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Captive breeding of the endangered San Esteban Chuckwalla, sauromalus varius: effects of a decade of captive breeding on maintaining genetic diversity.

Sauromalus varius, the San Esteban Chuckwalla, is a large, herbivorous lizard in the family Iguanidae (Etheridge, 1982; Grismer, 1994a; Grismer, 1994b) endemic to Isla San Esteban, Sonora, Mexico (Shaw, 1945). It is noteworthy for its large size (over 600 mm in length; Case, 1982; Petren and Case, 1997; Hollingsworth, 1998), extensive coloration, and the peculiar habit of retreating to rock crevices or hollows where a lizard will "swallow" air, wedging itself in place when threatened. The total number of individuals in the wild population dropped to as few as 4,500 (Case, 1982; Petren and Case, 1997) due to anthropogenic reasons; however, not for the reasons commonly associated with humans. Rather than habitat loss or hunting pressure, the greatest threat to this reptile has been illegal collection for the pet trade (Anonymous, 1979). With the listing of S. varius by the Convention on International Trade in Endangered Species of Wild Fauna and Flora in the early 1980s, the preservation of habitat by the Mexican government, and logistical difficulties of getting to the island, there is now a reasonable chance of ensuring the survival of this species. The Arizona Sonora Desert Museum (ASDM) has maintained a captive population of S. varius since 1977, and in 1981 an enclosure specifically designed for breeding this species was constructed (Lawler et al., 1994). This enclosure was created to imitate the natural habitat of this species with the idea of facilitating the eventual reintroduction of captive-bred individuals onto Isla San Esteban (Lawler et al., 1994). For these reasons, S. varius is an excellent

example of a captive breeding colony with a high probability of success should the need to reintroduce individuals into the wild population become necessary.

Captive breeding colonies, in the face of habitat loss, over-harvesting of animals, or both, can become the primary conservation method for many species (Bryant et al., 1999). While this can be important in the conservation of species, there are several associated problems with captive breeding colonies. These problems include but are not limited to a) impacts of diseases introduced by individuals placed in the colony, b) habituation to human contact, c) inbreeding depression, and d) genetic deterioration in captivity (Lande, 1988; Ostrowski et al., 1998; Frankham et al., 2002; Woodworth et al., 2002; Robert, 2009; Carmona-Catot et al., 2012). While all of these factors are of concern, there are methods that have been employed to reduce their effects (Mitchell et al., 2011; Landguth and Balkenhol, 2012). These include temporary isolation of individuals introduced into the colony, enclosures designed to mimic the wild habitat, introduction of individuals from the wild population, and maintenance of individuals in isolated enclosures (Woodworth et al., 2002). Studies designed to elucidate the effects of captive breeding on colonies have primarily utilized Drosophila species (Hartl and Clark, 1997; Goldstein and Schlotterer, 1999; Coyne and Orr, 2004) and, while effective at providing guidelines, there have been few direct studies examining the ability of captive breeding colonies to maintain genetic variation on vertebrate species over time (Leberg and Firmin, 2008; Carmona-Catot et al., 2012).

Introducing individuals back into wild populations requires careful consideration of the genetic health of the captive population as well as the wild population (Fisher, 1930; Gilpin and Soule, 1986; Nielsen et al., 2007; Robert, 2009). To address this issue, we have amplified and genotyped eight microsatellite loci in all individuals from the captive breeding colony at the ASDM in 2004. We also have isolated DNA samples from all individuals located in that same breeding colony in 1993 (including 13 individuals present in the 2004 population) and have typed them as well. The inclusion of two sets of samples covering a decade of captive breeding provides us with the ability to directly examine the temporal effects that captive breeding has had on maintaining genetic variation within this vertebrate species. Herein we discuss the temporal effects of captive breeding on maintaining genetic variation within an endangered vertebrate species and implications for introducing animals to this colony.

MATERIALS AND METHODS--Sample Collection--Blood samples were collected from S. varius maintained at ASDM from the years 1993 (n = 63) and 2004 (n = 38). Approximately 1-2 mL of blood was drawn from the caudal vein (Gorzula et al., 1976) of each individual in the population during the 2 y sampled. Half of the blood collected was placed in a tube with acid-citrated-extrose formula-B to prevent coagulation (White et al., 1998) before being placed on ice. The remaining portion of the blood sample was placed in lysis buffer (Longmire et al., 1997).

Blood samples were returned to the lab of LDD (Texas Tech University, Lubbock, Texas) and maintained at -80[degrees]C. DNA was then isolated from whole blood using the Gentra Puregene rapid prep for cells and tissues (Gentra, Inc., Minneapolis, Minnesota) following the manufacturer's instructions. Quality of the extracted DNA was determined by running 4 [micro]L of DNA on a 1% agarose gel, staining with ethidium bromide, and visualizing with the help of a Gel Logic 100 Illuminator (Eastman Kodak Co., Rochester, New York).

MOLECULAR METHODS--Eight polymorphic microsatellite loci (McAliley et al., 2006) were amplified for each individual in the captive breeding program during both years sampled. The eight loci analyzed include six di-nucleotide repeats (Sauro 2A5, Sauro 2F5, Sauro 7A9, Sauro 10H2, Sauro 13B12, and Sauro 18A4), one tri-nucleotide repeat (Sauro 1G8), and one tetranucleotide repeat (Sauro 9B10). Polymerase chain reaction (PCR) amplifications were performed using Thermus aquaticus DNA polymerase (Saiki et al., 1986; Saiki et al., 1988) in reaction volumes of 25 [micro]L following protocols described by Allard et al. (1991). PCR was performed using locus-specific fluorescently labeled primers and an Eppendorf Mastercycler[R] gradient thermocycler (Brinkmann Instruments Inc., Westbury, New York). Each reaction consisted of 1.5 [micro]L of each primer (10 [micro]M), 1.5 [micro]L of Mg[Cl.sub.2] (25 mM), 2.0 [micro]L of dNTP (1 mM each), 0.5 [micro]L of Taq (5 U/[micro]l), 2.5 [micro]L of 10 x buffer, and d[H.sub.2]O to a final volume of 25 [micro]L. Amplification began with an initial denaturing step of 94[degrees]C for 2 min, and 25 cycles were then performed with the following parameters: 45 s denaturation at 94[degrees]C; 30 s annealing at 53.5[degrees]C; and a 50-s extension at 72[degrees]C. Amplification ended with a 10-min extension step of 72[degrees]C followed by a 4[degrees]C hold. PCR products were analyzed on an ABI 3100 Genetic analyzer (PE Applied Biosystems[R], Foster City, California) and analyzed for length using Genemapper 3.0 (PE Applied Biosystems). Assigned fragment lengths were then verified by eye.

Genetic Analysis--There were two goals to this project: one was to determine the temporal effects a decade of captive breeding have had on genetic variation within a captive breeding colony and the second was to determine how effective breeding strategies have been on maintaining that variation within offspring located in the colony. Subsequently, data were analyzed in two fashions with three identified "datasets." Datasets were designated as individuals in the colony in 1993, individuals in the colony in 2004, and finally as progeny located in the colony in 2004. Progeny were defined as any animals in the 2004 colony that were not present in the colony in 1993.

Temporal Effects on Genetic Variation Within the Captive Breeding Colony at ASDM--Statistical analyses were conducted using those individuals in the colony in 1993 (n = 63) and those in the colony in 2004 (n = 38). Estimates of genetic variation within each year and comparisons between the two colonies were made using the software programs FSTAT (http::\\www.unil. ch/softwares/fstat.html) and Arlequin 2.0 (http://cmpg.unibe. ch/software/arlequin35/). FSTAT (http:\\ softwares/fstat.html) was used to calculate Weir and Cockerham's (1984) F-statistics (F, f, and 9), allelic diversity, and deviation from Hardy Weinberg Equilibrium (HWE). Weir and Cockerham's (1984) F, f, and 9 are equivalent to Wright's FIT, FIS, and Fst, respectively. F statistics have been shown to be useful for identifying levels of genetic variation between populations, with high f values signifying potential inbreeding. Deviations from HWE provide evidence for deviations from a randomly breeding population. When appropriate, all analyses were subjected to a Bonferroni correction. To test for population-level variation, we conducted an analysis of molecular variance (AMOVA, Excoffier et al., 1992) using Arlequin 2.0. Variation was tested within the two colonies, between the two colonies, and within individuals in the two colonies utilizing calculated [F.sub.ST] values.

Temporal Effects on Genetic Variation of Offspring Located at the ASDM Colony--Statistical analyses were carried out utilizing individuals in the colony in 1993 (n = 63) and progeny in the colony in 2004 (n = 25). Estimates of genetic variation within and between the 1993 colony and the 2004 progeny were made using the programs and methods described above.

RESULTS--Genetic Variation--The majority of loci were polymorphic, the one exception being locus Sauro 9B10, which was found to be monomorphic in both colonies and subsequently removed from further analyses. Three of the seven loci examined in this study contained an equal number of alleles in the 2004 colony and the 1993 colony, though there was a shift in the proportion of alleles between the two colonies (Table 1). The remaining four loci exhibited a loss of one allele. Observed heterozygosity (Ho) values ranged from 0.026-0.316 while expected heterozygosity (He) values ranged from a low of 0.052 to a high of 0.401 (Table 2). In all cases, deviations from HWE were due to an excess of homozygotes within the colonies.

Values for f ranged from a low of 0.200 in locus Sauro 13B12 to a high of 0.792 in Sauro 2F5 (Table 3). Calculated over all loci, f was 0.405. FIT values were high in all loci ranging from 0.194-0.791 with an overall value of 0.416. The 6 values for each locus were low, with a range of 0.000-0.068 in Sauro 7A9 and Sauro 18A4, respectively, and a 6 value over all loci low at 0.019.

Temporal Effects on Genetic Variation Within the Captive Breeding Colony at ASDM; Allelic Variation--Number of alleles ranged from a low of 2 to a high of 7 (Table 2). Allelic variation was reduced between 0-33% with an average loss of variation of 12.2% between the 1993 and 2004 colonies. In most cases, we observed a shift in allelic variation (Table 1).

Population Structure--Genetic structure within colonies was examined utilizing an AMOVA (Table 4). AMOVA results demonstrate that variation is found primarily within individuals rather than within colonies (Table 4). Variation within colonies then contributed the second-most to variation (26.47%) between the two colonies sampled. This finding strongly suggests the presence of few private alleles in the two colonies sampled.

Temporal Effects on Progeny in the ASDM Colony; Allelic Variation--Numbers of alleles ranged from a low of one in Sauro 2A5 in the 2004 progeny to a high of seven in the 1993 colony for Sauro 10H2 (Table 2). Allelic variation was reduced between 0-57% in the 2004 progeny, with an average reduction of allelic diversity of 30.5%. In all loci where a decrease in allelic diversity was observed, there was a corresponding increase in the proportion of one allele over the others (Table 1).

Genetic Variation--The majority of loci surveyed were polymorphic; the one exception was locus Sauro 2A5, which was found to be monomorphic in the 2004 progeny but polymorphic in the 1993 colony (Table 2). Observed heterozygosity values ranged from 0.000-0.304 and He values ranged from 0.093-0.502 (Table 2). In all cases where deviations from HWE were observed, excess homozygotes present in the population were the cause.

Values for f ranged from a low of 0.235 in locus Sauro 13B12 to a high of 0.542 in Sauro 7A9 (Table 5). Calculated over all loci, f was 0.420. FIT values were high in all loci, ranging from 0.224-0.787, with an overall value of 0.435. The 6 values for each locus were low with a range of 0.004-0.078 in Sauro 7A9 and Sauro 18A4, respectively, and a 6 value over all loci low at 0.025.

Population Structure--Genetic structure within the 1993 colony and the 2004 progeny was conducted with an AMOVA (Table 6). Using the AMOVA, we found variation to be contained primarily within individuals rather than within the datasets (Table 6). Variation within datasets then contributed the second-most to variation (25.05%) between the 1993 colony and the 2004 progeny. This is indicative of few private alleles in the two datasets.

DISCUSSION--Microsatellite Variation--In a captive breeding colony such as the one at the ASDM, the goal is to maintain as much of the founder population genetic signature as possible (Frankham et al., 1986; Ballou and Lacy, 1995; Mitchell et al., 2011) for potential reintroduction to the wild. However, allelic variation was low within these datasets for the seven loci analyzed in this study (Table 2). Indeed, if we consider the Ho levels detected in this dataset, our results are clearly outside the range considered "typical" for microsatellite loci in most species (Lau et al., 2004; Martin and Wilcox, 2004; Monsen and Blouin, 2004; Manier and Arnold, 2005). However, given the circumstances surrounding these populations (i.e., a captive breeding colony), these numbers are less surprising. Previous studies have reported lower genetic diversity in founder populations (Nielson et al., 2007; Tzika et al., 2008; Lallias et al., 2010; Mitchell et al., 2011). While variation is low within the colony, there is not a significant change in variation between the 1993 colony and the 2004 colony. Even though allele numbers are unusually low, there is still sufficient variation to determine that there has been a negative effect on genetic diversity in the offspring in this colony. In nearly all loci examined, there has been a reduction of almost half the original alleles observed in the population (Table 2). Loss of allelic diversity is often associated with the effects of inbreeding (Hartl and Clark, 1997; Primack, 1998) and is one of the first signs of potential problems within a population.

Comparisons Between Populations--A low [theta] value (0.019) is indicative of little or no genetic structure in the population. In this case, where we are examining the temporal effects of captive breeding, it is most likely an indication that we are observing the maintenance of a small population (Weeks et al., 2013). This finding is what we would expect from a captive breeding colony (Robert, 2009; Lallias et al., 2010; Carmona-Catot et al., 2012; Weeks et al., 2013). If this value were to increase, then we would suspect that there must have been an introduction of individuals from other captive breeders. This low level of structure viewed in conjunction with high estimates of F (0.416) and f (0.405) are more suggestive of populations that are experiencing the effects of inbreeding (Mitchell et al., 2011; Weeks et al., 2013). This conclusion is further enforced by examining the results from the AMOVA. Most variation is found within individuals in the colonies rather than within the colonies. Again this is noteworthy in that inbreeding would result in most of the variation being found within individuals. A moderate amount of variation (26.47%) within the colonies is most-likely explained by the fact that there is one allele found in the 2004 colony that is absent from the 1993 colony. We view these changes within the colony and deviations of F, f, and 9 as a sign of inbreeding taking place within this captive breeding colony.

Two possible alternative explanations for the high F and f values and low 9 are a Wahlund effect or the presence of null alleles. However, given the initial founder population (n = 23; Lawler et al., 1994) and 11 y of subsequent captive breeding, we do not believe either of these to be the case. There were few loci that failed to amplify and be detected, leading us to reject the idea that null alleles are significant in the dataset. While a Wahlund effect is certainly a potential candidate to explain this population, our examination of the values reported herein do not support this hypothesis. There is very little reduction in heterozygosity in the 2 y sampled. Rather, there seems to be a slight increase in observed heterozygosity values in most loci examined, indicating that there has not been a Wahlund effect in this population. This is most-likely due to drift and a smaller population size in 2004.

Understanding the temporal effects of captive breeding within a colony is important. It is also important to examine future effects of captive breeding on the colony. The examination of diversity within the two sampled years (1993 and 2004) found there to be differences, but these appear to be minimal at this time. However, examination of effects on the offspring located at ASDM provided a different result. FIT and FIS values were higher in the offspring as compared to the 2004 colony, which might be interpreted as an increase in inbreeding, but more critical was the reduction in allelic variation. In five of the seven loci, there was a reduction in the number of alleles, with one locus (Sauro 2A5) going to fixation. While the loss of allelic variation is unwanted at any level, it is not unexpected in a captive breeding colony. However, the level of loss here is surprising given the careful breeding structure maintained at the ASDM.

Conservation Implications for S. varius--It is clear that the captive breeding colony located at the ASDM has lost genetic variability in its progeny. While to date there has not been an apparent loss of fitness in S. varius maintained at the ASDM, now is the time to address this issue before it becomes a serious problem. Likely, the most appropriate solution is the introduction of individuals from a) wild populations or b) from other captive-bred individuals. These introductions would increase genetic diversity in the colony, thereby increasing the fitness of individuals in this colony. However, before introductions are made, careful consideration must be taken to minimize the potential effects of outbreeding depression. While we may wish to increase the genetic diversity within this important colony, we do not wish to replace the variation currently found but would rather augment that variation so as to increase the fitness of the colony as a whole. It is also essential to determine the genetic variation within the wild population so that we may better design protocols for the captive management of this species. This would also be important for identifying animals in the wild that are best suited for introduction into the captive colony, should that prove to be the best course of action. The observation that allelic diversity in progeny of the colony is reduced, even given careful breeding by the staff at ASDM, clearly demonstrates the need for careful monitoring of genetic health in this and other captive breeding colonies.

A healthy captive breeding colony is of utmost importance in the continued survival of S. varius, but for reasons beyond those that led to its listing. While the threat on Isla San Esteban is not critical at this point in time, and anthropogenic reasons will most likely not have an impact on the demise of this species, there is always the continued threat of natural catastrophes. The island serving as natural habitat for this species is small and is subject to natural disasters such as drought, hurricanes, or a fire. Should a disaster befall individuals on Isla San Esteban, the only resource for reintroduction will be those animals located in captive breeding colonies such as the one maintained by the ASDM.

We would like to thank the efforts of the staff at the Arizona Sonora Desert Museum for their assistance in the completion of this project. As well we would like to thank H. Lawler for his contribution to samples from the Arizona Sonora Desert Museum in 1993. We would also like to thank the members of R. Bradley's lab for their patience and assistance with us as we took time on their automated sequencer during the course of this project. Finally, we would like to thank anonymous reviewers for their comments and suggestions on this manuscript and J. P. C. Estupifian for his translation of the resumen.


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Submitted 22 October 2014.

Acceptance recommended by Associate Editor, Felipe de Jesus Rodriguez Romero, 13 September 2016.


Department of Natural Sciences, Northwest Missouri State University, Maryville, MO 64468 (LRM) College of Science and Mathematics, Midwestern State University, Wichita Falls, TX 76308 (REW) Arizona Sonora Desert Museum, Tucson, AZ 85743 (CI) Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409 (LDD)

* Correspondent:
TABLE 1--Allele sizes and proportion of each allele for each
locus (described by McAliley et al., 2006) for three groups of
Sauromalus varius from the Arizona Sonora Desert Museum:
colony in 1993 (n = 63), colony in 2004 (n = 38), and the 2004
progeny (n = 25). Dashes represent loss of that allele in the

                        1993     2004     2004
Locus         Allele   Colony   Colony   Progeny

Sauro 1g8      199     0.960    0.895     0.880
               202     0.040    0.105     0.120
Sauro 2A5      160     0.032    0.013      --
               162     0.968    0.987     1.000
Sauro 7A9      161     0.087    0.053     0.004
               163     0.008      --       --
               165     0.905    0.947     0.960
Sauro 10H2     180     0.009    0.013      --
               184     0.009      --       --
               186     0.009    0.013      --
               192     0.036    0.026      --
               194     0.714    0.776     0.840
               196       --     0.013     0.020
               198     0.214    0.145     0.140
               204     0.009    0.013      --
Sauro 2F5      156     0.066    0.026     0.040
               158     0.934    0.974     0.960
Sauro 13B12    144     0.825    0.868     0.860
               146     0.016      --       --
               148     0.159    0.132     0.140
Sauro 18A4     143     0.008      --       --
               145     0.278    0.069     0.043
               151     0.040    0.014      --
               153     0.659    0.819     0.804
               155     0.016    0.097     0.152

TABLE 2--Number of individuals of Sauromalus varius from the captive
colony housed at the Arizona Sonora Desert Museum (1993-2004) that
were genotyped for each locus by group, number of individuals
genotyped, number of alleles, heterozygosity measures, and
significance test for deviation from HWE are provided for each locus
for each group. Due to fixation of alleles at loci Sauro 2A5 in the
2004 progeny values are reported as NA (not applicable). Hq is
observed heterozygosity, He expected heterozygosity, and f is Wright's

Locus           N    Alleles     f

1993 Colony
  Sauro 1g8     63      2       0.795
  Sauro 2A5     63      2       0.490
  Sauro 7A9     63      3       0.639
  Sauro 10H2    56      7       0.442
  Sauro 2F5     61      2       1.000
  Sauro 13B12   63      3       0.250
  Sauro 18A4    63      5       0.549
2004 Colony
  Sauro 1g8     38      2       0.175
  Sauro 2A5     38      2       0.000
  Sauro 7A9     38      2      -0.042
  Sauro 10H2    38      7       0.171
  Sauro 2F5     38      2      -0.014
  Sauro 13B12   38      2       0.092
  Sauro 18A4    36      4       0.217
2004 Progeny
  Sauro 1g8     25      2      -0.116
  Sauro 2A5     25      1       NA
  Sauro 7A9     25      2      -0.021
  Sauro 10H2    25      3       0.145
  Sauro 2F5     25      2      -0.021
  Sauro 13B12   25      2       0.189
  Sauro 18A4    23      3       0.094

Locus            Ho      He     P-value    SD

1993 Colony
  Sauro 1g8     0.016   0.093   0.001     0.000
  Sauro 2A5     0.032   0.077   0.046     0.001
  Sauro 7A9     0.063   0.191   0.000     0.000
  Sauro 10H2    0.250   0.459   0.000     0.000
  Sauro 2F5     0.000   0.140   0.000     0.000
  Sauro 13B12   0.222   0.309   0.002     0.000
  Sauro 18A4    0.222   0.502   0.000     0.000
2004 Colony
  Sauro 1g8     0.158   0.217   0.336     0.002
  Sauro 2A5     0.026   0.052   1.000     0.000
  Sauro 7A9     0.105   0.126   1.000     0.000
  Sauro 10H2    0.316   0.401   0.022     0.000
  Sauro 2F5     0.053   0.078   1.000     0.000
  Sauro 13B12   0.211   0.254   0.491     0.002
  Sauro 18A4    0.250   0.341   0.012     0.000
2004 Progeny
  Sauro 1g8     0.240   0.251   1.000     0.000
  Sauro 2A5      NA      NA       NA       NA
  Sauro 7A9     0.800   0.117   1.000     0.000
  Sauro 10H2    0.240   0.280   0.491     0.001
  Sauro 2F5     0.080   0.117   1.000     0.000
  Sauro 13B12   0.200   0.285   0.385     0.002
  Sauro 18A4    0.304   0.371   0.037     0.001

TABLE 3--Weir and Cockerham's F-statistics for the 1993 colony and the
2004 colony of Sauromalus varius housed at the Arizona Sonora Desert
Museum. F is analogous to Wright's FIT, 9 is Wright's FST, and f is
Wright's FIS (Weir and Cockerham, 1984). Numbers in parentheses are
95% confidence intervals.

Locus                  F                  9                f

Sauro 1g8            0.435               0.02            0.423
Sauro 2A5            0.387              -0.008           0.391
Sauro 7A9            0.522               0.000           0.522
Sauro 10H2           0.340              -0.004           0.343
Sauro 2F5            0.791              -0.003           0.792
Sauro 13B12          0.194              -0.007           0.200
Sauro 18A4           0.497               0.068           0.460
All loci      0.416 (0.306-0.538)       0.019            0.405
                                    (-0.005-0.048)   (0.307-0.527)

TABLE 4--Analysis of molecular variance for the 1993 colony and the
2004 colony of Sauromalus varius housed at the Arizona Sonora Desert

Source of variation   Degrees   Sum of     Variance    Percentage
                        of      squares   components       of
                      freedom                          variation

Among colonies            1      0.696    0.00212         0.54
Among individuals        99     49.047    0.10415        26.47
  within colonies
Within individuals      101     29.000    0.287          72.99

TABLE 5--Weir and Cockerham's F-statistics for the 1993 colony and the
2004 progeny of Sauromalus varius housed at the Arizona Sonora Desert
Museum. F is analogous to Wright's FIT, 9 is Wright's FST, and f is
Wright's FIS. Numbers in parentheses are 95% confidence intervals.

Locus                  F            9                      f

Sauro 1g8            0.341              0.036            0.316
Sauro 2A5            0.493              0.002            0.492
Sauro 7A9            0.540              -0.004           0.542
Sauro 10H2           0.384              0.009            0.378
Sauro 2F5            0.787              -0.02            0.792
Sauro 13B12          0.224              -0.014           0.235
Sauro 18A4           0.502              0.078            0.460
All loci      0.435 (0.327-0.549)       0.025            0.420
                                    (-0.008-0.058)   (0.325-0.536)

TABLE 6--Analysis of molecular variance for the 1993 colony and the
2004 progeny of Sauromalus varius housed at the Arizona Sonora Desert

Source of variation   Degrees   Sum of     Variance    Percentage
                        of      squares   components       of
                      freedom                          variation

Among datasets           1       1.197    0.01001         2.55
Among individuals       86      41.343    0.09832        25.05
  within datasets
Within individuals      88      25.000    0.284          72.40
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Author:McAliley, L. Rex; Willis, Ray E.; Ivanyi, Craig; Densmore, Llewellyn D., III
Publication:Southwestern Naturalist
Article Type:Report
Date:Dec 1, 2016
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