present and the green graduated circles represent location and size of known populations that have been genetically sampled. The grey shading represents the species’ historic range (IUCN 2012).
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CHAPTER 1
Species Background and Purpose of the Study
Kangaroo rats (Dipodomys sp.) are rodents in the family Heteromyidae, which consists of six genera. The Heteromyidae is a group of small to medium sized rodents that occur primarily in deciduous thorn-scrub and arid-adapted habitats in North America (Alexander and Riddle
2005), and they exhibit an array of ecomorphological types (Hafner et al. 2007). The six genera are traditionally placed into three subfamilies: Heteromyinae, Perognathinae, and Dipodomyinae.
Dipodomyinae is made up of two genera: Dipodomys, the kangaroo rats, and Microdipodops, the kangaroo mice. There are 21 recognized species of Dipodomys (Goldingay et al. 1997; Cooper and Randall 2007; Prugh and Brashares 2010) and California has the greatest diversity of kangaroo rats of any state (Grinnell 1922) with 13 species, of which there are 23 subspecies endemic to the state (Goldingay et al. 1997). This high diversity is likely due to the climatic history and geomorphic processes along the crustal plates extending the length of the state
(Goldingay et al. 1997).
Using genetic data, Hafner et al. (2007) affirmed the monophyly of Heteromyidae.
Alexander and Riddle (2005) used maximum-likelihood, Bayesian, and maximum parsimony analyses of sequence data from two mitochondrial DNA genes, cytochrome oxidase subunit 3 gene, and cytochrome-b gene to determine phylogenetic relationships among 55 species-level taxa. They found support for monophyly of Dipodomys, Microdipodops, Chaetodipus, and
Perognathus. Both the maximum parsimony and 50% majority-rule consensus Bayesian trees showed a polytomy (a section of the phylogeny with unresolved evolutionary relationships; more than two descending branches) between Dipodomys ingens (Fig. 1), D. microps, D. gravipes; however, the maximum-likelihood tree gave support for D. microps as being the sister taxon of
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Figure 1. The Giant Kangaroo Rat (Dipodomys ingens). This individual was caught at a study site (North Lokern) west of Buttonwillow, Kern County, California, in the southern San Joaquin Valley. (Photographed by Nicole Blackhawk).
D. ingens. According to Stock (1974), the heermanni subgroup arose in the early Pleistocene and D. ingens shared a common ancestor with D. heermanni in the late Pleistocene.
Dipodomys ingens, the Giant Kangaroo Rat has been listed federally as endangered since
1987 and state-listed as endangered since 1980 (Williams et al. 1995; Williams and Kilburn
1991). The Giant Kangaroo Rat is an endemic to the western San Joaquin Valley and adjacent areas to the southwest (Kays and Wilson 2009). It now inhabits significantly less area than its estimated historic range and has experienced increased fragmentation and isolation from
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agricultural development (Good et al. 1997; Loew et al. 2005). Its numbers began decreasing in
the early 1900s, coincident with poisoning of the California Ground Squirrel, Spermophilus
beecheyi (Williams 1992; Whisson 1999). Historically, the Giant Kangaroo Rat was distributed
on a narrow band on gently sloping hills along the western San Joaquin Valley (Williams and
Kilburn 1991). Records show that they used to extend from the base of the San Emidio
Mountains on the south up to about 16 km south of Los Banos, Merced Coounty (Williams and
Kilburn 1991). They also were distributed on areas to the west of the San Joaquin Valley
including the Carrizo and Elkhorn Plains west of the Temblor Mountains, and the upper Cuyama
Valley adjacent to the Carrizo Plain (Williams and Kilburn 1991). There were also scattered
colonies in Ciervo, Kettleman, Panoche, and Tumey hills (Williams and Kilburn 1991).
Up until the late 1960s and the early 1970s, very little land within the geographic range
of the Giant Kangaroo Rats was cultivated or developed and they were wide spread in their
historic range (Loew et al. 2005). However, with the completion of the canals of the State Water
Project and the San Luis unit of the Central Valley Project, water for irrigation became available
to the west side of the Tulare Basin (the main habitat for Giant Kangaroo Rats; Williams and
Kilburn 1991). When the irrigation development began, much of the Tulare Basin was converted
to agriculture and this restricted the occurrence of most species in the valley including the Giant
Kangaroo Rat (Good et al. 1997; Loew et al. 2005; Williams and Kilburn 1991). The Giant
Kangaroo Rat then became isolated to areas in and around the Elk Hills in Kern County; the
Kettleman Hills in Fresno County; the southern portions of the Carrizo and Elkhorn Plains in San
Luis Obispo County; the Panoche Valley and Tumey Hills in Fresno and San Benito counties; as well as other small units scattered in the hills west of the Tulare Basin (Williams and Kilburn
1991).
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The habitat of Giant Kangaroo Rats typically consists of gently sloping piedmont plains without much shrub coverage located on sandy loam soils (Shaw 1934; Williams and Kilburn
1991). It is possible that the presence and absence of shrubs is related to the texture of the soils and the intensity of livestock grazing (Williams and Kilburn 1991), although past fires that only occurred once non-native grasses invaded the valley may also explain some areas devoid of shrubs (Germano et al. 2001). Some common plants that reside in the areas where Giant
Kangaroo Rat colonies are located include: annual Fescues (Festuca megalura and F. microstachys), Arabian Grass (Schismus arabicus), Bandergee (Eastwoodia elegans), California
Ephedra (Ephedra californica), Cheesebush (Hymenoclea salsola), Desert Thorn (Lycium andersonii), Fiddleneck (Amsinckia douglasiana), Filaree (Erodium cicutarium), Golden Bush
(Haplopappus acradenius), Malpais Blue Grass (Poa scabrella), Peppergrass (Lepidium nitidum), Red Brome (Bromus rubens), San Joaquin Saltbush, Snake-weed (Gutierrezia californica), and Winterfat (Eurotia lanata; Shaw 1934; Williams and Kilburn 1991). Giant
Kangaroo Rats are not found in irrigated fields, but do reestablish colonies in fallow dry grain fields when left uncultivated (Williams and Kilburn 1991). A fallow field site just south of
Buttonwillow was one of the sites at which I sampled Giant Kangaroo Rats. Predators of Giant
Kangaroo Rats include Barn Owls (Tyto alba), Great Horned Owls (Bubo virginianus), Coyotes
(Canis latrans), San Joaquin Kit Foxes (Vulpes macrotis mutica), Badgers (Taxidea taxus),
Prairie Rattlesnakes (Crotalis viridis), Gopher Snakes (Pituophis melanoleucus), Common King
Snakes (Lampropeltis getulus), and Coachwhips (Masticophis flagellum; Williams and Kilburn
1991).
Kangaroo rats play important ecological roles in the ecosystem structure and composition
(Goldingay et al. 1997). Prugh and Brashares (2012) described kangaroo rats as being keystone
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ecosystem engineers because of the important role they play in altering soil characteristics
(Williams and Kilburn 1991), changing habitat structure through grazing, and creating extensive networks of burrows that are used by many other species such as the threatened San Joaquin
Antelope Squirrel (Ammospermophilus nelsoni), and the endangered Blunt-nosed Leopard Lizard
(Gambelia sila). Kangaroo rats are important in seed dispersal of grasses (Brown et al. 2001) and play an important role when they reach high densities in areas where they are located
(Germano et al. 2001). Giant Kangaroo Rats can dramatically transform entire landscapes by their burrow development and foraging (Hawbecker 1944; Prugh and Brashares 2012). Giant
Kangaroo Rats constantly alter their precincts (burrow system) by digging and clipping paths, which result in chronic soil disturbance that has promoted the establishment of exotic and early successional plant species (Schiffman 1994). The exotic grasses that have established themselves on and around the precincts pose a management dilemma to conserving the native plant species (Schiffman 1994). In areas with Giant Kangaroo Rats where cattle and sheep are present, the grazers will trim the vegetation very close to the ground; whereas, when the grazers are absent, the Giant Kangaroo Rats will clip the vegetation around their precincts, and along runways leading from one precinct to another. It is thought that this helps them to evade predators by providing them with a clear escape path (Schiffman 1994). However, successive years of high growth of non-native grasses can overwhelm the abilities of Giant Kangaroo Rats to clear their habitat, and their numbers decline if large grazers do not keep herbaceous cover low (Germano et al. 2001, 2012).
Their very prominent burrow mounds (precincts) often are 7–10 m in diameter and are cleared of vegetation, even in years with dense plant cover (Shaw 1934; Prugh and Brashares
2012). Precincts will usually be composed of one to five separate burrow openings with three
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being most common (Williams and Kilburn 1991). Giant Kangaroo Rats will occasionally plug
their burrow entrances. Openings consist of two types: (1) a vertical shaft with a circular
opening approximately 50 mm in diameter without a well-worn trail leading away and (2) a
larger horizontally opening shaft with a well-worn path leading from the mouth (Williams and
Kilburn 1991). Giant Kangaroo Rats will clip the ripening heads of grasses and forbs and cure
them in surface pits on and around their precincts. These pit caches usually are similar in depth
and diameter at approximately 2.5 cm (Shaw 1934; Fig. 2A). At my North Lokern site, the Giant
Kangaroo Rats were experiencing a shortage of food during fall 2012 because of a preceding dry
winter and high numbers of kangaroo rats at the site (Germano, pers. comm.). Here the soil was
entirely disturbed without distinguishable pits (Fig. 2B). At this site, several of the kangaroo rats
that were captured had injuries that may have been due to competition over food (Jenkins and
Peters 1992).
Giant Kangaroo Rats will also drum their hind feet against the substrate
(“footdrumming”). This type of behavior usually occurs after an encounter with an intruder
(Williams and Kilburn 1991; Randall 1997). Footdrumming can also play a role in female mate choice. Meshriy et al. (2011) found that females were more likely to mate with nearby neighboring males that were familiar to them. Familiarity can be based on olfactory senses as well as a distinct footdrumming pattern (Murdock and Randall 2001). Giant Kangaroo Rats are
territorial and live solitarily among evenly spaced precincts (Meshriy et al. 2011; Randall 1997;
Shaw 1934; Williams and Kilburn 1991). The size of Giant Kangaroo Rat home ranges varies
between 60 and 350 m2 with no differences between males and females have been found (Braun
1985; Williams and Kilburn 1991).
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A
B
Figure 2. The pictures show the differences in appearance of a normal seed cache (A) of the Giant Kangaroo Rat (Dipodomys ingens) compared to a population that has been experiencing food shortage (B). (Photographed by Nicole Blackhawk).
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Because the Giant Kangaroo Rat has experienced agricultural and residential
development encroaching on its natural habitat and its historic range (Loew et al. 2005; Fig. 3),
populations of the species have become increasingly fragmented and isolated recently.
Understanding the genetic structure of these remaining, isolated populations is important to
conserving and recovering the species. Good et al. (1997) and Loew et al. (2005) completed genetic analyses of populations of Giant Kangaroo Rats at three sites in the northern part of the range (Panoche Valley, Tumey Hills, and Ciervo Hills) and on the Elkhorn and Carrizo Plains in the southern end of the range west of the San Joaquin Valley (Fig. 3). The purpose of my study was to determine the genetic structure of populations of Giant Kangaroo Rats in the southern
San Joaquin Valley compared to populations on the Carrizo and Elkhorn Plains and those in the northern part of the range. Dr. David Germano and I collected samples from six populations in the southern San Joaquin Valley, a part of the range encompassing a significant portion of remaining populations of Giant Kangaroo Rats and for which genetic analysis has not been published (Fig. 3).
Over the last few decades, population genetics has become a well-developed field as a result of advances in DNA sequencing technologies, and which can give insight into the conservation status of species threatened by human activities. The population structure of a species affects loci across the genome in similar ways; whereas, natural selection can affect a single locus (Wakeley 2004). Most models that examine population structure assume that populations are in equilibrium with respect to mutation, drift, and migration (Good et al. 1997).
This assumption may not be met if populations have been recently subdivided and/or expanded or if the mutation rate of a certain locus being examined is high (Good et al. 1997). This makes reconstructing the factors causing the natural structure difficult. DNA sequencing allows
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Figure 3. Locations of Giant Kangaroo Rat (Dipodomys ingens) populations sampled for this project (orange triangles). The green triangles indicate the populations sampled previously by Good et al. (1997) and Loew et al. (2005). The grey shading indicates the species’ historic range (IUCN 2012).
biologists to gain information about the evolutionary history of a population such as size,
fragmentation, and selection history (Frankham et al. 2009).
The coalescent theory and reconstructing gene trees has enhanced our ability to examine
the genealogical relationships among individuals and their population processes influencing their
current genetic structure (Good et al. 1997; Frankham et al. 2009). Wright’s F-statistic assumes
that genealogical processes occur; however, until recently we have not had the molecular tools to
estimate some of the key parameters that can influence a population. Microsatellite genotyping
is the current method for analyzing genetic structure in animal populations because it reflects the
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forces of mutation, migration, genetic drift, and inbreeding (Loew et al. 2005; Frankham 2009).
High rates of change at microsatellite loci allow the reconstruction of changes in the population structure (Loew et al. 2005). As a means to understanding the genetic structure, if any, of populations of Giant Kangaroo Rats in the southern San Joaquin Valley, I measured the genetic diversity of the six populations described above (Fig. 3) including the number of alleles per locus, the observed heterozygosity, and the expected heterozygosity under Hardy-Weinberg
assumptions using primers characterized by Davis et al. 2000. I also determined the degree of
genetic differentiation within and among my predefined geographical populations with pairwise
Fst measures and AMOVA using GENALEX v6.5 (Peakall and Smouse 2006; 2012). I then
compared my data to the published information by Good et al. (1997) and Loew et al. (2005).
Three of the sites I sampled were located in the Lokern area of the southern San Joaquin
Valley, west and south of Buttonwillow, Kern County, California. The northernmost Lokern site
was just west of the California aqueduct (Fig. 4A) and was surrounded by agricultural fields.
This site was a 48 ha (120 ac) parcel of land with a native cover of saltbush (Atriplex sp.) shrubs
(Fig. 4A). It was managed by the U.S. Bureau of Land Management (BLM). This site was
isolated from nearby natural lands supporting other populations of Giant Kangaroo Rats. The
Lokern site south of the BLM study area was not isolated and was part of a large expanse of
natural land rising up from the valley floor to the base of the Elk Hills (Fig. 4B). It was private
land that had been grazed by cattle in the recent past, and oil drilling operations occurred nearby.
Similar to the BLM site (about 9 km north), this site also was a mixture of open patches of
herbaceous plants and saltbush shrubs (Fig. 4B). The third sampling area on the Lokern was just
south of Buttonwillow past the agricultural fields on Elk Hills Road and just north of the
California aqueduct (Fig. 4C). This site was the only one that was sampled to the east of the
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A
B
C
Figure 4. Three sites in the Lokern area of the southern San Joaquin Valley where I collected genetic samples of Giant Kangaroo Rats (Dipodomys ingens). A: North Lokern study site. B: Study site south of the North Lokern site. C: Study site south of Buttonwillow, California on the southeast portion of the Lokern. (A and C photographed by David J. Germano; B photographed by Nicole Blackhawk).
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aqueduct. The aqueduct could serve as a recent genetic barrier. This site was about 12 km east of the second site. The area once had been used for agriculture, but might have been left fallow for a number of years and was returning to its natural state (Fig. 4C). Giant Kangaroo Rats had reinvaded the previously plowed ground and numbers seemed to be increasing based on many recently constructed precincts. The fourth site (Midway) I sampled was located just north of
Midway Road about halfway between Highways 119 and 33 (Fig. 5A). This was on the property of Occidental Petroleum. The area was an active oilfield with numerous pipelines crossing the area. As with the other sites, the habitat was open herbaceous plant covered areas with strands of saltbush shrubs interspersed (Fig. 5A). Northwest of this site but southwest of the Lokern sites was my fifth sampling area (Derby Acres site). It was located about 3 km east of Hwy 33 past oil fields east of the town of Derby Acres (Fig. 5B). Unlike the other sites, there was no shrub cover at this site (Fig. 5B). The most northernmost site I sampled was the Northwest Belridge site, which was located west of Hwy. 33 approximately 8 km north of Lokern Road (Fig. 6).
This site was near some well-developed oilfields and cattle were grazing on the land when I sampled the site. This site also had no shrubs of any kind (Fig. 6).
This study will be important in the conservation of this species as well as others that have experienced similar genetic isolation. Understanding the genetic structure of a species, especially of an endangered one, is important for identifying, protecting, and managing populations that could serve for future colonizations and assess the risk of local extinctions. The most at-risk populations can be determined and a genetically similar population could be used to help the at-risk population. If the genetic structure of these populations is better understood, then conservation action such as translocation projects or establishing corridors can be taken to help promote the populations’ genetic diversity. The next chapter is a manuscript of the data I
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collected and analyzed that I will be submitting to the peer-reviewed journal Conservation
Genetics.
A
B
Figure 5. Two sites in the southern San Joaquin Valley where I collected genetic samples of Giant Kangaroo Rats (Dipodomys ingens). A: Midway study site. B: Study site northwest of Midway site and southwest of the Lokern sites. (Photographed by David J. Germano).
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Figure 6. The northern most site (Northwest Belridge study site) where I collected genetic samples from Giant Kangaroo Rats (Dipodomys ingens) in the southern San Joaquin Valley. (Photographed by David J. Germano).
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Randall, J. A. 1997. Species-specific footdrumming in kangaroo rats: Dipodomys ingens, D. deserti, D. spectabilis. Animal Behavior 54:1167–1175.
Schiffman, P. M. 1994. Promotion of exotic weed establishment by endangered giant kangaroo rats (Dipodomys ingens) in a California grassland. Biodiversity and Conservation 3:524– 537.
Shaw, W. T. 1934. The ability of the giant kangaroo rat as a harvester and storer of seeds. Journal of Mammalogy 15:275–286.
Stock, A. D. 1974. Chromosome evolution in the genus Dipodomys and its taxonomic and phylogenetic implications. Journal of Mammalogy 55:505–526.
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Wakeley, J. 2004. Recent trends in population genetics: More data! More math! Simple models? Journal of Heredity 95:397–405.
Whisson, D. A. 1999. Modified bait stations for California Ground Squirrel control in endangered kangaroo rat habitat. Wildlife Society Bulletin 27:172–177.
Williams, D. F. 1992. Geographic distribution and population status of the giant kangaroo rat. Pages 301 – 328 in D.F. Williams, S. Byrne, and T. A. Rado, editors. Endangered and Sensitive Species of the San Joaquin Valley, California. California Energy Commission, Sacramento.
Williams, D. F., and K. S. Kilburn. 1991. Dipodomys ingens. Mammalian Species 377:1–7.
23
CHAPTER 2
Population Genetics of the Endangered Giant Kangaroo Rat, Dipodomys ingens, in the
Southern San Joaquin Valley
Nicole C. Blackhawk, David J. Germano, and Paul T. Smith
Department of Biology, California State University, Bakersfield, 9001 Stockdale Highway,
Bakersfield, CA, 93311
Abstract
Populations of the endangered giant kangaroo rat (Dipodomys ingens) have decreased because of habitat fragmentation and isolation over the past 100 years. Changes in the population structure due to habitat fragmentation can significantly affect the population size and the dispersal of these animals. We collected small ear clippings from male and female giant kangaroo rats from six sites in the southern San Joaquin Valley to determine the genetic population structure of this species in this part of their range. We predicted that geographic distance and isolation of populations would decrease genetic relatedness compared to populations closer together. Having a better understanding of the genetic structure in this species will help with conservation actions.
We compared these data to published estimates of genetic diversity of giant kangaroo rats in the
Carrizo Plain to the west and the Panoche area to the north, the other large population centers of this species.
Key words: Dipodomys ingens, endangered species, genetic structure, population fragmentation
24
Introduction
The giant kangaroo rat (Dipodomys ingens) is listed both federally and by California as an endangered species and has been since the 1980s, making its conservation of the utmost importance. The giant kangaroo rat now inhabits only a small portion of its historic range (Good et al. 1997; Loew et al. 2005). This species is endemic to the San Joaquin Valley, Carrizo and
Elkhorn Plains, and the Cuyama Valley. The giant kangaroo rat was once widespread, but populations have decreased since the early 1900s, in part due to secondary exposure to rodenticides when ranchers attempted to eliminate the California ground squirrel (Spermophilus beecheyi) on grazing land (Whisson 1999; Williams 1992). Populations have decreased further due to agricultural development since the 1960s (Williams and Kilburn 1991). Giant kangaroo rats occur on shrublands of saltbush (Atriplex sp.) and California ephedra (Ephedra californica;
Shaw 1934; Williams and Kilburn 1991; Williams 1992) on sandy loam soils that do not tend to flood. The habitat is usually dominated by a herbaceous layer of exotic grasses (Williams and
Kilburn 1991; Schiffman 1994).
Good et al. (1997) and Loew et al. (2005) completed genetic analyses of giant kangaroo rats in the Panoche Valley, Tumey Hills, and Ciervo Hills in the northern portion of the species range and in the southern end in the Carrizo Plains, which is west of the Temblor Mountains
(Fig. 1). Mitochondrial DNA suggested that population sizes have fluctuated over time and/or that populations have not been isolated from one another (Good et al. 1997). Their southern- most populations were not genetically subdivided, but there was significant subdivision between the northern and southern-most populations and among some of the northern populations (Good et al. 1997). There was more genetic distance between some northern haplotypes than between any northern and southern haplotypes despite greater geographic distance north to south. Also,
25
one northern population in the Panoche Valley contained old allelic lineages and shared ancestral
polymorphisms with several other populations (Good et al. 1997). Further, there was evidence
that there had been a recent increase in population size in the remaining populations in the north
and suggested that the Panoche Valley could play an important role in the expansions. This
increase in population size is consistent with what Williams et al. (1995) found when estimating
population size of giant kangaroo rats in the north. It was found that the space occupied by
colonies in the northern populations during the early 1990’s was 6.6 times greater than what was
calculated in the 1980s (Williams et al. 1995).
Using microsatellites, Loew et al. (2005) also found significant genetic subdivision for
the northern and southern range, but that there was still considerable gene flow among the
southern populations (Fig. 1). Gene flow was relatively high for an endangered species, the
northern populations in Fresno and San Benito counties suggested non-random mating and
genetic drift within subpopulations. They concluded that effective dispersal along with genetic
distances between populations is better predicted by ecological conditions and topography of the
environment than by linear geographic distance between populations.
Because of the endangered status of the giant kangaroo rat, the fragmentation of its range
can be especially dangerous to its long-term survival (US Fish and Wildlife Service 1998;
Germano et al. 2001). It also is important to understand the genetic structure of fragmented
populations and determine the most at-risk populations so conservation action can be taken if the
population structure indicates decline. This is not only important to this species, but to others that have been faced with similar fragmentation. We used microsatellites to determine the genetic structure of six populations of giant kangaroo rats in the southern San Joaquin Valley.
26
Using microsatellites has become a popular choice among ecologists over the last decade
(DeSalle and Amato 2004; Selkoe and Toonen 2006) because this multilocus approach allows
for a more precise and statistically powerful way to compare populations and individuals (Selkoe
and Toonen 2006). Microsatellites allow for estimates of migration and relatedness. Wright was
able to show that the variation in gene frequencies can be used to determine variation within and
among subpopulations (Wright 1943; 1951; 1965). Microsatellite genotyping is the current
preferred method for analyzing genetic structure and pedigrees in animal populations (Selkoe
and Toonen 2006).
Although the studies by Good et al. (1997) and Loew et al. (2005) were fairly
comprehensive for the populations they studied, they did not analyze populations of giant
kangaroo rats in the southern San Joaquin Valley. The southern San Joaquin Valley historically
and currently has supported large numbers of giant kangaroo rats. We conducted a genetic study
among populations of the giant kangaroo rat from six sites in the southern San Joaquin Valley
and compared our results to populations of this species to the west of the Temblor Mountains
(Carrizo Plains) and to the Panoche Valley in the north. We measured the genetic diversity,
including the number of alleles per locus, observed heterozygosity, and expected heterozygosity
under Hardy-Weinberg assumptions. We predicted that there would be some differentiation among populations in the southern San Joaquin Valley that we collected but that there would be less difference among populations within the southern San Joaquin than to populations across the
Temblor Mountains to the west and to populations farther north in the Panoche.
Methods and Materials
Sampling locations and populations – We collected tissue samples from six populations of giant kangaroo rats in the southern part of the range in the San Joaquin Valley (Fig. 2). All
27
sites were in Kern County and included populations at the North Lokern (n=20), Lokern (n=20),
Midway (n=10), Derby Acres (n=20), Buttonwillow (n=20), and Northwest Belridge (n=11).
We collected tissue samples fall 2011 and spring 2012. We used Sherman live traps baited with bird seed to catch kangaroo rats. We set approximately 3 traps around each active precinct to ensure catching the most individuals per night. Once an individual was caught at a precinct, we would move traps to new precincts if additional trapping was needed. We collected individual samples of small ear clips for DNA extraction from males and females from each site. All samples were stored in 90% ethanol and taken back to the lab for freezing until DNA extractions could be completed.
DNA extraction – We extracted genomic DNA from 2–4 mm2 ear clippings cut into small pieces using the DNeasy Tissue Kit (Qiagen, Valencia, California). Each sample was lysed in
150 µl of PBS Buffer by cutting the ear clippings into several small pieces and ground up with a mortar and pestle. We then added 20 µl of proteinase K (20 mg/ml) and 200 µl of Buffer AL.
Each sample was then mixed immediately by vortexing and then incubated at 70°C for 10 min.
We then added 200 µl of 100% ethanol to each sample and mixed by vortexing. We pipetted each of these mixtures into the DNeasy Mini Spin Column and placed them into a 2 ml collection tube. These samples were centrifuged at 8,000 rpm for 1 min. We discarded the flow- through and collection tubes and we used new 2 ml collection tubes. We added 500 µl Buffer
AW1 and centrifuged the samples again at 8,000 rpm for 1 min. The flow-through and collection tubes were discarded again and replaced by new ones. We then added 500 µl of
Buffer AW2 and centrifuged the samples at 14,000 rpm to dry the DNeasy membrane. We discarded the flow-through and collection tubes a third time and we replaced them with 1.5 ml microcentrifuge tubes. We added 50 µl Buffer AE onto the DNeasy membrane and incubated
28
the samples at room temperature for 1 min. The samples were then centrifuged at 8,000 rpm for
1 min to elute. We discarded the spin columns and we closed and froze the 1.5 ml
microcentrifuge tubes until the samples could undergo polymerase chain reaction (PCR)
amplification.
Amplification and sequencing – The microsatellite primers used in this study were
characterized in banner-tailed (D. spectabilis) and giant kangaroo rats by Davis et al. (2000). We
performed all (PCR) amplifications in 50 μl volume that contained 200 μM dNTP, 0.2 μM of
each primer, 1.5 mM MgCl2, 2.5 units of HotStarTaq DNA polymerase, and 1x PCR buffer
(10mM Tris buffer, pH 8.8, 0.1% Triton X-100, 50mM KCl and 0.16 mg/mL BSA). The
temperature profile for the microsatellite PCR amplification consisted of an initial heat activation
step of 95° C for 15 min followed by three cycles of 94° C 0.5–1 min, 50–68° C for 0.5–1 min,
and 72° C for 1 min. The number of cycles we used was 25–35. A final extension step of 72° C
for 10 min was also added. We electrophoresed PCR products on a 1.0% agarose gel to detect
successful amplification. We ran negative controls for all amplifications. We then submitted
PCR products to the DNA Sequencing Core Facility of the University of Florida for fragment
analysis using ABI instrumentation.
Statistical analyses – We imported and read the ABI fragment analysis files to predict
allele sizes to infer individual genotypes using GeneMarker v1.97 The Biologist Friendly
Software (Softgenetics, USA). We used these genotypes to measure genetic diversity, including
the number of alleles per locus (NA), observed heterozygosity (HO) and expected heterozygosity
(HE) under Hardy-Weinberg assumptions (Nei 1978), using the program GENALEX v6.5
(Peakall and Smouse 2006; 2012). We also used GENALEX to determine the degree of genetic differentiation among the predefined geographical populations with pairwise FST measures and
29
AMOVA (Peakall and Smouse 2006; 2012). Due to the recent fragmentation of giant kangaroo rat populations, FST values can provide much insight (Peakall and Smouse 2006; 2012). All significance tests were done using 999 permutations. A Nei’s genetic distance matrix was expressed using an UPGMA tree constructed in MEGA v5 (Tamura et al. 2011). We also analyzed the variation within the genetic distance using principal coordinate analysis (Peakall and Smouse 2006; 2012). Lastly, we tested these hypotheses using a Mantel test using
GENALEX and used spatial autocorrelation analysis to explore the relationship between ecological and/or genetic variables and geographic location.
Results
Initially, we evaluated all of the microsatellite loci except for Di12F characterized by
Davis et al. (2000) for amplification success from 101 giant kangaroo rats from six populations.
Of these, Ds46 was not successful and Ds19 was only successful for 17 individuals. Despite this, the other loci amplified successfully for all of the 101 individuals except for one individual from the Derby Acres site for Di5. The comparison of genetic diversity between populations was based on seven loci (Ds1, Ds3, Ds19, Ds28, Ds30, Di5, Di12E). The size of Ds1, including primers, ranged from 185 to 231 bp, Ds3 ranged from 169 to 197 bp, Ds19 ranged from 117 to
145 bp, Ds28 ranged from 196 to 226 bp, Ds30 ranged from 233 to 283 bp, Di5 ranged from 184 to 193 bp, and Di12E ranged from 210 to 237 bp (Table 1). The ranges of these allele sizes all were greater than those found by Loew et al. (2005) with a few of the alleles being shared at each locus.
Polymorphism and allele frequency distributions – Loci Ds1, Ds19, Ds28, and Ds30 were polymorphic for each subpopulation, whereas the Buttonwillow site was monomorphic for locus
Ds3 and Northwest Belridge was monomorphic at Di5 (Table 1). The monomorphism at Di5
30
was not surprising because only two alleles in D. ingens were seen in Davis et al. 2000 and was monomorphic at one of the subpopulations by Loew et al. (2005). The Northwest Belridge site was monomophic at Ds19; however, this should be interpreted with extreme caution because this is based on only one successful PCR amplification for this site at this locus (Table 1). The maximum number of alleles detected per polymorhphic locus was 14 at locus Ds30 (Table 1).
This was fairly consistent with what Loew et al. (2005) found in their southern populations. The mean number of alleles per locus (NA) ranged from 3 to 5 (Table 2). This is slightly less than those found by Loew et al. (2005) in the south, but consistent with the northern populations.
Hardy-Weinberg equilibrium and heterozygosity – Observed heterozygosities ranged
from 0.38 to 0.51, and expected heterozygosities ranged from 0.42 to 0.56 (Table 2). All
populations showed random mating with no heterozygote deficits, but they were less than those
found by Loew et al. (2005). Expected heterozygosity was not relatively high, ranging from 0.42
in Northwest Belridge to 0.56 in Buttonwillow (Table 2). An AMOVA, based on 999
permutations of the distance matrix for calculation of ΦST, revealed significant heterogeneity
among the six populations (Φ=0.183, P<0.001; Table 3). It was also seen by the AMOVA that
there was a greater variation within populations (82%) than among populations (18%; Table 3).
Population differentiation and genetic distances – FIS values for single loci were
significantly positive for Ds3, Ds19, and Ds28 (Table 4), which was consistent with the
deviation from Hardy-Weinberg frequencies within populations. The multilocus FIS was
significantly higher than zero at the 1% level, which indicates an extreme deficit in
heterozygotes. The multilocus FST values were significantly different from zero, which is not
surprising because the single locus analysis revealed significantly positive values for each locus
at the 1% level (Table 4).
31
The relationship between the matrices of the genetic distances (delta mu)2 and linear distances between populations was not significant (Rxy=0.257; P=0.220; Fig. 3). Pairwise FST,
ΦST, and Nei’s genetic distance analyses showed significant differentiation between all populations except for Lokern and Northwest Belridge (Table 5). Most all of these were significant at the 1% level except for Lokern and Buttonwillow, which was significant at 5%
(Table 5). The principle coordinates analysis, which gives a visual representation of groupings, showed that the Midway population and the Derby Acres population were different than the others; whereas, the other four grouped together (Fig. 4).
The populations spanned across approximately 35 km from the northernmost to the southernmost with no obvious natural geographic barriers. On the other hand, some populations were highly fragmented due to agricultural development (i.e., North Lokern) and separated by the California aqueduct (i.e., Buttonwillow). Pairwise comparisons did reveal FST values that were significantly different from zero (Table 5). Along with the high FIS and FST values among the populations the overall multilocus heterozygote deficit (FIT) was also significantly different from zero with the exception of loci Di5 and Di12E (Table 4).
The UPGMA tree revealed that the Lokern and North Lokern sites grouped together, these two sites then grouped with the Buttonwillow site (Fig. 5). The Midway site was more similar to the Northwest Belridge site (Fig. 5). The Midway and Derby Acres sites did not have kangaroo rats that were most similar (Fig. 5). Because we were not able to obtain genetic samples for the Carrizo Plains and the Panoche, we were not able to evaluate how populations in the southern San Joaquin Valley compared to these other regions in the range of the giant kangaroo rat.
32
Discussion
Local extinctions of the giant kangaroo rat have been occurring as a result of these
natural communities becoming highly fragmented due to agricultural development (Loew et al.
2005) as well as a poisoning that occurred in the early 1900s when the California ground squirrel
was targeted (Whisson 1999; Williams 1992). The remnant populations are fragmented and
limited to suboptimal habitat on the western edge of the Tulare and Carrizo Basins, and the
Cuyama and Panoche Valleys as well as the adjacent foothills (Grinnell 1932; Goldingay et al.
1997; Williams 1992). The UPGMA tree revealed that the Lokern and North Lokern sites
grouped together, which was expected by their close geographic distance. These two sites then
grouped with the Buttonwillow site, which was not expected due to the California aqueduct
being a recent geographic barrier to a relatively new population that was reinvading this fallow
field. The Midway site was more similar to the Northwest Belridge site which was also
unexpected. We predicted that the populations with less geographic distance would be more
differentiated. We would have expected the Northwest Belridge site to be more closely related
to the Lokern and North Lokern sites. We also expected that because the Midway and Derby
Acres sites were relatively close to each other that they would have been more closely related,
but this was not supported.
The significant genetic subdivision among populations was consistent with the increasing
habitat fragmentation among the six subpopulations examined. F-statistics confirmed a
significant decrease in observed heterozygosity, which suggests non-random mating and genetic drift within populations. The multilocus F-statistics also indicated that the six populations of giant kangaroo rat are not composed of randomly mating populations and that they are experiencing significant amounts of genetic drift and inbreeding.
33
The genetic drift these populations are experiencing can result in random fixation and
loss of alleles within populations and thus increase the amount of differentiation among
populations (Loew et al. 2005). On the other hand, gene flow among populations can counteract
the detrimental effects of drift and preserve genetic diversity within subpopulations (Harrison
and Hastings 1996). Endangered species have experienced declining populations; therefore, it is
expected to see decreased genetic diversity compared to non-endangered species. Endangered species generally only have an average of 65% molecular heterozygosity of taxonomically related non-endangered species (Frankham et al. 2002). This suggests that the populations of giant kangaroo rats in the southern San Joaquin Valley have been isolated long enough for them to have significantly differentiated. The pairwise FST values indicate that the Lokern and
Northwest Belridge sites are not completely isolated from each other but that the other
populations are genetically subdivided.
It was concluded by Good et al. (1997) that the southern populations on the Carrizo Plain
to the west of the Temblor Mountains effectively act as one large population, but that they had
experienced some fluctuations in size that can affect the genetic structure. The fluctuations in
size were consistent with what Loew et al. (2005) found in that the populations on the Carrizo
Plain deviated from Hardy-Weinberg expectations, which could be due to random genetic drift.
The populations that we sampled in the southern San Joaquin Valley are east of the Temblor
Mountains and also deviated from what would be expected under Hardy-Weinberg expectations
without having any long term natural barriers. It is possible that because of these deviations, if
these values could have been compared to those on the western side, they too would be
significant because the Temblor Mountains would serve as a natural geographic barrier.
Heterozygote frequencies at the Midway, Buttonwillow, and the North Lokern sites suggested
34
that though they did deviate from Hardy-Weinberg expectations, they were slightly higher than those of the other populations, which can be evidence of population growth (Loew et al. 2005).
It was observed while trapping at the North Lokern site in the spring of 2012 the population numbers were at an all-time high (D. Germano, unpubl data). Also, numbers of giant kangaroo rats at the Buttonwillow site were high on this once fallow field, which suggests rapid reestablishment and population growth. This population size fluctuation could have had an effect on the genetic structure that we found in these populations at the time of sampling, which would be consistent with what Good et al. (1997) found with their southern populations.
Population fluctuations and turn over within metapopulations can accelerate genetic drift
(Harrison and Hastings 1996). Also, these fluctuations can decrease genetic diversity within populations and increase differentiation among fragmented populations (Harrison and Hastings
1996; Toro and Caballero 2005). Population differentiation can be slowed, halted, or even reversed as a result of even a few migrants per generation (Lacy 1987). The number of migrants occurring per generation can be affected by geographic distance, topography and ecological conditions (Good et al. 1997; Loew et al. 2005) with ecological conditions having a greater effect than geographic distance (Hokit et al. 2010).
It was determined by both Good et al. (1997) and Loew et al. (2005) that the Ciervo Hills population was founded by one or several recent migrations from the Panoche Valley and isolated from other populations by distance and geographic barriers. The topography within the range of the giant kangaroo rat is complex and the remaining populations are separated by unsuitable habitat that can limit dispersal (Loew et al. 2005), and the area is becoming even more fragmented due to agricultural and residential development limiting dispersal even more. It was
35
shown that linear geographic distances are less likely to predict dispersal than ecological conditions (Good et al. 1997; Loew et al. 2005).
For our populations, Northwest Belridge showed the lowest proportion of polymorphic loci, as well as being on the lower end of expected heterozygosity, exhibiting a significant deficit of heterozygotes and significant isolation from all populations except for Lokern. In contrast, the
Midway site showed greater genetic diversity. There were no natural geographic barriers between our populations that we sampled, but if compared to those of Loew et al. (2005) they would likely be significant due to the major geographic barrier, the Temblor Mountains.
Because all six of the populations that we sampled were significantly differentiated, it is likely that they would also be significantly different from all of the populations to the west of the
Temblor Mountains with the possible exception of one. Because the genetic data from the Loew et al. (2005) study was not entered into GenBank and the authors would not permit us access to their data, we could not make a comparison to the northern populations in the Panoche Valley.
Ideally, it would be best to test all of the data from these populations using GENALEX.
The relationship between genetic distance and geographic distance was consistent with what Loew et al. (2005) found for both their northern and southern populations. This isolation by distance could be due to varying evolutionary histories for the different populations; however, the fragmentation among the populations is a more likely cause. The differences seen among the populations separated by the greatest linear distance, Northwest Belridge and Midway, were significant as were the populations closest together including North Lokern and Lokern. This difference over such a short linear distance is likely due to the high isolation of these sites caused by agricultural fields and the California aqueduct. The genetic differentiation seen in the
Buttonwillow site is likely due to its isolation because of the agricultural fields and being on the
36
opposite side of the aqueduct from all of the other populations. It is also likely that because this
was once a fallow field, the site was probably reinvaded by giant kangaroo rats from remnant
populations found along the edge of the Elk Hills, just across the aqueduct. The Buttonwillow
site is geographically closest to and not as genetically distant from the Lokern site. If giant
kangaroo rats could get across the California aqueduct (roads cross the aqueduct at several
locations), individuals from this site could have also dispersed to found the newer population at
the Buttonwillow site. Because of the fragmented nature of these populations, we conclude that
these populations in the southern San Joaquin Valley are highly structured.
Demographic factors, such as the populations being isolated from each other, could be
the cause for the significant FIS and FST values of giant kangaroo rat populations. Inbreeding can
result in heterozygote deficits relative to expected Hardy-Weinberg frequencies as well as provide the positive FIS values exhibited (Loew et al. 2005). Also, genetic drift in small
populations will decrease the observed heterozygote frequencies, which can increase inbreeding reinforcing the decrease in heterozygosity (Frankham et al. 2002). Moderated inbreeding was supported within our populations. There were no negative FIS values that were significant,
which is consistent with a low degree of polygyny and suggests insignificant sex bias in natal
dispersal (Metcalf et al. 2001).
Gene diversity within populations was relatively high and even small populations can add
to the overall genetic diversity of the species. Land managers should focus on adding more
corridors in an effort to increase connectivity among isolated populations. This can help increase
the genetic diversity among populations and population size fluctuations should be monitored.
Small population sizes with high heterozygosity and genetic isolation can be important to genetic
diversity that could be at risk for local extinctions. Loew et al. (2005) found that the Soda Lake
37
population, which was newly founded by translocation in 1989, showed significant population
growth and showed the highest levels of observed heterozygosity with no evidence of
inbreeding. This is not surprising as the population was made up of individuals captured across
the Carrizo and Elkhorn Plains to the south. This means that if translocations among our
populations to the east of the Temblor Mountains were implemented, it could greatly benefit
these isolated populations. Translocation can be a viable option for increasing genetic diversity
and improve population demographics (Loew et al. 2005). It would be to the utmost importance
for the most geographically isolated populations that are at risk of local extinction. For locations
where dispersal corridors are impractical, translocation can be helpful in preserving genetic
contributions among isolated populations.
Our study has demonstrated the importance of understanding the population structure in
the context of habitat fragmentation. Metcalf et al. (2001) demonstrated using mitochondrial
DNA that Dipodomys species can colonize previously occupied habitat through long distance dispersal or in a stepping stone fashion through shorter dispersal events. The Soda Lake population studied by Loew et al. (2005) is a good example that when translocations are implemented, genetically diverse founders and the resulting gene flow among neighboring populations can result in beneficial population growth and the maintenance of high genetic diversity.
In future genetic studies of this species, it would be beneficial to resample the populations in the Panoche Valley and those on the Carrizo Plain to determine their relation to those in the southern San Joaquin Valley. Also, future studies on natal and breeding dispersal would provide better insight on the differentiation within populations and help select individuals that would help to increase genetic diversity without having detrimental effects to the
38
populations. Monitoring in these cases would be important because this would allow for more
information to be gained for better implementing management plans that will allow long-term
survival of the giant kangaroo rat.
Acknowledgements
This research was funded by Student Research Scholars Program, San Joaquin Valley
Chapter of The Wildlife Society, and the Graduate Student-Faculty Collaborative Initiative. We thank L. Saslaw, D. Noce, R. Kelty, and J. Parker for assistance with fieldwork and support.
Also, thanks to K. White for the assistance with the GIS maps. Thanks to C. Kloock for reading an earlier version of this manuscript, and for several helpful discussions. Giant kangaroo rats were captured under permits (California SC-000955 and Federal Permit No. TE749872-2) held by the second author and the study protocol was approved by the Institutional Animal Care and
Use Committee (11-01) of California State University, Bakersfield.
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Williams DF, Kilburn KS (1991) Dipodomys ingens. Mamm Species 377:1–7
Wright S (1943) Isolation by distance. Genetics 28:114–138
Wright S (1951) The genetical structure of populations. Ann Eugenics 15:323–354
Wright S (1965) The interpretation of population structure by f-statistics with special regard to systems of mating. Evolution 19:395–420
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Table 1 Observed allele frequencies by locus and population of the giant kangaroo rat (Dipodomys ingens) from the southern San Joaquin Valley, California Size range (bp)/no. of Group n alleles ––––––––––––––––––––––––––––––––– Allele size (bp) –––––––––––––––––––––––––––––––––– Ds1 185 189 193 196 213 215 217 219 221 223 227 229 231 Lokern 20 185-229/9 0.150 0.125 0.050 0.000 0.025 0.050 0.200 0.000 0.050 0.000 0.200 0.150 0.000 North Lokern 20 185-229/6 0.125 0.275 0.000 0.000 0.000 0.000 0.075 0.000 0.025 0.000 0.375 0.125 0.000 Midway 10 189-231/4 0.000 0.100 0.000 0.000 0.000 0.300 0.000 0.000 0.000 0.050 0.250 0.000 0.300 Derby Acres 20 185-231/7 0.100 0.025 0.000 0.000 0.000 0.025 0.000 0.025 0.000 0.100 0.700 0.000 0.025 Buttonwillow 20 185-229/9 0.125 0.075 0.000 0.150 0.050 0.150 0.025 0.000 0.075 0.000 0.175 0.175 0.000 Northwest Belridge 11 185-229/8 0.136 0.182 0.045 0.045 0.091 0.000 0.227 0.000 0.000 0.000 0.091 0.180 0.000 Ds3 169 189 193 197 Lokern 20 169-197/3 0.075 0.850 0.000 0.075 North Lokern 20 189-193/2 0.000 0.975 0.025 0.000 Midway 10 169-193/3 0.100 0.850 0.050 0.000 Derby Acres 20 169-193/3 0.100 0.425 0.475 0.000 Buttonwillow 20 189/1 0.000 1.000 0.000 0.000 Northwest Belridge 11 169-193/3 0.045 0.773 0.182 0.000 Ds19 117 123 127 129 131 139 145 Lokern 7 129-145/3 0.000 0.000 0.000 0.286 0.000 0.286 0.429 Derby Acres 2 117-127/2 0.500 0.000 0.500 0.000 0.000 0.000 0.000 Buttonwillow 7 123-131/4 0.000 0.286 0.143 0.357 0.214 0.000 0.000 Northwest Belridge 1 123/1 0.000 1.000 0.000 0.000 0.000 0.000 0.000 Ds28 196 206 208 212 216 222 226 Lokern 20 206-222/4 0.000 0.200 0.000 0.650 0.100 0.050 0.000 North Lokern 20 196-226/5 0.125 0.025 0.000 0.700 0.125 0.000 0.025 Midway 10 206-212/3 0.000 0.150 0.150 0.700 0.000 0.000 0.000 Derby Acres 20 206-226/3 0.000 0.025 0.000 0.950 0.000 0.000 0.025 Buttonwillow 20 206-2224 0.000 0.125 0.000 0.675 0.125 0.075 0.000 Northwest Belridge 11 206-216/3 0.000 0.045 0.000 0.864 0.091 0.000 0.000 Ds30 233 235 246 252 254 256 258 261 263 265 267 269 271 283 Lokern 20 235-283/8 0.000 0.100 0.000 0.150 0.075 0.000 0.150 0.000 0.275 0.000 0.050 0.125 0.000 0.075 North Lokern 20 233-283/9 0.100 0.000 0.000 0.200 0.050 0.075 0.000 0.000 0.325 0.025 0.100 0.075 0.000 0.050 Midway 10 233-267/7 0.400 0.000 0.050 0.000 0.000 0.000 0.150 0.050 0.050 0.150 0.150 0.000 0.000 0.000 Derby Acres 20 233-271/5 0.425 0.000 0.000 0.000 0.000 0.000 0.000 0.375 0.000 0.150 0.000 0.025 0.025 0.000 Buttonwillow 20 233-283/9 0.075 0.125 0.000 0.275 0.050 0.050 0.000 0.175 0.100 0.000 0.000 0.025 0.000 0.125 Northwest Belridge 11 233-283/9 0.045 0.000 0.000 0.136 0.273 0.000 0.091 0.000 0.091 0.182 0.045 0.045 0.000 0.091
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Di5 184 190 193 Lokern 20 190-193/2 0.000 0.025 0.975 North Lokern 20 184-193/3 0.150 0.050 0.800 Midway 10 184-193/3 0.150 0.150 0.700 Derby Acres 19 190-193/2 0.000 0.132 0.868 Buttonwillow 20 190-193/2 0.000 0.150 0.850 Northwest Belridge 11 193/1 0.000 0.000 1.000 Di12E 210 220 224 227 233 237 Lokern 20 220-237/4 0.000 0.300 0.125 0.025 0.000 0.550 North Lokern 20 210-237/4 0.050 0.500 0.100 0.000 0.000 0.350 Midway 10 210-237/4 0.050 0.200 0.500 0.000 0.000 0.250 Derby Acres 19 210-237/4 0.150 0.675 0.025 0.000 0.000 0.150 Buttonwillow 20 210-237/5 0.025 0.250 0.225 0.000 0.075 0.425 Northwest Belridge 11 220-237/4 0.000 0.364 0.045 0.091 0.000 0.500
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Table 2 Descriptive statistics for six populations across seven loci, including the mean sample size (n), proportion of polymorhphic loci (P), number of alleles (NA), number of effective alleles (Ne), information index (I), observed heterozygosity (Ho), expected heterozygosity (HE), the mean number of private alleles, and the fixation (F) indices of the giant kangaroo rat (Dipodomys ingens) from the southern San Joaquin Valley, California Mean # of Private Population n P Na Ne I HO(SE) HE Alleles F Lokern 18.14 1.00 4.71 3.26 1.10 0.46(0.15) 0.54 0.30 0.16 Midway 8.57 0.86 3.57 2.30 0.92 0.51(0.13) 0.48 0.18 -0.06 Derby Acres 17.27 1.00 3.71 1.95 0.78 0.38(0.11) 0.44 0.20 0.10 Buttonwillow 18.14 0.86 4.86 3.57 1.16 0.50(0.13) 0.56 0.18 0.08 Northwest Belridge 9.57 0.71 4.14 2.88 0.88 0.44(0.15) 0.42 0.00 -0.06 North Lokern 17.14 0.86 4.14 2.36 0.89 0.50(0.14) 0.43 0.14 -0.15 Total 14.81 0.88 4.19 2.72 0.96 0.47(0.05) 0.48 0.02
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Table 3 Population comparisons of six populations of the giant kangaroo rat (Dipodomys ingens) in the southern San Joaquin Valley using an analysis of molecular variance (AMOVA) df SS MS % variation Φ P Among populations 5 84.30 16.86 18 Within populations 95 340.20 3.58 82 0.183 <0.001 Total 100 424.50
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Table 4 Estimates of F-statistics across six populations of the giant kangaroo rat (Dipodomys ingens) in the southern San Joaquin Valley by locus FIS: standard genetic variation within populations; FST: standard genetic variation between populations; FIT: to partition genetic variation Locus FIS FST FIT Ds1 -0.013 0.099** 0.087* Ds3 0.138* 0.256** 0.359** Ds19 0.930** 0.085** 0.936** Ds28 0.143* 0.043** 0.180** Ds30 -0.057 0.110** 0.060* Di5 -0.078 0.054** -0.020 Di12E -0.146 0.100** -0.031 Total 0.056** 0.106** 0.156** Significant difference at *P < 0.05; **P < 0.01
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Table 5 Pairwise values of FST, ΦPT, and Nei’s genetic distance for six populations of the giant kangaroo rat (Dipodomys ingens) in the southern San Joaquin Valley, California Populations Pairwise FST value Pairwise ΦPT Nei’s pairwise distance Lokern vs. Midway 0.100** 0.169** 0.247 Lokern vs. Derby Acres 0.202** 0.311** 0.443 Lokern vs. Buttonwillow 0.018* 0.031* 0.121 Lokern vs. Northwest Belridge 0.018 0.031 0.247 Lokern vs. North Lokern 0.047** 0.089** 0.119 Midway vs. Derby Acres 0.171** 0.284** 0.344 Midway vs. Buttonwillow 0.074** 0.128** 0.192 Midway vs. Northwest Belridge 0.107** 0.193** 0.353 Midway vs. North Lokern 0.096** 0.189** 0.175 Derby Acres vs. Buttonwillow 0.191** 0.298** 0.387 Derby Acres vs. Northwest Belridge 0.170** 0.277** 0.452 Derby Acres vs. North Lokern 0.170** 0.296** 0.304 Buttonwillow vs. Northwest Belridge 0.043** 0.074** 0.175 Buttonwillow vs. North Lokern 0.054** 0.101** 0.121 Northwest Belridge vs. North Lokern 0.049** 0.100** 0.229 Significant difference at *P < 0.05; **P < 0.01
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List of Figures
Fig. 1 Locations of giant kangaroo rat (Dipodomys ingens) populations sampled for this project (orange triangles). The green triangles indicate the populations sampled previously by Loew et al. (2005). The grey shading indicates the historic range of the species (IUCN 2012)
Fig. 2 Locations of giant kangaroo rat (Dipodomys ingens) populations sampled for this project (orange triangles). The grey shading indicates the historic range of the species (IUCN 2012)
Fig. 3 Visualization of Mantel test for geographic distance (GGD) vs. pairwise population ΦPT (PhiPTP) for six populations of the giant kangaroo rat (Dipodomys ingens) in the southern San Joaquin Valley, California
Fig. 4 Visualization of the percentage variation explained by the principal coordinate analysis using pairwise ΦPT values of six populations of the giant kangaroo rat (Dipodomys ingens) in the southern San Joaquin Valley, California
Fig. 5 UPGMA tree based on a pairwise population matrix of Nei’s genetic distances for six populations of the giant kangaroo rat (Dipodomys ingens) in the southern San Joaquin Valley, California
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Figure 1
49
Figure 2
50
Figure 3
0.35
0.30
0.25
0.20
PhiPTP 0.15
0.10
0.05
0.00 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 GGD
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Figure 4
Midway Coord. 2 Coord. Buttonwillow
Lokern North Lokern Derby Acres
Northwest Belridge Coord. 1
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Figure 5
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CHAPTER 3
Conservation Actions and the Reassessment of Giant Kangaroo Rat Habitat Occupancy
Gaining a better understanding of the population structure of the Giant Kangaroo Rat is
important due to its endangered status. I found that all of the populations that I sampled were
genetically distinct except the Lokern and Northwest Belridge populations. This was consistent
with what Loew et al. (2005) found with the northern populations, but not for their southern
ones. It is probable that because most of the six populations that I sampled were significantly
different, these populations would be different from the southern populations sampled by Loew
et al. (2005) with the possible exception of one. The strong differentiation that I observed among
the populations in the southern San Joaquin Valley is likely due to the highly fragmented nature
of the remaining populations within the Giant Kangaroo Rat’s range. Because these populations
are highly structured, it is important to understand the steps that should be taken to conserve this
species as well as others like it that have also experienced similar fragmentation.
The number of species that have now been listed on the endangered species list as a result of human activity over the last two centuries is overwhelming (Bijlsma et al. 2010; DeSalle and
Amato 2004; Frankham 2003). Major factors causing the extinction and endangerment of species include habitat loss, introduced species, over exploitation, habitat fragmentation, and
pollution (Frankham 2003). These factors are related to human population growth and make
ecological communities especially susceptible to environmental, demographic, and/or genetic
effects. Because of this, conservation biology has been defined as a crisis discipline (DeSalle
and Amato 2004). Ecological restoration is a process that attempts to repair damage caused by
humans to the diversity and dynamics of ecosystems (Jackson et al. 1995). Restoration ecology
is a recent discipline that shares many of the biodiversity goals with conservation biology
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(Young 2000). Conservation biology has been more focused on zoological and population and
genetic studies than restoration ecology, which has largely focused on botanical, experimental,
population, and community and ecosystem studies (Young 2000). Restoration practices can
enhance conservation efforts, but should not be relied on in the long run because a “restored”
habitat may never function as well as the original habitat (Young 2000) or sustain the same
amount of diversity. Therefore, restoration mitigation should not be misused and relied on to fix
problems that could have been prevented, but viewed as a valuable tool to enhance conservation
efforts in situations where habitats and species have been compromised. If conservation and
restoration mitigation is carried out well, it could be possible to help increase the genetic flow
among the highly structured populations of Giant Kangaroo Rats. This could help increase the
general wellbeing of the Giant Kangaroo Rat and help establish more self-sustainable populations in the remainder of its habitat.
Over the past decade, there has been more integration of genetics into conservation biology. With the technological advances in sequencing and microsatellite analysis of DNA and non-invasive DNA sampling, incorporating genetic tools into conservation biology has become simpler and more accessible. These techniques make it easy to sample the genetics of the Giant
Kangaroo Rat in a non-invasive manner to gain more information about its genetic structure.
Population genetics are especially important to gaining an understanding of the population structure of endangered species so that conservation action can be taken and do so in an efficient manner. Conservation biology has expanded into many sub-disciplines allowing the urgent
problems involved with managing endangered species and critical areas to be studied and aided
(DeSalle and Amato 2004).
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Endangered species already experience small/declining populations, so inbreeding and loss of genetic diversity are virtually unavoidable (Frankham 2003). Genetic changes occurring in populations can dramatically affect their survival rates and inbreeding has resulted in extinctions in many natural populations (Frankham 2003). I found in my study that the populations that I sampled were inbred. Small populations are especially vulnerable to genetic drift and inbreeding, which is associated with decreased fitness resulting from loss of genetic diversity (Waite et al. 2005; Bijlsma et al. 2010). This loss of genetic diversity makes these types of populations highly susceptible to extinction (Frankham 2003; Tallmon et al. 2004;
Waite et al. 2005; Frankham et al. 2009). The amount of inbreeding in the remaining populations of the Giant Kangaroo Rat should be closely monitored.
Individuals in natural populations are constantly faced with environmental change, including disease, pests, parasites, competitors and predators, pollution, climatic cycles, and global warming (Frankham 2003; Frankham et al. 2009; Bijlsma et al. 2010). To cope with these pressures, species must evolve or face extinction. For species to evolve, high levels of genetic diversity are required (Frankham 2003). Naturally outbred species usually have greater genetic diversity and are able to cope with changes better (Frankham 2003; Frankham et al. 2009).
When populations are fragmented, they start losing their genetic diversity and inbreeding increases, increasing the loss of genetic variation. The populations of Giant Kangaroo Rat sampled were highly fragmented with low genetic diversity and inbreeding was observed.
Populations experiencing loss of genetic diversity and increased inbreeding are more susceptible to local extinctions. If small populations are isolated, they will experience reductions in individual fitness as a result of inbreeding depression (Hogg et al. 2006). If genetic factors are
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ignored, recovery programs for susceptible populations may not be successful. Therefore, genetic considerations are especially important when trying to manage fragmented populations.
Genetic Rescue
Genetic rescue is when the genetic load expressed through inbreeding depression in small populations is removed (Bouzat et al. 2009). Genetic rescue is considered to have occurred when the fitness of a population increases and there are new alleles introduced into a population from forces other than what can be attributed to the demographic contribution by immigrants
(Tallmon et al. 2004). Genetic rescue can play an important role in the evolution of small populations and can be an effective tool in conservation (Tallmon et al. 2004; Bijlsma et al.
2010). The goal of genetic rescue is that when a local population is experiencing inbreeding depression, immigrants can introduce new genetic variation that can positively affect fitness
(Waite et al. 2005). Genetic rescue can be measured by positive population growth over multiple generations (Tallmon et al. 2004; Hogg et al. 2006). Population growth can be a result of immigrant alleles masking some of the deleterious recessive alleles, which have high local frequencies due to genetic drift (Tallmon et al. 2004). Also, immigrants mating with local individuals tend to produce heterozygous offspring, which are usually favored by natural selection (Tallmon et al. 2004). Genetic rescue could be a promising technique to help increase genetic diversity in the Giant Kangaroo Rat.
Heterosis (increased fitness of offspring produced by genetically divergent individuals;
Tallmon et al. 2004) is maximized when gene flow and selection intensity is low in small populations (Bijlsma et al. 2010). Genetic and non-genetic factors (demography, behavior, and environment) can be determining factors in whether immigrants can increase or decrease a population’s fitness (Tallmon et al. 2004). These factors can make it very difficult to predict
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how any one immigration event will affect genetic rescue, especially the interactions that occur among genes at different loci (epistasis; Tallmon et al. 2004). Researchers must be selective when considering which individuals would be most beneficial to use as immigrants. Immigrants have a decreased opportunity to provide genetic rescue to a local population if they are immature and have a decreased chance of survival to reproductive age or are too old and have little chance of future reproduction (Tallmon et al. 2004). Even after this selective process based on genetics and demography, other factors can have an impact on genetic rescue. Once an immigrant is selected for introduction, there may be behavioral subtleties that would decrease mating opportunities and ultimately decreasing their evolutionary impact. Some of these include mate choice, dominance hierarchies, and infanticide (Tallmon et al. 2004). Ultimately, immigrants can have a range of reproductive success based on the interactions they have with local individuals. Furthermore, immigrants could be potential vectors for parasites and pathogens that would have detrimental effects on the local population (Tallmon et al. 2004). To add to this complexity, economic, social, legal, and political ramifications must be considered too (Tallmon et al. 2004).
There are some potential risk factors associated with taking genetic rescue measures to help restore heterozygosity in susceptible populations. While genetic rescue can increase fitness, it can also result in an increase in the frequency of deleterious alleles carried by the immigrants
(Tallmon et al. 2004; Bijlsma et al. 2010). This can ultimately put the population at risk again when it undergoes recurrent inbreeding (Bijlsma et al. 2010). Steps can be taken to minimize these risks, though. If a sufficient number of immigrants are used with the appropriate demographic measures, then this will help to minimize the problems associated with genetic rescue (Bijlsma et al. 2010). For this method to be successful, there must be a substantial
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increase in the population size after the immigration event occurs (Bijlsma et al. 2010). If the population does not experience positive growth and stays small and genetic drift and inbreeding are still significant forces, it is expected that the fitness of the population will decrease again in subsequent generations (Bijlsma et al 2010). Furthermore, if the deleterious alleles present in the immigrants reach high frequencies in the rescued populations, inbreeding and genetic drift may lead to the population experiencing even more severe inbreeding depression (Tallmon et al.
2004; Bijlsma et al. 2010). Therefore, in the case of the Giant Kangaroo Rat, it would be beneficial to attempt genetic rescue during their breeding season with a sufficient amount of reproductive individuals so that they have an increased chance to begin mating once they reach the new location. Also, the population would need to be monitored to ensure that the population size does increase after the immigration event.
As mentioned previously, the effects of genetic rescue can be difficult to predict. Several studies have tested the effects of genetic rescue on small inbred populations. However, most of these studies were completed on small inbred populations in the laboratory (Waite et al. 2005;
Bijlsma 2010). While it is useful to gain information about this subject using laboratory studies first, it still makes it difficult to predict the outcome on wild populations. Genetic rescue can be extremely risky, especially when implementing these methods on endangered species. Also most of these studies have been conducted on insects (Waite et al. 2005; Bijlsma 2010). How vertebrates respond to genetic rescue is not well understood.
Translocation as a Conservation Tool
Translocation is defined as relocating wild individuals from one site to another in an effort to create, reestablish, or augment wild populations in an attempt to conserve them by human intervention (Storfer 1999; Frankham 2009; Germano et al. 2013). Researchers must be
59
selective about the potential immigrants that are selected for translocations. Individuals to be moved must be within the appropriate age bracket to reproduce and introduce their genetic material into the population (Tallmon et al. 2004). Their successful reproduction can depend on the interactions that they have with the local individuals and their parasite loads (Tallmon et al.
2004). It is expected that the use of translocations will increase as a response to dealing with rapid environmental change (Shier and Swaisgood 2011).
The goals of translocations include increasing heterogeneity of small populations, reducing the risk of species loss when catastrophes occur, and to speed the recovery of species after their habitat has been restored from negative effects (Griffith et al. 1989). The definition of a successful translocation is that it would result in a self-sustaining population (Griffith et al.
1989). As extinction rates increase and biodiversity decreases, it is important to determine how well translocations work including methodology, results, and strategies (Griffith et al. 1989).
Translocations are becoming used more often as a method of managing genetic restoration of endangered species and populations (Tallmon et al. 2004; Bouzat et al. 2009). Some of the goals of genetic restoration include removing the genetic load expressed through inbreeding depression in small populations (genetic rescue), retaining local adaptive variation, and restoring genetic diversity so that neutral variation may be beneficial (Bouzat et al. 2009).
Several recent examples of translocations of kangaroo rat populations have occurred in
California. Shier and Swaisgood (2011) conducted a translocation study on the Stephens’
Kangaroo Rat, Dipodomys stephensi, which has been listed as an endangered species since 1988 by the U.S. Fish and Wildlife Service (Shier and Swaisgood 2011). The Stephen’s Kangaroo Rat occurs in southern California. Another translocation study was conducted by Germano et al.
(2013) on the endangered Tipton Kangaroo Rat, Dipodomys nitratoides nitratoides, a species
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found on the floor of the southern San Joaquin Valley. Both these studies have shown some of the requirements needed for success when planning translocations of other kangaroo rats, especially endangered species in which the individuals are solitary and territorial such as the
Giant Kangaroo Rat.
Translocation projects are often unsuccessful because the mortality rates of post-release animals are extremely high (Shier and Swaisgood 2011). Mortality is usually highest in the first days to weeks following the release when the animals attempt to decide where to settle in the new unfamiliar territory and search for resources (Shier and Swaisgood 2011). There are few translocation studies that consider the intraspecific source-site social relationships among solitary territorial animals (Shier and Swaisgood 2011). This is an important aspect to understand because the Giant Kangaroo Rat is both solitary and territorial. Territorial animals will be less aggressive towards familiar neighbors than to unfamiliar neighbors (Randall 1984; 1994). Once animals establish relationships with neighbors, there is reduced aggression (Randall 1984; 1994) that can conserve time and energy reducing the risk of injury (Shier and Swaisgood 2011).
Translocated animals may have to invest more time in establishing social relationships with their neighbors if these neighbor relationships are disrupted. Kangaroo rats are capable of distinguishing familiar and unfamiliar neighbors (Randall 1989; Murdock and Randall 2001) and these considerations should be taken into account when carrying out translocation projects.
Translocations of kangaroo rats have been largely ineffective, but have been attempted with several species of kangaroo rats (Germano 2010). Unfortunately, there are no documented cases where a translocation study has been carried out in which the population was able to sustain itself over the long-term (Shier and Swaisgood 2011). The reason that so many translocations have been ineffective is largely unknown, but none of the current methods take
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into consideration social relationships with neighbors (Shier and Swaisgood 2011). Germano et al. (2013) suggested that the low success rate is due to the translocated animals being unfamiliar with the release site. Shier and Swaisgood (2011) set out to determine if keeping neighboring groups together during translocation could be more effective, and found positive results that supported this method. The neighbor groups had increased settlement, survival, and reproductive success when compared to the individuals that were not translocated with neighbors
(Shier and Swaisgood 2011). Individuals were able to spend more time foraging and experienced less aggression than kangaroo rats that were translocated without neighbors.
Furthermore, the individuals translocated with neighbors were able to spend more time excavating burrows than the kangaroo rats without neighbors. This behavior can be crucial in avoiding predators. If they can develop a decent burrow to seek cover in, then there would be a decrease in mortality against predators. Also, snakes, weasels, and foxes can be attracted to the new soil turned up by a newly established burrow (Germano et al. 2013). If these burrows are not intricate, then a fleeing kangaroo rat may not be able to escape an animal that can enter their burrow.
Another method that may improve translocation success is soft-releasing animals into their new location (Germano et al. 2013). Soft-releasing is when some method of holding individuals at a site prior to release to allow them to acclimate to their new environment and protect the animals from predation (Germano et al. 2013). However, a study done on Tipton
Kangaroo Rats found no significant difference between animals that were hard-released and soft- released (Germano et al. 2013). It is still unclear as to how many individuals should be translocated to establish a new population (Storfer 1999). Germano et al. (2013) believed that their success with the Tipton Kangaroo Rat was due to the large number of individuals moved.
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It is possible that when conducting translocations for the Giant Kangaroo Rat, they could be more successful if (1) large numbers of animals are moved (Germano et al. 2013) along with their familiar neighbors (Shier and Swaisgood 2011), (2) are soft-released (Germano et al. 2013),
(3) if competing species are removed from the habitat (Germano et al. 2013; Griffith et al. 1989),
(4) the release site is of high habitat quality (Griffith et al. 1989), and (5) translocations from sites should be done when the population is expanding and numbers are relatively high (Griffith et al. 1989). Also, it must be assessed as to how translocations can effect genetic restoration and long-term viability of natural populations by assessing the historical levels of genetic diversity before a population decline, document levels of genetic diversity when effectively restored to historic levels as a result of managed translocations, and demographic trends that indicate changes in fitness and population size following the translocations when there have not been any changes to the habitat (Bouzat et al. 2009). Once the effects of translocations on genetic restoration can be better understood and actions can be taken to ensure long-term success, management of small fragmented populations should become more effective. Bouzat et al.
(2009) showed that controlled translocations can be an effective management strategy for genetic restoration in endangered populations by removing detrimental variation associated with inbreeding depression and by restoring neutral variation to historical levels. The authors go on to say that demographic recovery and long-term viability may not be effective unless complemented with management strategies aimed at the overall conservation of the available habitat for endangered species.
Effects of Habitat Fragmentation
Populations, such as those of endangered species like the Giant Kangaroo Rat that have experienced habitat fragmentation have restricted gene flow, which results in the loss of genetic
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variation. Management strategies that are directed to alleviate these pressures include translocations of individuals that can serve as beneficial immigrants with high fitness (Storfer
1999). However, gene flow can also limit local adaptation (Slatkin 1987). When local populations have adapted to local conditions, genetic mixture among them can sometimes lead to outbreeding depression (Storfer 1999). If long-term genetic mixture continues among the populations, it can lead to different selection regimes occurring and lead to the development of new species (Storfer 1999). If Giant Kangaroo Rats were translocated, the populations would need to be monitored to ensure that they do not begin experiencing outbreeding depression.
Due to the continuing habitat fragmentation, there is a definite need for managing these populations to restore historical patterns of between-population gene flow (Hogg et al. 2006).
Storfer (1999) makes three important management recommendations to restore gene flow: First, when considering artificial enhancement of gene flow, biologists should consider the historical relationships of the managed populations (Storfer 1999). If populations with no historical connection are mixed, homogenization can occur (Storfer 1999). Second, there should be more studies conducted on gene flow to provide a better understanding of population structure, species’ colonization ability, and evolutionary potential (Storfer 1999). Third, ecological surveys must be conducted on different habitat types and the selection pressures that affect different populations of endangered species must be monitored (Storfer 1999). Hogg et al.
(2006) say that more studies on genetic decline and rescue on natural populations would provide valuable information on whether the costs and risks should be taken. Pedigree-based studies of natural populations can also provide insight as to whether the translocations did indeed result in genetic rescue (Hogg et al. 2006).
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Reassessment of Habitat Occupancy
It is important when managing endangered species that there is an accurate and relatively recent population estimate along with an understanding of the metapopulation dynamics.
Predictive species models play an important role in conservation biology and wildlife management and planning (Marmion et al. 2009). The use of distribution models has been increasing especially from the increased availability of remote sensing (RS) and the development of geographical information systems (GIS) integration with statistical models (Marmion et al.
2009). These predictive models can sometimes be more accurate than published range maps
(Bustamante and Seoane 2004).
The Giant Kangaroo Rat has experienced range reduction and estimates of currently occupied habitat are important for correctly managing the species and for making decisions affecting its listing as an endangered species. It has been estimated that the Giant Kangaroo Rat now inhabits only 3% of its historic range (Grinnell 1932; Williams 1992; Goldingay et al 1997).
Williams and Kilburn (1991) went as far to say that their distribution as of 1979 was less than
2%. Although clearly endangered, it seems that the estimates of the amount of habitat occupied by Giant Kangaroo Rats in its current distribution are too restrictive. Their historic range encompasses the western edge of the San Joaquin Valley and areas to the west of the Temblor
Mountains including the Carrizo and Elkhorn Plains and colonies in the Ciervo, Kettleman,
Panoche, Tumey Hills and the Panoche Valley (Fig. 1; Grinnell 1922; Williams and Kilburn
1991). However, it is not likely that all of this historic range was appropriate habitat before the arrival of Europeans to California.
An early paper by Grinnell (1932) shows the patchiness of this species distribution in areas that likely had not been greatly affected by people. In discussing surveys across the
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Figure 1. The historic range of the Giant Kangaroo Rat (Dipodomys ingens). The red triangles represent the places that I traveled to that did not have Giant Kangaroo Rats present and the green graduated circles represent location and size of known populations that have been genetically sampled. The grey shading represents the species’ historic range (IUCN 2012).
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Carrizo Plains and Cuyama Valley in the south part of the species range, he stated “In parts of both valleys I found extensive populations of Dipodomys ingens,” and referring to his own notebook about the distribution in the Carrizo Plains “D. ingens occupies hundreds of acres of the higher level or hummocky ground here, now green, but it does not extend down into the alkali ground of the lowest ‘sink’.” Between Coalinga and Panoche he stated “I did not find signs of Dipodomys ingens continuously, indeed nowhere in the immediate vicinity of Coalinga; but they were common here and there south of Panoche Creek for about 9 miles, at altitudes between 400 and 600 feet.” Most species do occupy all of the area circumscribed on a map.
Although precise estimates of habitat occupancy are subject to many assumptions, I estimate that the Giant Kangaroo Rat currently occupies about 30% of its historic range (Fig. 1). As a state and federally listed endangered species, I suggest that the amount of range currently occupied should be reassessed to improve management and conservation actions.
Conclusions
To complete the genetic study on the Giant Kangaroo Rat, I had to travel to potential habitat in Kern and Kings Counties to locate populations for genetic sampling (Fig. 1). I found that the six populations that I sampled from in the southern San Joaquin Valley were highly structured and experiencing inbreeding depression. Loew et al. (2005) found that the southern populations they sampled on the Carrizo and Elkhorn Plains were not significantly different and function as one large population with relatively high genetic diversity for an endangered species.
They did, however, find that the northern populations in the Panoche Valley were highly structured which was likely due to the complex topography in that area. It needs to be assessed how all of these populations in the entire range of the Giant Kangaroo Rat compare. I was
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unable to access the data used by Loew et al. (2005). Gaining this information is of the utmost importance before management throughout the entire range can begin.
I would recommend implementing management strategies for the Giant Kangaroo Rat in the southern San Joaquin Valley first to help increase the genetic diversity and decrease the inbreeding that they are currently experiencing in this area. High numbers of individuals of the appropriate age should be selected that possess genotypes that would help increase the genetic diversity in the populations that they are translocated. Also, it is very important to consider the neighbor relationships when translocating Giant Kangaroo Rats because they are solitary and territorial. This could greatly increase their survival rates at the new location by having familiar neighbors in which the social hierarchy has already been established. I would also recommend that when the individuals are moved to their new site that they be soft-released and that any other competing species are removed from the area. The translocations need to be followed by the monitoring of generations thereafter, to ensure that the genetic diversity and population size continues to increase using pedigree-based studies. I would recommend managing the southern
San Joaquin Valley populations as one group and the populations in the north as a separate group until these methods have shown positive outcomes. This is because the northern and southern populations in the San Joaquin Valley are likely very different from each other and these differences should not be taken lightly, at least until the genetics of the species can be compared throughout the entire range.
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Acknowledgements
I would like to thank Dr. David Germano for all of the field training, data collection, support, and guidance that he has provided over the last two years. I would also like to thank Dr.
Paul Smith for the laboratory training, help with the data analyses, and guidance. I am also grateful to Dr. Carl Kloock for his support, feedback, and for serving on my committee. In addition, I would like to thank my fellow graduate student and friend Donna Noce for providing countless hours of help in the field, valuable feedback, and enthusiastic support over the last two years. In addition, I want to thank Robert Kelty and Catherine Estheranne for the assistance in the field. Also, I would like to thank Kevin Erbas-White for assistance with the GIS maps.
Also, I thank Larry Saslaw for assistance with some of the field work. I would like to thank my father Edward Blackhawk and mother Cheryl Blackhawk for their love and support. I would like to thank my boyfriend Jason Parker for assisting me in the field as well as providing emotional support. I also thank the Biology Department Faculty and Staff for their enthusiastic support over the last six years and for providing me with multiple employment opportunities while completing my graduate work. Lastly, I would like to thank the organizations that helped fund my research: Student Research Scholars Program, San Joaquin Valley Chapter of The Wildlife
Society, and the Graduate Student-Faculty Collaborative Initiative.
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