# A molecular evaluation of conservation units, translocations, and habitat fragmentation for a threatened species: the White Sands pupfish

J.S. Heilveil\* and C.A. Stockwell

Department of Biological Sciences, Stevens Hall, North Dakota State
University, Fargo, ND 58105; e-mail: Jeffrey.Heilveil@ndsu.edu;
Craig.Stockwell@ndsu.edu.

\* Current address: Department of Natural Sciences, 1200 Murchison Rd.
Fayetteville State University, Fayetteville, NC 28301,
jheilveil@uncfsu.edu

Keywords: conservation genetics, fragmentation, microsatellites,
*Cyprinodon tularosa*, translocation, ESU

Correspondence to:

Craig A. Stockwell

Department of Biological Sciences, Stevens Hall, North Dakota State
University, Fargo, ND 58105-5517

Phone: (701) 231-8449, Fax: (701) 231-7149, e-mail:
Craig.Stockwell@ndsu.edu

# Running Title: Conservation genetics of White Sands pupfish 

**Abstract**

*Cyprinodon tularosa*, a New Mexico state-Threatened fish, is restricted
to four populations worldwide and has been subjected to both
translocations and habitat fragmentation. Two native populations at
Malpais Spring and Salt Creek were previously recognized as ESUs and two
non-native populations at Lost River and Mound Spring were recently
derived by translocation from the Salt Creek ESU. Further, the latter
three habitats are fragmented. Here we use a suite of fourteen recently
developed microsatellite markers to evaluate overall genetic structure,
ESU designations, introduction history, and effects of habitat
fragmentation. Overall F~ST~ was high (0.382); however, removing fish
from Malpais Spring (a *sensu lato* ESU) reduced F~ST~ (0.058),
confirming a previous ESU designation made based on a limited number of
markers. Genetic assignment tests (both Bayesian and Likelihood) suggest
Salt Creek Lower (SCLW) as the source population for both the Lost River
and Mound Spring populations. Lost River showed the most divergence from
SCLW, supporting anecdotal reports of a small number of founders (n=30).
Populations from both Salt Creek and Lost River show significant
differences between upper and lower populations, but no barrier effects
were seen in either Mound Spring or Malpais Spring. Based on these data,
management recommendations for the species are discussed.

**Introduction**

The patterns of temporal and spatial genetic structure are of critical
importance to the conservation of rare and endangered species (e.g.
Waples 1991; Moritz 1994; Moran 2002; Moritz 2002; Bouzat and Johnson
2004). For example, the designation of conservation units is an
important first-step in the genetic management of rare species (Waples
1991; Moritz 1994; Crandall *et al*. 2000). This information can direct
management decisions to preserve distinct evolutionary lineages, and
help prioritize management actions such as the establishment of refuge
populations (Stockwell *et al.* 1998).

Molecular data can also be useful for evaluating the effects of historic
transplants and habitat fragmentation on genetic variation. Historical
translocation events can be important drivers in altering genetic
structure, causing reductions in genetic diversity in recently
introduced populations (e.g. Stockwell *et al*. 1996; Helenurm and
Parsons 1997) and potentially compromising their evolutionary potential.
Landscape factors, such as anthropogenic or natural barriers, may
further reduce genetic diversity (*e.g*. Smith *et al*. 1983; Templeton
*et al*. 1990; Keller and Largiadèr 2003; Yamamoto *et al*. 2004).

High levels of genetic diversity in a population can increase the
ability to respond to novel threats (Frankham 1995; Amos and Balmford
2001). It is therefore important, when designing the conservation plan
for a species, to maintain as much genetic diversity as possible within
and among populations. The maintenance of genetic diversity is
influenced by gene flow; as only a few migrants between populations are
necessary to ameliorate the negative effects of genetic drift (e.g.
Wright 1931, 1970; Mills and Allendorf 1996). When physical barriers are
present, however, gene flow can be severely limited or eliminated. In
aquatic systems, the damming of rivers has been shown to result in
reduced genetic diversity for above-dam populations (*e.g.* Smith *et
al*. 1983; Yamamoto *et al.* 2004). These barrier-induced reductions in
genetic diversity can be especially important in species with limited
geographic distribution.

Collectively, the issues of conservation units, translocations and
habitat fragmentation are of particular importance to the conservation
of western fishes (Minckley and Deacon 1991; Waples 1991; Minckley 1995;
Allendorf and Waples 1996; Vrijenhoek 1996; Stockwell and Leberg 2002).
Identification of conservation units has been particularly important for
the conservation of pacific salmon (Waples 1991; Waples 1995) and desert
fishes (Quattro *et al.* 1996; Stockwell *et al.* 1998; Parker *et al*.
1999; Echelle *et al*. 2000). Further, habitat fragmentation is of
particular concern for many western fishes such as pacific salmon and
various trout species (Nehlsen *et al.* 1991; Moyle 1994). Finally,
translocations have been extensively used, especially for the
conservation of many protected fish species (Wiliams *et al.* 1988;
Hendrickson and Brooks 1991; Minckley 1995; Vrijenhoek 1996).

Historically, many studies of fish species have been constrained by
relatively low genetic variation at traditional markers (Echelle 1991);
however, the development of hypervariable markers, *e.g*.
microsatellites, has allowed genetic structure to be studied in such
species (Parker *et al*. 1999; Martin and Wilcox 2004).

One species of particular concern is the White Sands pupfish
(*Cyprinodon tularosa*; Miller and Echelle 1975), a New Mexico
state-listed Threatened species. This species, which is restricted to
two native and two introduced populations in southern New Mexico, has
limited variation at 37 allozyme loci and a short segment of mtDNA
d-loop (Echelle *et al*. 1987; Stockwell *et al*. 1998). Recently, a
battery of microsatellites has been developed for *C. tularosa* and its
congeners (Jones *et al*. 1998; Stockwell *et al*. 1998; Burg *et al*.
2002; Iyengar *et al*. 2004), which allows us to evaluate the variation
between native populations, as well as the effects of historic
translocations and habitat fragmentation on genetic variation in this
species.

**[Background]{.underline}**

Malpais Spring and Salt Creek, which harbor native populations of *C.
tularosa* (Pittenger and Springer 1999), are located on White Sands
Missile Range (WSMR; Fig. 1). In 1970, a population was established at
Lost River on Holloman AFB, and a second population was established
between 1967 and 1973 at Mound Spring on WSMR (Pittenger and Springer
1999). The source population(s) for the introductions at Lost River and
Mound Spring was not documented, but 30 fish were reportedly used to
establish the Lost River population (Pittenger and Springer 1999).

Stockwell *et al*. (1998) used genetic and ecological data to recognize
two Evolutionarily Significant Units (ESUs; *sensu lato*) of White Sands
pupfish, the Malpais Spring ESU and the Salt Creek ESU. The Salt Creek
ESU was shown to include the native populations at Salt Creek as well as
the Lost River and Mound Spring populations both of which were descended
from the Salt Creek population (Stockwell *et al.* 1998). The
designation of the two ESUs was based on fixed and nearly-fixed
differences at a microsatellite marker and an allozyme marker,
respectively. Further, Malpais Spring and Salt Creek differ ecologically
in terms of salinity, flow and parasite communities (Stockwell and
Mulvey 1998; Stockwell *et al*. 1998; Collyer and Stockwell 2004;
Rogowski 2004; Collyer *et al*. 2005). The earlier study did not
evaluate the retention/loss of genetic variation in the introduced
populations at Lost River and Mound Spring. The genetic effects of
habitat fragmentation on this species have not been well studied due to
a lack of variable markers.

Within each population, there are varying degrees of habitat
fragmentation (Figure 1). Malpais Spring experiences the least
fragmentation, with continuous habitat between the springhead and a
nearby playa. Although Malpais Spring lacks physical barriers to
migration, non-obvious barriers have been shown to significantly deter
gene flow between sub-populations in other animals (*e.g.* Hitchings and
Beebee 1997). Indeed, Stockwell and Mulvey (1998) found a significant
difference in PGDH frequencies between upper and lower Malpais Spring
that was correlated with a sharp gradient in environmental salinity.
Earlier workers reported distinct ponds (Miller and Echelle 1975) in the
southern end of the Malpais Spring complex, though water is currently
diverted to the south where these isolated ponds would have existed;
leaving the hydrological relationship between upper and lower Malpais
Spring poorly understood.

Salt Creek is fragmented by a head-cut waterfall approximately 1.2 m in
height (Stockwell and Mulvey 1998; Pittenger and Springer 1999). Salt
Creek is further fragmented by a road culvert, but bi-direction
migration is possible during and after high water events (J. Pittenger,
NMDFG, pers. comm.).

Lost River is intermittent, with permanent water in three different
reaches. The upper reach is characterized by a series of deep pools that
are periodically connected following high water events. The middle reach
is bounded by a road culvert upstream and a dry playa downstream. Below
the playa, Lost River re-emerges and flows until it reaches the White
Sands dune fields. During high water events, the three segments are
connected, but the road culvert prevents fish from moving upstream.

Mound Spring is composed of upper and lower pools, which differ in
elevation by approximately 3 meters. A small amount of water trickles
from the upper to lower pools, but it is unclear if fish migrate to the
lower pools. Upstream fish movement is not possible.

This study employed microsatellite DNA markers to assess the
distribution of genetic variation in the species, with the goal of
informing future conservation and management efforts. Specifically, we
attempted to evaluate the ESU designation of Stockwell *et al.* (1998)
and the effects of historic introductions and habitat fragmentation on
genetic diversity in this species.

**Materials and Methods**

*Sampling and molecular techniques*

Forty fish were caught by minnow-trapping and seining during March,
2003, at each of the following eight locations: 1) Malpais Spring below
the USGS flow gauge (MLUP), 2) Malpais Spring, lower pool (Jet Playa)
(MLLW), 3) Salt Creek, above the waterfall and at terminus of upper road
(SCUP), 4) Salt Creek, below Range Road 316 (SCLW), 5) Mound Spring,
upper pool (MDUP), 6) Mound Spring, lower pool (MDLW), 7) Lost River,
above the confluence of Ritas Draw and Malone Draw (LRUP), 8) Lost
River, near its terminus at the dune fields (LRLW). Fish were
subsequently sacrificed, frozen, and stored at -80 ^o^C upon return to
the laboratory.

Whole genomic DNA was extracted from fin tissue using DNeasy kits
(Qiagen) and stored at 4 ^o^C. Fourteen microsatellite loci previously
shown to be polymorphic in *C. tularosa* were used to assess genetic
differentiation: WSP11 (Jones *et al*. 1998); WSP2 (Stockwell *et al*.
1998); WSP20, WSP23, WSP24, WSP25, WSP26, WSP30, WSP32, WSP33, WSP34
(Iyengar *et al*. 2004); AC23, C509, and GATA02 (Burg *et al.* 2002)
(See Table 1).

Amplification reactions were performed in 25 ul volumes using 2.5 ul 10x
PCR buffer, 1ul 5 mM dNTP mix, 0.175 ul AmpliTaq Gold polymerase
(Applied Biosystems), 1 ul template DNA, 0.25 uM unlabeled reverse
primer, and dye-labeled forward primer (for concentration, see Table 1).
The annealing temperatures and number of cycles varied for each primer
set (Table 1). Automated fragment analysis was performed on a Beckman
Coulter CEQ8000, using 600 size-standard (0.5 ul), for the following
four groups of pool-plexed PCR products: **1)** WSP2 (2.0 ul), WSP20
(0.25 ul), WSP26 (1.0 ul), C509 (1.0 ul); **2)** AC23 (0.5 ul), WSP23
(0.5 ul), WSP24 (1.0 ul), WSP33 (0.25 ul); **3)** WSP11 (1.0 ul), WSP30
(0.5 ul), WSP32 (0.25 ul); **4)** GATA02 (1.0 ul), WSP25 (0.5 ul), WSP34
(0.5 ul).

*Data Analyses*

Measurements of Hardy-Weinburg Equilibrium (HWE), linkage
disequilibrium, F-statistics, a locus-by-locus AMOVA, exact tests of
sample differentiation, and genotype assignment tests were performed for
each subpopulation using Arlequin (ver. 3.0, Excoffier *et al*. 2005).
One of the loci, WSP11, was found to consist of a complex compound
microsatellite and was therefore input into Arlequin as allele sizes,
because the number of repeats could not be determined. The ESU
designation and source of the introduced populations were determined
using the results of the genotype assignment tests. For ESU designation,
the average log-likelihood for each ESU was compared for all 320 fish.
Earlier work suggested that the Mound Spring and Lost River populations
were established with fish from lower Salt Creek (as opposed to upper
Salt Creek; Stockwell and Mulvey 1998). To further evaluate this
hypothesis, we compared log-likelihood scores for SCUP and SCLW for each
fish from Mound Spring and Lost River populations, pooling the upper and
lower sub-populations together within each population.

Overall and locus-by-locus tests of population differentiation were
performed using polymorphic loci to examine whether barrier-separated
populations differed genetically. Additionally, the number of alleles
per locus and the specific alleles present in each population were used
to examine the loss/retention of alleles for the introduced populations
at Lost River and Mound Spring.

To corroborate the results from Arlequin, genotype data were analyzed
using STRUCTURE (ver. 2.0, Pritchard *et al*. 2000; MCMC = 1,100,000
generations, burnin = 100,000 generations). This analysis uses Bayesian
methods to assign individuals to population-groups disregarding
geographic origin, allowing natural population divisions to be
determined.

**Results**

*Overall genetic structure and ESU designation*

None of the loci showed consistent linkage disequilibrium, and therefore
were assumed to be unlinked for all further analyses. After bonferroni
correction was performed, no population was significantly out of HWE.
For all populations, across all loci, F~ST~ = 0.382, indicating a high
level of structure (Table 2a). When the Malpais Spring fish were removed
from the analysis, F~ST~ dropped to 0.058 (Table 2b). Fixed differences
between the two ESUs were observed for WSP-11 and WSP-20, and commonly
occurring (frequency \> 0.20) private alleles were observed for GATA02,
WSP-24, WSP-25, WSP-26, and WSP33.

According to the genotype assignment tests, all fish were correctly
assigned to their ESU of origin. When fish from all eight subpopulations
were analyzed in STRUCTURE, Malpais Spring Fish were clearly different
from all other populations (Fig. 2a), supporting the ESU designation
(Stockwell *et al*. 1998). Within the Salt Creek ESU, fish were assigned
to their specific habitats most of the time; Salt Creek -- 77.5%; Lost
River -- 83.8%; and Mound Spring -- 75%.

*Introduction History*

According to the genotype assignment test, 85% (n = 80) and 87.5% (n =
80) of all fish were more likely to have originated in SCLW than SCUP
for Lost River and Mound Spring, respectively. We therefore considered
SCLW to be the source population for the fish introduced to Mound Spring
and Lost River. To test for genetic divergence of introduced
populations, we compared each subpopulation at Mound Spring and Lost
River to their putative founding stock (SCLW) in terms of gene
frequencies and allelic richness.

The exact tests of sample differentiation showed both subpopulations at
Lost River to be significantly different from SCLW (Table 3), while
neither subpopulation at Mound Spring (MDUP and MDLW) was significantly
different from SCLW. When the Salt Creek ESU was analyzed alone in
STRUCTURE, fish from Lost River were assigned the highest likelihood of
coming from a single population, while fish from Salt Creek and Mound
Spring were relatively indistinguishable (Fig. 3).

Compared to the SCLW subpopulation, LRLW lost 0.9 alleles per locus,
LRUP lost 1 allele / locus, MDLW lost 0.6 alleles / locus, and MDUP lost
0.8 alleles / locus (Table 4). The lost alleles ranged in frequency at
SCLW as follows: LRLW, 1.75% - 3.75%; LRUP, 1.75% - 15%; MDLW, 3.75% -
6.25%; and MDUP, 1.75% - 10%. Not all of the SCLW alleles lost at LRLW
were missing in LRUP, despite LRLW being the most likely source of
introduction at Lost River.

*Barrier effects*

The upper and lower subpopulations of Lost River and Salt Creek
populations were found to be significantly different at 63% of the
polymorphic loci, while significant differences were only seen in 8% of
polymorphic loci at Malpais Spring, and 0 loci at Mound Spring (Fig. 4).
Allelic richness was lower for LRUP at 3 loci and SCUP at 6 loci (though
higher for 1 locus; Table 5), as compared to their lower counterpart.

In both Mound and Malpais Springs, the loss of allelic richness above
the barrier was balanced with an equal number of alleles present above
the barrier that were absent in the lower sub-population (two and four
alleles, respectively; Table 5).

The probability of correctly assigning a fish to its own sub-population
was generally high. When genotypic assignment was restricted by
drainage, correct assignment occurred for 83% of MLLW fish, 78% of MLUP
fish, 85% of SCLW fish, 95% of SCUP fish, 80% of LRLW fish, 78% of LRUP
fish, 80% of MDLW fish, and 85% of MDUP fish (Fig. 4).

**Discussion**

This multilocus microsatellite dataset revealed a number of interesting
patterns, with important consequences for the conservation and
management of *Cyprinodon tularosa*. The high F~ST~ and correct
assignment of fish supports the ESU designations of Stockwell *et al.*
(1998). The genetic signature of these differences was evident in over
half of the loci with either fixed differences or commonly occurring
alleles that were restricted to one ESU.

We echo earlier recommendations (Stockwell *et al*. 1998) that
additional efforts be made to secure both the Malpais Spring ESU and the
Salt Creek ESU of *C. tularosa.*

If refuge populations are to be established, then the Malpais ESU should
be given a top priority for "replication", as it is genetically distinct
from all other *C.* *tularosa* populations. Although the Salt Creek ESU
appears to be at lower risk, we suggest guidelines for the genetic
management of this population to mediate the effects of historic
translocations and barriers.

Within the Salt Creek ESU, we observed significant drift in the Lost
River population as evidenced by exact tests of differentiation and by a
21.4 -- 23.8% reduction in allelic richness. These data suggest that a
bottleneck occurred during the founding of this population, consistent
with anecdotal reports of the Lost River population resulting from a
translocation of 30 fish (Pittenger and Springer 1999). Additionally,
the significant differences between LRLW and LRUP suggest a second
bottleneck occurred in the LRUP subpopulation, a conclusion supported by
an 11% reduction in allelic richness between LRLW and LRUP. The reduced
allelic richness, often cited as a signature of population bottlenecks
(e.g. Leberg 1992), is especially noteworthy because of the limited
number of loci examined.

In contrast, the Mound Spring population did not significantly diverge
from SCLW; however, allelic richness has been compromised for this
population (14.3 -- 19% reduction). The loss of alleles at Mound Spring
and Lost River is consistent with theoretical expectations (Allendorf
1986) and earlier empirical studies (Stockwell *et al*. 1996).

The loss of allelic richness is of some concern, as it may compromise
evolutionary potential. One solution may be to use artificial migration
to increase genetic diversity in both Lost River and Mound Spring. This
type of "genetic restoration" (*sensu* Hedrick 1995; Westemeier *et al*.
1998) would increase the value of these populations as "genetic
replicates" for the Salt Creek population; however, this approach should
be balanced against the possibility that migrants may compromise local
adaptation in the "refuge" populations (see Storfer 1999; Stockwell *et
al.* 2006).

Within the Salt Creek ESU, rapid divergence at an allozyme locus and in
body shape for the Mound Spring population appears to be due to altered
selection from reduced salinity and rate of flow (Stockwell and Mulvey
1998; Collyer *et al.* 2005). Because gene flow can compromise local
adaptation (Storfer 1999; Stockwell *et al.* 2006), we repeat the
recommendation of Stockwell *et al.* (1998) that Mound Spring population
be treated as a separate Management Unit.

By contrast, the ecological similarity of Lost River to Salt Creek
reduces the likelihood that Lost River has rapidly diverged from Salt
Creek. In fact, divergence at an allozyme locus has been modest, and
there has been no divergence for life history traits (Rogowski 2004) or
body shape (Collyer *et al.* 2005). In terms of ecological replication,
Lost River is very similar to Salt Creek in both salinity and flow
(Stockwell and Mulvey 1998; Rogowski 2004). Although the possibility
that Lost River has diverged for some unsurveyed trait can not be ruled
out, we suggest that the advantages of genetic restoration outweigh the
unlikely costs of gene flow.

While theory predicts that as few as one migrant per generation (Wright
1931; Mills and Allendorf 1996) should keep populations from diverging,
this may not be sufficient to restore allelic diversity in a timely
fashion. As an alternative, translocating 100 fish from Salt Creek
should theoretically allow alleles as rare as 0.5% in Salt Creek to be
introduced into Lost River, greatly increasing the replication value of
the population.

We therefore recommend one of two approaches be taken to restore genetic
variation at Lost River. One approach would be a one-time movement of
100 fish followed by movement of one migrant per generation (once a
year, see Rogowski 2004).

Although 1-10 migrants per generation is a more traditional rule of
thumb, higher levels of gene flow may be necessary when a population has
been isolated for an extended period, (Mills and Allendorf 1996), such
as the case with Lost River. A less aggressive approach would be to
relocate 10 fish per generation (year) over at least 10 generations,
followed by one migrant per generation (see Wright 1931; Mills and
Allendorf 1996); however, the effects of genetic restoration would be
delayed by at least one decade.

In both cases, we recommend that fish introductions be taken in a series
of steps. For instance, the first migration pulse could be targeted for
lower Lost River with later additions progressing upstream. This
iterative approach would also allow managers to evaluate if the
translocations are resulting in genetic restoration.

As previously mentioned, the genetic effects of fragmentation were only
observed at Lost River and Salt Creek; in both cases, allelic richness
was reduced for the upstream sub-populations. Changes in genetic
structure due to dams are common in fish (e.g. Yamamoto *et al*. 2004;
Neraas and Spruell 2001). Gene flow directly from Salt Creek to the
three segments of Lost River should be sufficient for managing the
divergence between the habitat fragments. The reduced variation of SCUP
is likely due to the headcut waterfall. It is not clear, however, if
this waterfall is of recent anthropogenic origin, or whether the Salt
Creek population has been historically fragmented. In terms of
population management, we believe the replication of fish in other
habitats should offer sufficient insurance and thus do not recommend any
movement of fish within Salt Creek.

The lack of significant divergence between MDLW and MDUP and the
moderate (8.1%) reduction in allelic diversity, leads us to conclude
that one migrant per generation (Wright 1931, 1970; Mills and Allendorf
1996) could be applied in bidirectional fashion to keep these
subpopulations from diverging from each other.

These data combined with information from previous studies (Stockwell
and Mulvey 1998; Stockwell *et al.* 1998; Collyer and Stockwell 2004;
Rogowski 2004; Collyer *et al.* 2005), have allowed us to better
evaluate the genetic management of White Sands pupfish. A similar
iterative approach to management has previously been taken with other
desert fishes such as the Gila topminnow (*Poeciliopsis occidentailis
occidentalis*; Parker *et al*. 1999). This approach is especially useful
for species with complicated histories that are actively managed.
Maintaining the genetic diversity of threatened populations should
enhance both the short and long-term prospects for such species.

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**Acknowledgements**

Special thanks are due to David Layfield for his assistance with
collecting these data. The authors would also like to thank Jeanne Dye
and Hildegard Reiser(CES/CEV, Holloman AFB) and Robert Myers
(Environmental Stewardship, Environmental Division in the Directorate of
Public Works, WSMR), for arrangement of range visitation. Janice Terfehr
assisted with collecting fish. This research was funded by DOD Legacy
Resource Program Grant no. DACA87-00-H-0014 administered by H. Reiser
and J. Dye, (CES/CEV, Holloman AFB) and North Dakota EPA-STAR EPSCoR
Grant to CAS. Pupfish were collected on White Sands Missile Range under
New Mexico State collecting permit 2887. R. Myers, S. Carman and Y. Chen
commented on an earlier version of this manuscript.

**Figure Legends**

Figure 1. Map of *Cyprinodon tularosa* habitats. Solid lines represent
permanent barriers to fish movement, while dashed lines represent
barriers that do not block fish movement during high precipitation
events. Modified from Stockwell *et al.* 1998.

Figure 2. Population assignment for *C. tularosa*. A STRUCTURE analysis
for all sampled *C. tularosa*, where individuals were assigned Bayesian
likelihood scores for belonging to one of four populations. LR = Lost
River, ML = Malpais Springs, MD = Mound Spring, SC = Salt Creek.

Figure 3. Population assignment within the Salt Creek ESU. A STRUCTURE
analysis for individuals sampled from the Salt Creek ESU, where
individuals were assigned Bayesian likelihood scores for belonging to
one of three populations. Drainage designations as per Figure 2.

Figure 4. Percent of polymorphic alleles with significant barrier
effects. Barrier effects were considered present when populations were
significantly differentiated by exact tests of sample differentiation in
Arlequin.

Figure 5. Genotype assignment assessing barrier effects. Assignment
likelihood scores were plotted to determine how well individuals were
assigned to their subpopulation of origin. In all graphs, grey triangles
refer to individuals sampled from the lower subpopulation, while black
circles indicate individuals sampled from the upper subpopulation. a)
Malpais Spring, b) Salt Creek, c) Lost River, d) Mound Spring.

Table 1

Table 2a

Sum of Variance Percentage

Source of Variation Squares Components of variation

Among populations 709.514 1.44248 37.096

Among sub-populations

/within populations 22.831 0.04129 1.062

Within sub-populations 1519.800 2.40475 61.84

Total 2252.145 3.88852

Fixation indices

F~SC~ 0.01688

F~ST~ 0.38158

F~CT~ 0.37096

Table 2b

Sum of Variance Percentage

Source of Variation Squares Components of variation

Among populations 42.854 0.09719 3.98

Among sub-populations

/within populations 17.631 0.04473 1.83

Within sub-populations 1089.562 2.29866 94.19

Total 1150.048 2.44057

Fixation indices

F~SC~ 0.01909

F~ST~ 0.05815

F~CT~ 0.03982

Table 3

Table 4

Table 5

  ---------- ---------- ---------- ------------ ----------- ----------- ----------- ----------- ----------- ----------- ----------- ----------- ----------- ----------- ----------- -------------
  ** **      **AC23**   **C509**   **GATA02**   **WSP11**   **WSP02**   **WSP20**   **WSP23**   **WSP24**   **WSP25**   **WSP26**   **WSP30**   **WSP32**   **WSP33**   **WSP34**   **AVERAGE**

  **MLUP**   6          2          9            6           3           1           2           2           4           2           3           2           1           5           3.43

  **MLLW**   6          2          7            9           2           2           2           5           3           2           2           2           1           6           3.64

  **SCUP**   4          1          11           2           3           1           3           2           3           1           2           1           2           3           2.79

  **SCLW**   7          1          12           2           4           1           5           3           4           1           1           1           2           3           3.36

  **LRUP**   6          1          10           2           3           1           3           2           2           1           1           1           2           2           2.64

  **LRLW**   6          1          10           2           4           1           3           2           2           1           2           1           2           3           2.86

  **MDUP**   6          1          11           2           3           1           6           2           2           1           1           1           2           3           3.00

  **MDLW**   5          1          13           2           4           1           4           2           2           1           1           1           2           3           3.00
  ---------- ---------- ---------- ------------ ----------- ----------- ----------- ----------- ----------- ----------- ----------- ----------- ----------- ----------- ----------- -------------

Figure 1

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Figure 2

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height="1.8520833333333333in"}

![](media/image3.jpeg){width="6.0in" height="2.069291338582677in"}Figure
3

Figure 4

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Figure 5

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![](media/image7.wmf)![](media/image8.wmf)