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Genetic diversity assessment of in situ and ex situ Texas wild rice (Zizania texana) populations, an endangered plant
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Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Genetic diversity assessment of in situ and ex situ Texas wild rice (Zizania texana) populations, an endangered plant Wade D. Wilson a,∗ , Jeffrey T. Hutchinson b , Kenneth G. Ostrand b a b
U.S Fish & Wildlife Service, Southwestern Native Aquatic Resources and Recovery Center, PO Box 219, Dexter, NM 88230-0219, USA U.S Fish & Wildlife Service, San Marcos Aquatic Resources Center, 500 East McCarty Lane, San Marcos, Texas 78666, USA
a r t i c l e
i n f o
Article history: Received 24 June 2015 Received in revised form 2 December 2015 Accepted 21 December 2015 Available online xxx Keywords: Genetics Endangered species Refugia San Marcos River Texas wild rice
a b s t r a c t Texas wild rice (Zizania texana) is an endangered, aquatic perennial plant endemic to the upper section of the San Marcos River, Texas. Ex situ populations of Z. texana are maintained by the U.S. Fish and Wildlife Service in the event of a catastrophe event. We analyzed the genetics of in situ and ex situ populations of Z. texana to address the following questions: (1) are in situ populations adequately represented in ex situ population? (2) Is there genetic diversity among the current in situ population? (3) Has the current in situ genetic diversity increased or decreased from historical estimates in 2007. Results indicated that the overall ex situ populations were lower in genetic (allelic) diversity compared to the in situ population, with some in situ populations not present in the refugia. Overall, heterozygosity was moderate and ranged from HO = 0.530 to HO = 0.635 in the wild and HO = 0.549 to HO = 0.727 in the ex situ population. Inbreeding coefficients (FIS ) were near zero or negative indicating that inbreeding is not common within the current populations (in situ and ex situ) suggesting that some populations have an excess of heterozygotes (negative FIS ). Analysis of current in situ population structure indicated there are three unique genetic clusters in the San Marcos River. Comparison of the in situ population with historical analysis indicates the genetic diversity of the wild population is dynamic both temporally and spatially. The results indicate that Z. texana exhibits a plastic reproductive system utilizing both asexual (vegetative) and sexual (flowering and seed production) reproduction. Published by Elsevier B.V.
1. Introduction The present rate of habitat loss and landscape alteration has led to the decline and extinction of many species (Dobson et al., 2006; Wilcove et al., 1998). Habitat restoration, single species reintroductions, and species augmentations will become increasingly important in long-term protection of rare plants (Kramer and Havens, 2009). Worldwide, funding and research for rare plants remains limited while the number of plants threatened continues to increase (Havens et al., 2014). Ex situ conservation programs compliment in situ conservation programs with short to long-term storage of genetically representative samples and allow for long term research on species in which little is known of their life history (Heywood and Iriondo, 2003). Historically, most ex situ conservation genetic programs have focused on food crops. Moreover, most of these programs have concentrated on seed collection and less on living collections. For plants with recalcitrant seeds such as Texas
∗ Corresponding author. Fax: +1 575 734 6130. E-mail address: Wade [email protected] (W.D. Wilson).
wild rice (Zizania texana Hitchc.), living plant collections are the only option for long-term ex situ collections. Plant species maintained ex situ need to represent the population’s genetic diversity, follow protocols to ensure genetic diversity during reproduction, and ensure that two or more collective samples are maintained at different locations (Cohen et al., 1991). The main goal of managing ex situ living plant collections is to ensure that the highest level of genetic diversity is maintained at an economically feasible level (Cibrian-Jaramillo et al., 2013). Often this can best be accomplished through a collaborative effort between the organization managing the ex situ plants and a university or research facility with genetic analysis capabilities (Griffith et al., 2011). Maintaining ex situ populations of rare plants that represent the genetic diversity of in situ populations results in a safety net to prevent the extinction of rare species (Guerrant et al., 2004). Balancing genetic diversity and operational cost of living populations can be challenging, but can best be obtained with genetic analysis. Resources for maintaining living plants are finite and genetic analysis can help in allocation of funds for maintaining ex situ plant collections that represent the genetic diversity of in situ populations.
http://dx.doi.org/10.1016/j.aquabot.2015.12.005 0304-3770/Published by Elsevier B.V.
Please cite this article in press as: Wilson, W.D., et al., Genetic diversity assessment of in situ and ex situ Texas wild rice (Zizania texana) populations, an endangered plant. Aquat. Bot. (2015), http://dx.doi.org/10.1016/j.aquabot.2015.12.005
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Zizania texana is a federal endangered plant in the USA and is endemic to the upper portion of the San Marcos River, Hays County, Texas (Poole et al., 2007). Zizania texana prefers swift moving cool spring-fed runs and is most commonly found at water depths less than 1 m growing in coarse sandy soils (Poole and Bowles, 1999). It is a perennial C3 grass that occurs in the upper 5 km of the headwaters of the San Marcos River with 97% of the Z. texana population occurring within the upper 2.2 km. Threats to Z. texana include aquifer depletion, habitat destruction and alteration, invasive species, droughts, floods, unintended recreational impacts and its extremely small and limited range (Poole, 2002). Fragmentation of stands, large gaps between stands, and damaged stems also result in reduced pollination and seed production by Z. texana (Power and Oxley, 2004). Because of its limited distribution, Z. texana is listed (USFWS, 1978) as a G1 critically imperiled species due to its extreme rarity in five or fewer populations. The San Marcos Aquatic Resources Center (SMARC; 29◦ 50 23.72 N, 97◦ 58 33.78 W) is responsible for maintaining Z. texana in refugia as an ex situ population in the event a catastrophe or stochastic event destroys the San Marcos River in situ population (USFWS, 1995). An additional ex situ population is maintained at the U.S. Fish and Wildlife Service (USFWS) Uvalde National Fish Hatchery (29◦ 11 15.37 N, 99◦ 49 59.27 W). The ex situ population of Z. texana at the SMARC is comprised of specimens collected from 10 of the 14 Texas Parks and Wildlife Department (TPWD) monitoring segments within the San Marcos River (Table 1, Fig. 1). Specimens from these segments were collected using a stratified sampling scheme along a longitudinal gradient (see Richards et al., 2007). The in situ sampling design was implemented to avoid maintaining large redundant collections at the SMARC that may divert resources from other conservation priorities. Starting in 2005, collections representing allelic richness and diversity of Z. texana populations from the San Marcos River have been maintained at the SMARC (Richards et al., 2007); nevertheless, the spatial distribution and cover of various Z. texana stands have changed since their sample collection in 1998, 1999 and 2002. Many stands of Z. texana have increased in size, some new stands have become established, and some smaller stands have disappeared (Fig. 2). These demographic changes in Z. texana are due to the combined establishment of floating tillers, seeds and human mediated translocations. In order to ensure that conservation goals are met in accordance with the regulatory and guiding conservation documents (e.g. USFWS, 1995) the objectives of this project were: (1) to conduct genetic analyses to ensure that ex situ population held at the SMARC represent the extant in situ (wild) population, (2) determine if there is genetic diversity among the current in situ population, (3) and evaluate if historical estimates of Z. texana in situ population genetic diversity have changed compared to Richards et al. (2007).
2. Materials and methods 2.1. Tissues A total of 245 Z. texana samples were collected from both the San Marcos River population (N = 184) and the ex situ population at the SMARC (N = 61) in September and October 2012. In situ sample nomenclature follow TPWD segment nomenclature and are designated as A, B, C, etc. with ex situ refugia (R) samples designated as AR, BR, CR, etc. Sample collection of in situ individuals were conducted by swimmers who clipped fresh 10–12 cm leaf samples from most stands <2 m2 and all stands >2 m2 from eight of the eleven segments (Fig. 1). Samples of Z. texana were not collected from the other segments because either Z. texana did not occur in
these locations (I and M) or spatial coverage was <0.1 m2 (E, G, J, and L). The ex situ (i.e. refugia) samples were collected from the SMARC and only represented five (A, B, C, F and K) of the eleven segments. Collection methods differed from Richards et al. (2007) in that the exact location of each sample was not mapped either within or between stands. Leaf tissue samples were placed in plastic zip bags, packed on ice, and shipped to the Southwestern Native Aquatic Resources and Recovery Center (SNARRC). Upon arrival at the SNARRC, samples were stored at −20 ◦ C until Genomic DNA extraction. A sub-sample of each individual was freeze-dried and archived at −80 ◦ C for future reference. 2.2. Extraction, PCR, and genotyping Genomic DNA was extracted using Qiagen DNeasy® 96 Blood and Tissue Kits (Qiagen, Valencia, CA) using a modified method. Approximately 0.025 g of each leaf tissue sample was finely sliced in preparation for DNA extraction with the amount of proteinase K and Buffer ATL doubled from standard protocol amounts for tissue lysis. Leaf tissue was incubated overnight at 56 ◦ C after which each sample was disrupted with a small metal spatula at three points during lysis: once immediately following the addition of proteinase K and buffer ALT, once following the overnight incubation, and a third time two hours after the second. Undigested leaf material was removed with forceps following the third tissue disruption and DNA extraction was completed following the DNeasy spin-column protocols for animal tissues. Multiplex PCR amplifications (10 l) consisted of 3.0 l Qiagen Multiplex Mastermix® ; 0.5 l each, forward and reverse primer (10 M); 3.6 l ddH20; 0.4 l 100x (10 mg/ml) purified Bovine Serum Albumin (BSA; New England Biolabs); 2 l template DNA. Forward primers were labeled with one of four fluorescent dyes (6FAM, PET, NED, VIC). Touchdown cycling (temperature decreased by 0.2 ◦ C/cycle) using a GeneAmp® System 9700 (Applied Biosystems) consisted of one cycle at 95 ◦ C for 15 min (to activate the HotStar Taq DNA polymerase), followed by 33 cycles of 94 ◦ C for 45 s, an initial annealing temperature of 56 ◦ C for 45 s, and an extension temperature of 72 ◦ C for 60 s with a final extension of 30 min at 70 ◦ C. BSA was needed as PCR inhibitors initially prevented consistent amplification, initially. All PCR reagents were purchased from Applied Biosystems (Foster City, CA). The six microsatellite used were identical to Richards et al. (2007). PCR products were processed on an ABI 3130xl genetic analyzer using GeneScanTM 500 LIZ® size standard. Composite genotypes for individual samples were compiled with GeneMapperTM 4.0 software (Applied Biosystems). 2.3. Quality control/quality assurance To ensure accuracy in genotyping, samples were run through a Quality Control/Quality Assurance (QA/QC) process consisted of two actions: (1) rescoring of all runs by a second researcher, and (2) independently re-extracting, amplifying, and scoring 10% of the samples. Genotypes produced by the different researchers were compared and all inconsistencies were resolved, thus no samples were removed prior to analyses. 2.4. Data analysis A total of 245 individuals of Z. texana were genotyped using six microsatellite loci used by Richards et al. (2007). Departures from the Hardy–Weinberg (HW) equilibrium and global tests of linkage equilibrium (LD) among all pairs of loci and populations were tested using GENEPOP v4.0 (Raymond and Rousset, 1995; Rousset, 2008). The test for HW equilibrium used the heterozygote deficiency method (Raymond and Rousset, 1995), a global test that
Please cite this article in press as: Wilson, W.D., et al., Genetic diversity assessment of in situ and ex situ Texas wild rice (Zizania texana) populations, an endangered plant. Aquat. Bot. (2015), http://dx.doi.org/10.1016/j.aquabot.2015.12.005
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Table 1 Current inventory and future needs of the ex situ population of Zizania texana at the San Marcos Aquatic Resources Center. The recommended inventory is based on several factors including available space (capacity of 430), availability in the wild as determined by percent cover, and the number of alleles represented in each segment of the in situ population divided by the total number of alleles, based on current the 2012 analyses. Segment
Current inventory
Recommended inventory
Additional need
Wild% cover (NA /NAT )a
A B C D E F G H I X J K L M Unknown Total
6 45 26 3 8 19 1 3 0 0 13 6 0 0 27 157
50 205 68 10 8 60 5 5 – 0 13 6 0 0 0 430
44 160 42 7 0 41 4 2 – 0 0 0 0 0 – 300
9.2 (39/65) 62.7 (32/65) 16.7 (35/65) 0.4 (46/65) <0.1 (−) 8.0 (48/65) 0.4 (−) 1.2 (−) 0.0 (−) 0 <0.1 (−) 1.2 (32/65) 0.1 (−) 0
a
–
2
% Cover = % of the approximately 4996 m in the in situ population, NA = number of alleles found in each segment, NAT = total number of alleles (65) found in the in situ (current study), – = not genotyped in current study.
Fig. 1. Map showing the 11 Zizania texana monitoring segments (A–K) within the San Marcos River. Segments L, M (below K) are not shown because genetic samples were not analyzed from these segments.
Please cite this article in press as: Wilson, W.D., et al., Genetic diversity assessment of in situ and ex situ Texas wild rice (Zizania texana) populations, an endangered plant. Aquat. Bot. (2015), http://dx.doi.org/10.1016/j.aquabot.2015.12.005
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Fig. 2. Historical Zizania texana cover in selected segments of Texas Parks and Wildlife Department’s monitoring program. Segments A, B, C, and F are represented in top figure, and segments E, J, and K are represented in bottom figure.
examines either the population(s) or locus but not both simultaneously. The test of LD examines the relationship between genotypes at each pair of loci (i.e., composite LD; Weir, 1996). Deviations from HW equilibrium due to stuttering, null alleles, and large allele dropout were tested using MICROCHECKER 2.2.3 (van Oosterhout et al., 2004). Heterozygosity (HE ; gene diversity), observed heterozygosity (HO ) on a per locus basis, the number of alleles per locus (NA ), average inbreeding coefficients (FIS ), and private alleles were estimated using GenAlEx v6 (Peakall and Smouse, 2006). GenAlEx was also used to calculate the Probability of Identity (PI) that provides the average probability estimate that two unrelated individuals, drawn from the same randomly mating population, will have the same multilocus genotype. Allele frequencies and descriptive statistics, including allelic richness (AR ) were estimated using FSTAT v2.9.3.1 (Goudet, 1995). Allelic richness was calculated using the methods described by Petit et al. (1998) and uses rarefaction and repeated random subsampling to provide unbiased estimates of AR (Leberg, 2002). The number of genetic clusters (K) was calculated using the Bayesian clustering method of STRUCTURE v2.3.2 (Pritchard et al., 2000). The admixture model that assumes gene flow among
populations and allows for correlated allele frequencies across populations was applied. This model assigns a proportion of each individual’s genome to each of the genetic clusters pursuing solutions that maximize HW equilibrium and LD within clusters. Ten iterations were performed for each K with the true K assumed to be between 1 and 8. All runs had a burn-in of 100,000 preliminary iterations followed by 100,000 iterations of data collection. The method of Evanno et al. (2005) that uses the second order rate of change between K and K + 1 cluster (K) was used to estimate the number of genetic clusters, as implemented in STRUCTURE HARVESTER (Earl and vonHoldt, 2011). The K with the largest K value is assumed to be the best estimator of K. Pairwise FST was calculated using GENODIVE (Meirmans and van Tienderen, 2004). This distance is the FST statistic resulting from an Analysis of Molecular Variance (AMOVA) performed between each pair of populations (Excoffier et al., 1992; Michalakis and Excoffier, 1996). Identical genotypes that occurred in different segments were included in the analysis, while identical genotypes occurring within segments were removed from the final analysis to eliminate repetitive genotypes within a segment. The final dataset consisted of 203 individuals.
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3. Results The probability of identity (PI) was low and ranged from 2.25 × 10−6 to 7.42 × 10−3 (mean 1.06 × 10−3 ). This study found 40 of the 245 Z. texana samples grouped into 28 clusters with each cluster having multiple (mode = 2, mean = 3.2, range 2–9) identical genotypes (data not shown). Identical genotypes were found both within a segment (15 of 28 clusters) and among segments (13 of 28 clusters). For example, genetic cluster one had two samples with identical genotypes, one from segment F and the other from the refugia population that was originally collected from segment B. Genetic cluster seven had nine identical genotypes from five different segments; CR and FR (refugia) and C, H and K (wild). Observed heterozygosity was moderate and ranged from HO = 0.530 (segment A) to HO = 0.635 (segment D) for in situ and HO = 0.549 (BR) to HO = 0.727 (FR) for ex situ population (Table 2). Inbreeding coefficients (FIS ) were near zero or negative indicating that inbreeding is not common within the current populations (in situ and ex situ) and suggests that some populations have an excess of heterozygotes (negative FIS ). Only three segments (A, F, and H) were not in HW equilibrium and showed signs of homozygote excess (positive FIS ). Using MICROCHECKER, the presence of null alleles was suggested at segments A, F, and H at three loci (Zt13, Zt21, and Zt22); however, null alleles were not indicated in other segments or ex situ samples for these loci. Therefore, the data suggest that these three segments had higher inbreeding (heterozygote deficiencies) in the current sampling period and that the higher positive FIS values are not the result of the markers themselves. Excluding segments with <10 individuals, the mean number of alleles per locus for the in situ samples ranged from 8.0 (segment D) to 4.7 (segment H). Two of the segments represented in the in situ population had similar number of alleles per locus when compared to their ex situ counterparts, the exception being segment F, that had lower diversity in the in situ population (NA = 5.0 vs. NA = 8.0). Private alleles are those that are unique to a certain population or collection of samples. Within the San Marcos River, three segments (B, D, and F) contained private alleles at low frequency, usually occurring in one individual. Segment B had three private alleles (Zt23-164, Zt13-224, and Zt22-204), D had one (Zt13-218) occurring in three individuals, and F had five (Zt13-238, Zt13-214, Zt23-176, Zt18-90, and Zt1-258) occurring in seven individuals (Table 1). An analysis of population structure of the current dataset using the Bayesian clustering method of STRUCTURE and the K method of Evanno et al. (2005) as implemented in STRUCTURE HARVESTER (Earl and vonHoldt, 2011) indicated there are three genetic clusters with all three clusters occurring in each segment within the current in situ population (Fig. 3). Cluster 1 was most prevalent in segments A (76.8%), K (69.3%), and H (49.6%), cluster 2 in segments B (81.6%), C (63.7%), and D (46.8%), and cluster 3 in D (37.2%) and F (34.2%). The high prevalence of cluster 1 in segment A is most likely due to the addition of 10,000 seeds in the summer of 1996 with the source of the seeds being from a few plants obtained from segment F after which the number of stands increased from 8 to 28 (J. Poole, pers. comm.); currently segment F is comprised of all three genetic clusters in equal amounts (37.1%, 28.7% and 34.2%, respectively). AMOVA indicated that there was significant genetic differentiation: (1) among individuals within segments (FST = 0.047, P = 0.003) and (2) among segments (FST = 0.049, P = 0.001); however, 90.6% of the variation was within individuals not among segments. Pairwise FST values were significantly different between all in situ segments (Table 3) and consistent with the STRUCTURE results. In a comparison of the in situ samples with their ex situ counterparts, results differed (only in situ populations with N > 10 were used in the pairwise FST analysis). Segment B did not differ from the ex situ population (BR); however, F was significantly different from FR and is
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due to the in situ population having more allelic diversity than the ex situ population. This was apparent and evidenced at locus Zt21.
4. Discussion Captive conservation populations (ex situ) found at zoos, botanic gardens and hatcheries have been a necessary tool to protecting Threatened and Endangered species and preventing extinction (Fisch et al., 2015; Guerrant et al., 2004; Maunder et al., 2004). These conservation populations can be maintained as refuge populations (propagation is not conducted) or as broodstock populations used for production of offspring or seeds that are subsequently used for augmentation or restoration conservation activities. Establishing and maintaining ex situ conservation populations that are genetically unrepresentative of the source population can occur and go undetected unless genetic evaluations are periodically conducted (Christe et al., 2014). In addition, using unrepresentative broodstock populations or few individuals for propagation can alter the genetic diversity and effective size of the recipient wild population (Christie et al., 2012; Fisch et al., 2015). Although many of examples are from animal species such as fishes, which are well studied in terms of genetic consequences of small populations, the concepts apply across species (see Falk and Holsinger, 1991). In a recent genetic assessment of in situ and ex situ in two tree species (Zelkova abelica and Zelkova carpinifolia), Christe et al. (2014) found that only one of the species (Z. carpinifolia) was representative of the in situ population and although Z. abelica was represented in eight botanic gardens, the collections had originated from a single region; however, previously unidentified chloroplast DNA haplotypes were found, underscoring the utility of ex situ populations in preserving genetic diversity. In addition, several individuals identified as Z. carpinifolia in ex situ populations were found to be misidentified to both the wrong species and genus. Therefore, genetic evaluations of existing ex situ populations are vital for effective planning and implementation of recovery actions of species of conservation concern. To this end, the first goal of this study was to evaluate the Z. texana ex situ population at the SMARC and determine if it was representative of the in situ San Marcos River population. Of the five segments represented in the ex situ population (AR, BR, CR, FR, and KR), only two (BR and FR) had sample sizes adequate for in situ/ex situ comparisons. In cases where comparisons could be made the results suggest that overall the ex situ population (BR and FR) is lower in genetic (allelic) diversity compared to the in situ population suggesting that the ex situ population should be augmented. In the other ex situ segments (A, C, K), the number of individuals was either too low to compare genetically or be used for reintroductions because individuals may be identical and limited in diversity. For example, the ex situ population currently has six plants from segment A and two of the three plants genotyped were genetically identical. Given the ability of Z. texana to reproduce clonally, a genetic assessment of each individual added to the ex situ population is imperative. Segment A, differs in allelic frequency compared to the other segments, but this may be the result of human intervention and the addition of 10,000 seeds in 1996 from a few plants taken from segment F (J. Poole, pers. comm.), either way the ex situ segment A population is limited in diversity compared to the in situ segment. The ex situ population may also harbor unique and important individuals from two segments (E and J) that have few Z. texana compared to historical cover. Annual population monitoring has shown that the cover of Z. texana decreased substantially after 1998 with 2012 cover estimates being 2 m2 (segment E) and 1 m2 (J); both of these segments had cover of over 100 m2 in 1991. Flooding, droughts, and recreation activities can result in the decline of Z. texana in the San Marcos River (Poole et al., 2007). These stochastic and
Please cite this article in press as: Wilson, W.D., et al., Genetic diversity assessment of in situ and ex situ Texas wild rice (Zizania texana) populations, an endangered plant. Aquat. Bot. (2015), http://dx.doi.org/10.1016/j.aquabot.2015.12.005
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Table 2 Summary statistics of the 6 microsatellite loci used to screen Zizania texana in situ (wild-San Marcos River) and ex situ (refugia-San Marcos Aquatic Resources Center) samples collected in 2012. Refugia samples are designated with an R after site (e.g. BR). Locus
Statistic
In situ (wild) samples
Ex situ (refugia) samples
A n = 28
B n = 22
C n = 26
D n = 22
F n = 29
H n = 15
K n = 12
X n=2
AR n=2
BR n = 17
CR n=8
FR n = 11
KR n=2
Zt1
NA AR HO HE FIS
2.00 2.00 0.36 0.49 0.27
2.00 2.00 0.23 0.27 0.15
2.00 1.85 0.04 0.11 0.65
2.00 1.99 0.27 0.24 −0.16
3.00 2.33 0.24 0.22 −0.12
2.00 2.00 0.13 0.23 0.42
2.00 2.00 0.33 0.28 −0.20
1.00 – 1.0 0.63 −0.60
2.00 – 0.50 0.38 −0.33
2.00 2.00 0.35 0.46 0.23
2.00 – 0.25 0.22 −0.14
2.00 2.00 0.27 0.24 −0.16
2.00 – 0.50 0.38 −0.33
Zt13
NA AR HO HE FIS
6.00 5.68 0.57 0.78 0.27
6.00 4.83 0.82 0.66 −0.23
6.00 4.72 0.54 0.57 0.04
8.00 6.63 0.76 0.79 0.03
9.00 6.32 0.62 0.76 0.19
6.00 5.65 0.60 0.72 0.17
5.00 4.83 0.75 0.60 −0.25
2.00 – 0.50 0.38 −0.33
3.00 – 1.00 0.63 −0.67
5.00 4.57 0.47 0.57 0.18
6.00 – 0.75 0.74 −0.01
3.00 3.00 0.73 0.60 −0.21
4.00 – 1.00 0.75 −0.33
Zt18
NA AR HO HE FIS
10.00 6.84 1.00 0.81 −0.24
7.00 6.02 0.96 0.74 −0.30
7.00 5.61 0.96 0.76 −0.27
9.00 7.74 0.91 0.83 −0.09
10.00 7.25 0.97 0.81 −0.19
7.00 6.39 0.87 0.78 −0.11
6.00 5.83 0.92 0.74 −0.24
3.00 – 1.00 0.63 −0.60
4.00 – 1.00 0.75 −0.33
9.00 8.13 0.88 0.83 −0.07
7.00 – 1.00 0.83 −0.21
8.00 8.00 1.00 0.81 −0.24
3.00 – 1.00 0.63 −0.60
Zt21
NA AR HO HE FIS
10.00 7.41 0.57 0.80 0.29
7.00 5.89 0.73 0.72 −0.01
8.00 6.30 0.84 0.79 −0.06
14.00 11.22 0.86 0.90 0.04
13.00 9.58 0.69 0.88 0.22
5.00 4.45 0.60 0.61 0.02
9.00 8.58 0.92 0.82 −0.12
3.00 – 1.00 0.63 −0.60
3.00 – 1.00 0.63 −0.60
8.00 7.65 0.82 0.83 0.01
8.00 – 1.00 0.79 −0.27
6.00 9.58 0.91 0.74 −0.23
3.00 – 0.50 0.63 0.20
Zt22
NA AR HO HE FIS
4.00 2.57 0.07 0.17 0.57
3.00 2.00 0.09 0.09 −0.04
4.00 3.02 0.16 0.22 0.27
5.00 3.73 0.32 0.35 0.09
4.00 2.53 0.07 0.16 0.57
4.00 3.66 0.13 0.34 0.61
3.00 2.83 0.08 0.16 0.47
2.00 – 0.50 0.38 −0.33
1.00 – 0.00 0.00 N/A
2.00 1.65 0.06 0.06 −0.03
3.00 – 0.38 0.48 0.21
4.00 4.00 0.55 0.58 0.06
1.00 – 0.00 0.00 N/A
Zt23
NA AR HO HE FIS
7.00 5.27 0.61 0.63 0.03
7.00 5.69 0.73 0.69 −0.06
8.00 6.82 0.75 0.76 0.02
8.00 6.58 0.68 0.78 0.12
9.00 6.89 0.76 0.81 0.06
4.00 3.94 0.40 0.70 0.43
7.00 6.75 0.75 0.74 −0.02
3.00 – 1.00 0.63 −0.60
2.00 – 0.50 0.38 −0.33
7.00 5.94 0.71 0.77 0.08
4.00 – 0.38 0.60 −0.38
7.00 7.00 0.91 0.75 −0.21
4.00 – 1.00 0.75 −0.33
Mean
NA AR HO HE FIS PHWDeficit PHWExcess
6.50 4.96 0.53 0.61 0.20 P < 0.05 –
5.30 4.40 0.59 0.53 −0.08 – –
5.80 4.72 0.55 0.54 0.11 – –
7.70 6.31 0.64 0.65 0.01 – –
8.00 5.82 0.56 0.61 0.12 P < 0.05 –
4.70 4.35 0.46 0.57 0.26 P < 0.05 –
5.30 5.14 0.63 0.56 −0.06 – –
2.30 – 0.67 0.44 −0.49 – –
2.50 – 0.67 0.46 −0.44 – –
5.00 4.99 0.55 0.59 0.07 – –
5.00 – 0.63 0.61 −0.01 – –
5.00 5.00 0.73 0.62 −0.18 – –
2.80 – 0.67 0.52 −0.28 – –
demographic changes in segments E and J highlight the importance of having an established ex situ population that is representative of the in situ population. It also highlights how ex situ populations can protect populations and ultimately species; however, the ex situ population must be developed properly and have thorough planning, including regular monitoring to determine shifts in genetic structure and demographics. The second goal of the study was to evaluate temporal changes in population structure and diversity within the in situ San Marcos River population. The only other genetic evaluation of Z. texana within the San Marcos River was conducted by Richards et al. (2007) and although a comparison of the current study to their study is not possible for all metrics due to different sample collection methods, their raw data (allele nomenclature) when adjusted were comparable to the current study. A comparison, after adjustment, indicates that the genetic patterns of the in situ population is complex and dynamic on both temporal and spatial scales and indicates that tillers may move between segments, establishing new stands at a higher frequency as compared to Richards et al. (2007). That is, the number of identical multi-locus genotypes (clones) found had a wider distribution in the current study, occurred in multiple segments and grouped into a higher number of clusters (28 vs. 17). This suggests that movement and establishment of new stands within
the system is occurring more frequently; however, results may be biased due to varied sampling techniques. In terms of genetic diversity, the two studies were comparable in overall heterozygosity and total number of alleles per locus and similar to estimates within Zizania latifolia using microsatellites (Chen et al., 2012). The locus Zt13 did differ between the current study and Richards et al. (2007) as they reported 20 alleles at this locus, whereas the current study found 14. This is expected given that the number of samples genotyped was fewer in this study (245 vs. 471) and the number of alleles detected is correlated with sample size (Leberg, 2002). Correcting for sample size and estimating genetic diversity in terms of allelic richness (AR ), diversity within each segment has remained stable over time with estimates of AR slightly higher in 2012 compared to the combined 1998, 1999 and 2002 collections. For example, AR in segment F was 5.22 in the previous collection period and 5.82 in 2012; likewise, in segment C estimates were 4.21 and 4.72 respectively. Unlike genetic diversity estimates, the interpretation of inbreeding coefficients are more complex and linked to the varied reproductive strategies of Zizania. In general most in situ segments had negative FIS values indicating heterozygote excess between 1998 and 2002 (Richards et al., 2007) and were lower in the current study. The negative value of FIS in clonal organisms such as Z. texana is expected (Balloux et al., 2003) and is a strategy for an organism
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Fig. 3. Output of the Bayesian cluster analysis STRUCTURE from Richards et al., 2007 (right) and this study (left) from showing the probability (y-axis) of each of the sample (y-axis, individual bars) being part of the three genetic clusters (k = 3). Each cluster is represented by a different color (light grey, dark grey, and black).
to maintain high genetic diversity and is a pattern seen in other Zizania, such as Z. latifolia (Chen et al., 2012). If the clonal and pollination strategies of Z. texana (Power and Oxley, 2004) are taken into account, then the system may maintain high allelic diversity and negative FIS values through clonal reproduction and not random mating, but tillers do not seem to establish within stands (Richards et al., 2007) but may instead disperse and form new stands or become lodged within previously established stands. In contrast, segment F had positive FIS values in both studies which could be attributed to clonal reproduction of homozygous individuals. Significant excess of homozygotes as indicated by positive FIS was also found in segments A and H in the current study. These patterns could be explained by either of two hypotheses: (1) an increase in self-pollination or cross-pollination rates in certain segments or (2) a Wahlund effect; however, further research is needed to refute or confirm these hypotheses. The reproductive strategies of Z. texana are varied, with most plants being submerged most of their lifespan, which prevents flowering and pollination and only allows for clonal reproduction via tillers. At segment F it may be that when compared to other segments, flowering and pollination are more common overall due to ecological conditions (lower water levels), as the positive FIS values have been consistent temporally, indicating higher inbreeding. This is despite the
segment having more allelic diversity with the number of alleles ranging from 15 to 42 (1998–1999, Richards et al., 2007) and 48 (2012, current study). Although it is possible that increased flowering due to low water levels contributed to this pattern it is unlikely given that the plants in this reach did not show increased flowering in early 2000 when it was dewatered (J. Poole per. comm.). In addition, the backwater downstream from Cape’s Dam was enough to keep plants underwater (J. Poole per. comm.). It is more probable that the observed pattern in our study is due to a Wahlund effect. This occurs when two previously sub-divided populations (differing allele frequencies) come into contact skewing allele frequencies of all possible allele pairs (and thus HO ) indicating that there are fewer heterozygotes than would be expected (HE ) in a random mating situation. Given time and random breeding in a large population HO stabilizes and trends toward HE . There have been numerous plantings (sub-population mixing) in segments A, F and H both in the 1990’s and early 2000’s (J. Poole pers. comm.). These plantings along with a lack of random mating due to clonal reproduction may also explain the apparent excess of homozygotes as indicated by the positive FIS values. Analysis of population structures were also comparable between the two studies indicating that population structure has remained relatively stable over time. Based on the results of this
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Table 3 Zizania texana pairwise FST values (below diagonal) and associated P values (above diagonal) for each of the seven in situ and two ex situ (BR and FR) populations using the six microsatellite loci used by Richards et al., (2007). Significant P values, after Bonferroni correction for multiple comparisons, are in bold (P = 0.002).
A B BR C D F FR H K
A
B
BR
C
D
F
FR
H
K
– 0.079 0.033 0.101 0.071 0.086 0.100 0.087 0.054
0.000 – 0.040 0.019 0.031 0.061 0.039 0.026 0.069
0.016 0.003 – 0.042 0.038 0.034 0.046 0.033 0.005
0.000 0.018 0.001 – 0.036 0.048 0.027 0.043 0.038
0.000 0.001 0.002 0.000 – 0.015 0.018 0.034 0.037
0.000 0.000 0.004 0.000 0.043 – 0.057 0.037 0.024
0.000 0.004 0.008 0.037 0.072 0.001 – 0.050 0.059
0.000 0.026 0.019 0.003 0.007 0.003 0.014 – 0.037
0.002 0.000 0.299 0.004 0.003 0.023 0.005 0.021 –
study and Richards et al. (2007), it is important to continue genetically monitoring the in situ population of Z. texana in the San Marcos River to better understand temporal and spatial genetic patterns. Additional genetic analysis should lend support or refute the need to maintain ex situ samples from all segments or keep specimens that represent distinct genetic clusters. Plants should continue to be added to the ex situ population at the SMARC to match the relative proportions of genetic clusters and unique alleles found within the river. The formulation of a genetics management plan is warranted that includes methods and schedules for in situ and ex situ genetics monitoring, augmentation strategies, propagation methods, and a reintroduction protocol. Acknowledgements We thank Christopher Richards of the United States Department of Agriculture for providing us with data from the 2007 study. Doug Phillips, Daniel Huston, and Shannon Devine assisted with the collection of Texas wild rice samples from the San Marcos River. Meredith Bartron, Robert Doyle, Jackie Poole, and Christian Smith all provided helpful comments on earlier drafts. The conclusions in this article are those of the authors and do not necessarily represent the views of the U.S. Fish and Wildlife Service, and reference to trade names does not imply endorsement by the U.S. Government. This project was funded by the U.S. Fish and Wildlife Service. References Balloux, F., Lehmann, L., de Meeûs, T., 2003. The population genetics of clonal and partially clonal diploids. Genetics 164, 1635–1644. Chen, Y.Y., Chu, H.J., Liu, H., Liu, Y.L., 2012. Abundant genetic diversity of the wild rice Zizania latifolia in central China revealed by microsatellites. Ann. Appl. Biol. 161, 192–201. Christe, C., Kozlowski, G., Frey, D., Fazan, L., Bétrisey, S., Pirintsos, S., Gratzfeld, J., Naciri, Y., 2014. Do living ex situ collections capture the genetic variation of wild populations? A molecular analysis of two relict tree species, Zelkova abelica and Zelkova carpinifolia. Biodivers. Conserv. 23, 2945–2959. Christie, M.R., Marine, M.L., French, R.A., Waples, R.S., Blouin, M.S., 2012. Effective size of a wild salmonid population is greatly reduced by hatchery supplementation. Heredity 109, 254–260. Cibrian-Jaramillo, A., Hird, A., Oleas, N., Ma, H., Meerow, A.W., Francisco-Ortega, J., Griffith, M.P., 2013. What is the conservation value of a plant in a botanic garden? Using indicators to improve management of ex situ collections. Bot. Rev. 79, 559–577. Cohen, J.I., Williams, J.T., Plucknett, D.L., Shands, H., 1991. Ex situ conservation of plant genetic resources: global development and environmental concerns. Science 253, 866–872. Dobson, A., Lodge, D., Alder, J., Cummings, G.S., Keymer, J., McGlade, J., Mooney, H., Rusak, J.A., Sala, O., Wolters, V., Wall, D., Winfree, R., Xenopoulos, M.X., 2006. Habitat loss, trophic collapse, and the decline of ecosystem services. Ecology 87, 1915–1924.
Earl, D.A., vonHoldt, B.M., 2011. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the EVANNO method. Conserv. Genet. Resour. 4, 359–361. Evanno, G., Regnaut, S., Goudet, J., 2005. Selecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14, 2611–2620. Excoffier, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479–491. Falk, D.A., Holsinger, K.E., 1991. Genetics and Conservation of Rare Plants. Oxford University Press, UK. Fisch, K.M., Kozfkay, C.C., Ivy, J.A., Ryder, O.A., Waples, R.S., 2015. Fish hatchery genetic management techniques: integrating theory with implementation. N. Am. J. Aquacult. 77, 343–357. Goudet, J., 1995. FSTAT (Version 1: 2): a computer program to calculate F-statistics. J. Hered. 86, 485–486. Griffith, P., Husby, C., Lewis, C.E., Francisco-Ortega, J., 2011. Palm conservation at a botanical garden: a case for the Keys thatch palm. Palms 55, 93–101. Guerrant Jr., E.O., Havens, K., Mauder, M. (Eds.), 2004. Ex Situ Plant Conservation. Supporting Species Survival in the Wild. Island Press, Washington, D.C. Havens, K., Kramer, A.T., Guerrant Jr., E.O., 2014. Getting plant conservation right (or not): the case of the United States. Int. J. Plant Sci. 175, 3–10. Heywood, V.H., Iriondo, J.M., 2003. Plant conservation: old problems, new perspectives. Biol. Conserv. 113, 321–335. Kramer, A.T., Havens, K., 2009. Plant conservation genetics in a changing world. Trends Plant Sci. 14, 599–607. Leberg, P.L., 2002. Estimating allelic richness: effects of sample size and bottlenecks. Mol. Ecol. 11, 2445–2449. Maunder, M., Havens, K., Guerrant Jr., E.O., Falk, D.A., 2004. Ex situ methods: a vital but underused set of conservation resources. In: Guerrant Jr., E.O., Havens, K., Mauder, M. (Eds.), Ex Situ Plant Conservation. Supporting species survival in the wild. Island Press, Washington, D.C, pp. 3–20. Meirmans, P.G., van Tienderen, P.H., 2004. GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organisms. Mol. Ecol. Notes 4, 792–794. Michalakis, Y., Excoffier, L., 1996. A generic estimation of population subdivision using distances between alleles with special reference for microsatellite loci. Genetics 142, 1061–1064. Peakall, R., Smouse, P.E., 2006. GENALEX 6: genetic analysis in excel: population genetic software for teaching and research. Mol. Ecol. Notes 6, 288–295. Petit, R.J., El Mousadik, A., Pons, O., 1998. Identifying populations for conservation on the basis of genetic markers. Conserv. Biol. 12, 844–855. Poole, J.M., 2002. Historical distribution of Texas wild-rice (Zizania texana) from 1989 to 2001. Section 6 Final Report. Albuquerque, N.M.: U.S. Fish and Wildlife Service. Poole, J.M., Bowles, D.E., 1999. Habitat characterizations of Texas wild-rice (Zizania texana), an endangered aquatic macrophyte from the San Marcos River, Texas, USA. Aquat. Conserv. 9, 291–302. Poole, J.M., Carr, W.R., Price, D.M., Singhurst, J.R., 2007. Rare Plants of Texas. Texas A&M University Press College Station. Power, P., Oxley F.M., 2004. Assessment of factors influencing Texas wild-rice (Zizania texana) sexual and asexual reproduction. Final Report for Edwards Aquifer Authority, San Antonio, TX. Pritchard, J.K., Stephens, M., Donnelly, P., 2000. Inference of population structure using multilocus genotype data. Genetics 155, 945–959. Raymond, M., Rousset, F., 1995. GENEPOP (version 1: 2): population genetics software for exact tests and ecumenicism. J. Hered. 86, 248–249. Richards, C.M., Antolin, M.F., Reille, A., Pool, J., Walters, C., 2007. Capturing genetic diversity of wild populations for ex situ conservation: Texas wild rice (Zizania texana) as a model. Genet. Resour. Crop Evol. 54, 837–848. Rousset, F., 2008. Genepop’007: a complete reimplementation of the Genepop software for Windows and Linux. Mol. Ecol. Resour. 8, 103–106. U.S. Fish and Wildlife Service (USFWS), 1978, Determination that 11 plant taxa are endangered species and 2 plant taxa are threatened species. Federal Register 43, 81: 17910–17916. U.S. Fish and Wildlife Service (USFWS), 1995, San Marcos/Comal (Revised) Recovery Plan. Albuquerque, New Mexico. pp x + 93 with 28 pages of appendices. van Oosterhout, C., Hutchinson, W.F., Wills, D.P.M., Shipley, P., 2004. Micro-checker: software for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Notes 4, 535–538. Weir, B.S., 1996. Genetic Data Analysis II. Sinauer Associates Sunderland, Mass. Wilcove, D.S., Rothstein, D., Dubow, J., Phillips, A., Losos, E., 1998. Quantifying threats to imperiled species in the United States. Bioscience 45, 607–615.
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Genetic diversity assessment of in situ and ex situ Texas wild rice (Zizania texana) populations, an endangered plant Wade D. Wilson a,∗ , Jeffrey T. Hutchinson b , Kenneth G. Ostrand b a b
U.S Fish & Wildlife Service, Southwestern Native Aquatic Resources and Recovery Center, PO Box 219, Dexter, NM 88230-0219, USA U.S Fish & Wildlife Service, San Marcos Aquatic Resources Center, 500 East McCarty Lane, San Marcos, Texas 78666, USA
a r t i c l e
i n f o
Article history: Received 24 June 2015 Received in revised form 2 December 2015 Accepted 21 December 2015 Available online xxx Keywords: Genetics Endangered species Refugia San Marcos River Texas wild rice
a b s t r a c t Texas wild rice (Zizania texana) is an endangered, aquatic perennial plant endemic to the upper section of the San Marcos River, Texas. Ex situ populations of Z. texana are maintained by the U.S. Fish and Wildlife Service in the event of a catastrophe event. We analyzed the genetics of in situ and ex situ populations of Z. texana to address the following questions: (1) are in situ populations adequately represented in ex situ population? (2) Is there genetic diversity among the current in situ population? (3) Has the current in situ genetic diversity increased or decreased from historical estimates in 2007. Results indicated that the overall ex situ populations were lower in genetic (allelic) diversity compared to the in situ population, with some in situ populations not present in the refugia. Overall, heterozygosity was moderate and ranged from HO = 0.530 to HO = 0.635 in the wild and HO = 0.549 to HO = 0.727 in the ex situ population. Inbreeding coefficients (FIS ) were near zero or negative indicating that inbreeding is not common within the current populations (in situ and ex situ) suggesting that some populations have an excess of heterozygotes (negative FIS ). Analysis of current in situ population structure indicated there are three unique genetic clusters in the San Marcos River. Comparison of the in situ population with historical analysis indicates the genetic diversity of the wild population is dynamic both temporally and spatially. The results indicate that Z. texana exhibits a plastic reproductive system utilizing both asexual (vegetative) and sexual (flowering and seed production) reproduction. Published by Elsevier B.V.
1. Introduction The present rate of habitat loss and landscape alteration has led to the decline and extinction of many species (Dobson et al., 2006; Wilcove et al., 1998). Habitat restoration, single species reintroductions, and species augmentations will become increasingly important in long-term protection of rare plants (Kramer and Havens, 2009). Worldwide, funding and research for rare plants remains limited while the number of plants threatened continues to increase (Havens et al., 2014). Ex situ conservation programs compliment in situ conservation programs with short to long-term storage of genetically representative samples and allow for long term research on species in which little is known of their life history (Heywood and Iriondo, 2003). Historically, most ex situ conservation genetic programs have focused on food crops. Moreover, most of these programs have concentrated on seed collection and less on living collections. For plants with recalcitrant seeds such as Texas
∗ Corresponding author. Fax: +1 575 734 6130. E-mail address: Wade [email protected] (W.D. Wilson).
wild rice (Zizania texana Hitchc.), living plant collections are the only option for long-term ex situ collections. Plant species maintained ex situ need to represent the population’s genetic diversity, follow protocols to ensure genetic diversity during reproduction, and ensure that two or more collective samples are maintained at different locations (Cohen et al., 1991). The main goal of managing ex situ living plant collections is to ensure that the highest level of genetic diversity is maintained at an economically feasible level (Cibrian-Jaramillo et al., 2013). Often this can best be accomplished through a collaborative effort between the organization managing the ex situ plants and a university or research facility with genetic analysis capabilities (Griffith et al., 2011). Maintaining ex situ populations of rare plants that represent the genetic diversity of in situ populations results in a safety net to prevent the extinction of rare species (Guerrant et al., 2004). Balancing genetic diversity and operational cost of living populations can be challenging, but can best be obtained with genetic analysis. Resources for maintaining living plants are finite and genetic analysis can help in allocation of funds for maintaining ex situ plant collections that represent the genetic diversity of in situ populations.
http://dx.doi.org/10.1016/j.aquabot.2015.12.005 0304-3770/Published by Elsevier B.V.
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Zizania texana is a federal endangered plant in the USA and is endemic to the upper portion of the San Marcos River, Hays County, Texas (Poole et al., 2007). Zizania texana prefers swift moving cool spring-fed runs and is most commonly found at water depths less than 1 m growing in coarse sandy soils (Poole and Bowles, 1999). It is a perennial C3 grass that occurs in the upper 5 km of the headwaters of the San Marcos River with 97% of the Z. texana population occurring within the upper 2.2 km. Threats to Z. texana include aquifer depletion, habitat destruction and alteration, invasive species, droughts, floods, unintended recreational impacts and its extremely small and limited range (Poole, 2002). Fragmentation of stands, large gaps between stands, and damaged stems also result in reduced pollination and seed production by Z. texana (Power and Oxley, 2004). Because of its limited distribution, Z. texana is listed (USFWS, 1978) as a G1 critically imperiled species due to its extreme rarity in five or fewer populations. The San Marcos Aquatic Resources Center (SMARC; 29◦ 50 23.72 N, 97◦ 58 33.78 W) is responsible for maintaining Z. texana in refugia as an ex situ population in the event a catastrophe or stochastic event destroys the San Marcos River in situ population (USFWS, 1995). An additional ex situ population is maintained at the U.S. Fish and Wildlife Service (USFWS) Uvalde National Fish Hatchery (29◦ 11 15.37 N, 99◦ 49 59.27 W). The ex situ population of Z. texana at the SMARC is comprised of specimens collected from 10 of the 14 Texas Parks and Wildlife Department (TPWD) monitoring segments within the San Marcos River (Table 1, Fig. 1). Specimens from these segments were collected using a stratified sampling scheme along a longitudinal gradient (see Richards et al., 2007). The in situ sampling design was implemented to avoid maintaining large redundant collections at the SMARC that may divert resources from other conservation priorities. Starting in 2005, collections representing allelic richness and diversity of Z. texana populations from the San Marcos River have been maintained at the SMARC (Richards et al., 2007); nevertheless, the spatial distribution and cover of various Z. texana stands have changed since their sample collection in 1998, 1999 and 2002. Many stands of Z. texana have increased in size, some new stands have become established, and some smaller stands have disappeared (Fig. 2). These demographic changes in Z. texana are due to the combined establishment of floating tillers, seeds and human mediated translocations. In order to ensure that conservation goals are met in accordance with the regulatory and guiding conservation documents (e.g. USFWS, 1995) the objectives of this project were: (1) to conduct genetic analyses to ensure that ex situ population held at the SMARC represent the extant in situ (wild) population, (2) determine if there is genetic diversity among the current in situ population, (3) and evaluate if historical estimates of Z. texana in situ population genetic diversity have changed compared to Richards et al. (2007).
2. Materials and methods 2.1. Tissues A total of 245 Z. texana samples were collected from both the San Marcos River population (N = 184) and the ex situ population at the SMARC (N = 61) in September and October 2012. In situ sample nomenclature follow TPWD segment nomenclature and are designated as A, B, C, etc. with ex situ refugia (R) samples designated as AR, BR, CR, etc. Sample collection of in situ individuals were conducted by swimmers who clipped fresh 10–12 cm leaf samples from most stands <2 m2 and all stands >2 m2 from eight of the eleven segments (Fig. 1). Samples of Z. texana were not collected from the other segments because either Z. texana did not occur in
these locations (I and M) or spatial coverage was <0.1 m2 (E, G, J, and L). The ex situ (i.e. refugia) samples were collected from the SMARC and only represented five (A, B, C, F and K) of the eleven segments. Collection methods differed from Richards et al. (2007) in that the exact location of each sample was not mapped either within or between stands. Leaf tissue samples were placed in plastic zip bags, packed on ice, and shipped to the Southwestern Native Aquatic Resources and Recovery Center (SNARRC). Upon arrival at the SNARRC, samples were stored at −20 ◦ C until Genomic DNA extraction. A sub-sample of each individual was freeze-dried and archived at −80 ◦ C for future reference. 2.2. Extraction, PCR, and genotyping Genomic DNA was extracted using Qiagen DNeasy® 96 Blood and Tissue Kits (Qiagen, Valencia, CA) using a modified method. Approximately 0.025 g of each leaf tissue sample was finely sliced in preparation for DNA extraction with the amount of proteinase K and Buffer ATL doubled from standard protocol amounts for tissue lysis. Leaf tissue was incubated overnight at 56 ◦ C after which each sample was disrupted with a small metal spatula at three points during lysis: once immediately following the addition of proteinase K and buffer ALT, once following the overnight incubation, and a third time two hours after the second. Undigested leaf material was removed with forceps following the third tissue disruption and DNA extraction was completed following the DNeasy spin-column protocols for animal tissues. Multiplex PCR amplifications (10 l) consisted of 3.0 l Qiagen Multiplex Mastermix® ; 0.5 l each, forward and reverse primer (10 M); 3.6 l ddH20; 0.4 l 100x (10 mg/ml) purified Bovine Serum Albumin (BSA; New England Biolabs); 2 l template DNA. Forward primers were labeled with one of four fluorescent dyes (6FAM, PET, NED, VIC). Touchdown cycling (temperature decreased by 0.2 ◦ C/cycle) using a GeneAmp® System 9700 (Applied Biosystems) consisted of one cycle at 95 ◦ C for 15 min (to activate the HotStar Taq DNA polymerase), followed by 33 cycles of 94 ◦ C for 45 s, an initial annealing temperature of 56 ◦ C for 45 s, and an extension temperature of 72 ◦ C for 60 s with a final extension of 30 min at 70 ◦ C. BSA was needed as PCR inhibitors initially prevented consistent amplification, initially. All PCR reagents were purchased from Applied Biosystems (Foster City, CA). The six microsatellite used were identical to Richards et al. (2007). PCR products were processed on an ABI 3130xl genetic analyzer using GeneScanTM 500 LIZ® size standard. Composite genotypes for individual samples were compiled with GeneMapperTM 4.0 software (Applied Biosystems). 2.3. Quality control/quality assurance To ensure accuracy in genotyping, samples were run through a Quality Control/Quality Assurance (QA/QC) process consisted of two actions: (1) rescoring of all runs by a second researcher, and (2) independently re-extracting, amplifying, and scoring 10% of the samples. Genotypes produced by the different researchers were compared and all inconsistencies were resolved, thus no samples were removed prior to analyses. 2.4. Data analysis A total of 245 individuals of Z. texana were genotyped using six microsatellite loci used by Richards et al. (2007). Departures from the Hardy–Weinberg (HW) equilibrium and global tests of linkage equilibrium (LD) among all pairs of loci and populations were tested using GENEPOP v4.0 (Raymond and Rousset, 1995; Rousset, 2008). The test for HW equilibrium used the heterozygote deficiency method (Raymond and Rousset, 1995), a global test that
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Table 1 Current inventory and future needs of the ex situ population of Zizania texana at the San Marcos Aquatic Resources Center. The recommended inventory is based on several factors including available space (capacity of 430), availability in the wild as determined by percent cover, and the number of alleles represented in each segment of the in situ population divided by the total number of alleles, based on current the 2012 analyses. Segment
Current inventory
Recommended inventory
Additional need
Wild% cover (NA /NAT )a
A B C D E F G H I X J K L M Unknown Total
6 45 26 3 8 19 1 3 0 0 13 6 0 0 27 157
50 205 68 10 8 60 5 5 – 0 13 6 0 0 0 430
44 160 42 7 0 41 4 2 – 0 0 0 0 0 – 300
9.2 (39/65) 62.7 (32/65) 16.7 (35/65) 0.4 (46/65) <0.1 (−) 8.0 (48/65) 0.4 (−) 1.2 (−) 0.0 (−) 0 <0.1 (−) 1.2 (32/65) 0.1 (−) 0
a
–
2
% Cover = % of the approximately 4996 m in the in situ population, NA = number of alleles found in each segment, NAT = total number of alleles (65) found in the in situ (current study), – = not genotyped in current study.
Fig. 1. Map showing the 11 Zizania texana monitoring segments (A–K) within the San Marcos River. Segments L, M (below K) are not shown because genetic samples were not analyzed from these segments.
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Fig. 2. Historical Zizania texana cover in selected segments of Texas Parks and Wildlife Department’s monitoring program. Segments A, B, C, and F are represented in top figure, and segments E, J, and K are represented in bottom figure.
examines either the population(s) or locus but not both simultaneously. The test of LD examines the relationship between genotypes at each pair of loci (i.e., composite LD; Weir, 1996). Deviations from HW equilibrium due to stuttering, null alleles, and large allele dropout were tested using MICROCHECKER 2.2.3 (van Oosterhout et al., 2004). Heterozygosity (HE ; gene diversity), observed heterozygosity (HO ) on a per locus basis, the number of alleles per locus (NA ), average inbreeding coefficients (FIS ), and private alleles were estimated using GenAlEx v6 (Peakall and Smouse, 2006). GenAlEx was also used to calculate the Probability of Identity (PI) that provides the average probability estimate that two unrelated individuals, drawn from the same randomly mating population, will have the same multilocus genotype. Allele frequencies and descriptive statistics, including allelic richness (AR ) were estimated using FSTAT v2.9.3.1 (Goudet, 1995). Allelic richness was calculated using the methods described by Petit et al. (1998) and uses rarefaction and repeated random subsampling to provide unbiased estimates of AR (Leberg, 2002). The number of genetic clusters (K) was calculated using the Bayesian clustering method of STRUCTURE v2.3.2 (Pritchard et al., 2000). The admixture model that assumes gene flow among
populations and allows for correlated allele frequencies across populations was applied. This model assigns a proportion of each individual’s genome to each of the genetic clusters pursuing solutions that maximize HW equilibrium and LD within clusters. Ten iterations were performed for each K with the true K assumed to be between 1 and 8. All runs had a burn-in of 100,000 preliminary iterations followed by 100,000 iterations of data collection. The method of Evanno et al. (2005) that uses the second order rate of change between K and K + 1 cluster (K) was used to estimate the number of genetic clusters, as implemented in STRUCTURE HARVESTER (Earl and vonHoldt, 2011). The K with the largest K value is assumed to be the best estimator of K. Pairwise FST was calculated using GENODIVE (Meirmans and van Tienderen, 2004). This distance is the FST statistic resulting from an Analysis of Molecular Variance (AMOVA) performed between each pair of populations (Excoffier et al., 1992; Michalakis and Excoffier, 1996). Identical genotypes that occurred in different segments were included in the analysis, while identical genotypes occurring within segments were removed from the final analysis to eliminate repetitive genotypes within a segment. The final dataset consisted of 203 individuals.
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3. Results The probability of identity (PI) was low and ranged from 2.25 × 10−6 to 7.42 × 10−3 (mean 1.06 × 10−3 ). This study found 40 of the 245 Z. texana samples grouped into 28 clusters with each cluster having multiple (mode = 2, mean = 3.2, range 2–9) identical genotypes (data not shown). Identical genotypes were found both within a segment (15 of 28 clusters) and among segments (13 of 28 clusters). For example, genetic cluster one had two samples with identical genotypes, one from segment F and the other from the refugia population that was originally collected from segment B. Genetic cluster seven had nine identical genotypes from five different segments; CR and FR (refugia) and C, H and K (wild). Observed heterozygosity was moderate and ranged from HO = 0.530 (segment A) to HO = 0.635 (segment D) for in situ and HO = 0.549 (BR) to HO = 0.727 (FR) for ex situ population (Table 2). Inbreeding coefficients (FIS ) were near zero or negative indicating that inbreeding is not common within the current populations (in situ and ex situ) and suggests that some populations have an excess of heterozygotes (negative FIS ). Only three segments (A, F, and H) were not in HW equilibrium and showed signs of homozygote excess (positive FIS ). Using MICROCHECKER, the presence of null alleles was suggested at segments A, F, and H at three loci (Zt13, Zt21, and Zt22); however, null alleles were not indicated in other segments or ex situ samples for these loci. Therefore, the data suggest that these three segments had higher inbreeding (heterozygote deficiencies) in the current sampling period and that the higher positive FIS values are not the result of the markers themselves. Excluding segments with <10 individuals, the mean number of alleles per locus for the in situ samples ranged from 8.0 (segment D) to 4.7 (segment H). Two of the segments represented in the in situ population had similar number of alleles per locus when compared to their ex situ counterparts, the exception being segment F, that had lower diversity in the in situ population (NA = 5.0 vs. NA = 8.0). Private alleles are those that are unique to a certain population or collection of samples. Within the San Marcos River, three segments (B, D, and F) contained private alleles at low frequency, usually occurring in one individual. Segment B had three private alleles (Zt23-164, Zt13-224, and Zt22-204), D had one (Zt13-218) occurring in three individuals, and F had five (Zt13-238, Zt13-214, Zt23-176, Zt18-90, and Zt1-258) occurring in seven individuals (Table 1). An analysis of population structure of the current dataset using the Bayesian clustering method of STRUCTURE and the K method of Evanno et al. (2005) as implemented in STRUCTURE HARVESTER (Earl and vonHoldt, 2011) indicated there are three genetic clusters with all three clusters occurring in each segment within the current in situ population (Fig. 3). Cluster 1 was most prevalent in segments A (76.8%), K (69.3%), and H (49.6%), cluster 2 in segments B (81.6%), C (63.7%), and D (46.8%), and cluster 3 in D (37.2%) and F (34.2%). The high prevalence of cluster 1 in segment A is most likely due to the addition of 10,000 seeds in the summer of 1996 with the source of the seeds being from a few plants obtained from segment F after which the number of stands increased from 8 to 28 (J. Poole, pers. comm.); currently segment F is comprised of all three genetic clusters in equal amounts (37.1%, 28.7% and 34.2%, respectively). AMOVA indicated that there was significant genetic differentiation: (1) among individuals within segments (FST = 0.047, P = 0.003) and (2) among segments (FST = 0.049, P = 0.001); however, 90.6% of the variation was within individuals not among segments. Pairwise FST values were significantly different between all in situ segments (Table 3) and consistent with the STRUCTURE results. In a comparison of the in situ samples with their ex situ counterparts, results differed (only in situ populations with N > 10 were used in the pairwise FST analysis). Segment B did not differ from the ex situ population (BR); however, F was significantly different from FR and is
5
due to the in situ population having more allelic diversity than the ex situ population. This was apparent and evidenced at locus Zt21.
4. Discussion Captive conservation populations (ex situ) found at zoos, botanic gardens and hatcheries have been a necessary tool to protecting Threatened and Endangered species and preventing extinction (Fisch et al., 2015; Guerrant et al., 2004; Maunder et al., 2004). These conservation populations can be maintained as refuge populations (propagation is not conducted) or as broodstock populations used for production of offspring or seeds that are subsequently used for augmentation or restoration conservation activities. Establishing and maintaining ex situ conservation populations that are genetically unrepresentative of the source population can occur and go undetected unless genetic evaluations are periodically conducted (Christe et al., 2014). In addition, using unrepresentative broodstock populations or few individuals for propagation can alter the genetic diversity and effective size of the recipient wild population (Christie et al., 2012; Fisch et al., 2015). Although many of examples are from animal species such as fishes, which are well studied in terms of genetic consequences of small populations, the concepts apply across species (see Falk and Holsinger, 1991). In a recent genetic assessment of in situ and ex situ in two tree species (Zelkova abelica and Zelkova carpinifolia), Christe et al. (2014) found that only one of the species (Z. carpinifolia) was representative of the in situ population and although Z. abelica was represented in eight botanic gardens, the collections had originated from a single region; however, previously unidentified chloroplast DNA haplotypes were found, underscoring the utility of ex situ populations in preserving genetic diversity. In addition, several individuals identified as Z. carpinifolia in ex situ populations were found to be misidentified to both the wrong species and genus. Therefore, genetic evaluations of existing ex situ populations are vital for effective planning and implementation of recovery actions of species of conservation concern. To this end, the first goal of this study was to evaluate the Z. texana ex situ population at the SMARC and determine if it was representative of the in situ San Marcos River population. Of the five segments represented in the ex situ population (AR, BR, CR, FR, and KR), only two (BR and FR) had sample sizes adequate for in situ/ex situ comparisons. In cases where comparisons could be made the results suggest that overall the ex situ population (BR and FR) is lower in genetic (allelic) diversity compared to the in situ population suggesting that the ex situ population should be augmented. In the other ex situ segments (A, C, K), the number of individuals was either too low to compare genetically or be used for reintroductions because individuals may be identical and limited in diversity. For example, the ex situ population currently has six plants from segment A and two of the three plants genotyped were genetically identical. Given the ability of Z. texana to reproduce clonally, a genetic assessment of each individual added to the ex situ population is imperative. Segment A, differs in allelic frequency compared to the other segments, but this may be the result of human intervention and the addition of 10,000 seeds in 1996 from a few plants taken from segment F (J. Poole, pers. comm.), either way the ex situ segment A population is limited in diversity compared to the in situ segment. The ex situ population may also harbor unique and important individuals from two segments (E and J) that have few Z. texana compared to historical cover. Annual population monitoring has shown that the cover of Z. texana decreased substantially after 1998 with 2012 cover estimates being 2 m2 (segment E) and 1 m2 (J); both of these segments had cover of over 100 m2 in 1991. Flooding, droughts, and recreation activities can result in the decline of Z. texana in the San Marcos River (Poole et al., 2007). These stochastic and
Please cite this article in press as: Wilson, W.D., et al., Genetic diversity assessment of in situ and ex situ Texas wild rice (Zizania texana) populations, an endangered plant. Aquat. Bot. (2015), http://dx.doi.org/10.1016/j.aquabot.2015.12.005
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Table 2 Summary statistics of the 6 microsatellite loci used to screen Zizania texana in situ (wild-San Marcos River) and ex situ (refugia-San Marcos Aquatic Resources Center) samples collected in 2012. Refugia samples are designated with an R after site (e.g. BR). Locus
Statistic
In situ (wild) samples
Ex situ (refugia) samples
A n = 28
B n = 22
C n = 26
D n = 22
F n = 29
H n = 15
K n = 12
X n=2
AR n=2
BR n = 17
CR n=8
FR n = 11
KR n=2
Zt1
NA AR HO HE FIS
2.00 2.00 0.36 0.49 0.27
2.00 2.00 0.23 0.27 0.15
2.00 1.85 0.04 0.11 0.65
2.00 1.99 0.27 0.24 −0.16
3.00 2.33 0.24 0.22 −0.12
2.00 2.00 0.13 0.23 0.42
2.00 2.00 0.33 0.28 −0.20
1.00 – 1.0 0.63 −0.60
2.00 – 0.50 0.38 −0.33
2.00 2.00 0.35 0.46 0.23
2.00 – 0.25 0.22 −0.14
2.00 2.00 0.27 0.24 −0.16
2.00 – 0.50 0.38 −0.33
Zt13
NA AR HO HE FIS
6.00 5.68 0.57 0.78 0.27
6.00 4.83 0.82 0.66 −0.23
6.00 4.72 0.54 0.57 0.04
8.00 6.63 0.76 0.79 0.03
9.00 6.32 0.62 0.76 0.19
6.00 5.65 0.60 0.72 0.17
5.00 4.83 0.75 0.60 −0.25
2.00 – 0.50 0.38 −0.33
3.00 – 1.00 0.63 −0.67
5.00 4.57 0.47 0.57 0.18
6.00 – 0.75 0.74 −0.01
3.00 3.00 0.73 0.60 −0.21
4.00 – 1.00 0.75 −0.33
Zt18
NA AR HO HE FIS
10.00 6.84 1.00 0.81 −0.24
7.00 6.02 0.96 0.74 −0.30
7.00 5.61 0.96 0.76 −0.27
9.00 7.74 0.91 0.83 −0.09
10.00 7.25 0.97 0.81 −0.19
7.00 6.39 0.87 0.78 −0.11
6.00 5.83 0.92 0.74 −0.24
3.00 – 1.00 0.63 −0.60
4.00 – 1.00 0.75 −0.33
9.00 8.13 0.88 0.83 −0.07
7.00 – 1.00 0.83 −0.21
8.00 8.00 1.00 0.81 −0.24
3.00 – 1.00 0.63 −0.60
Zt21
NA AR HO HE FIS
10.00 7.41 0.57 0.80 0.29
7.00 5.89 0.73 0.72 −0.01
8.00 6.30 0.84 0.79 −0.06
14.00 11.22 0.86 0.90 0.04
13.00 9.58 0.69 0.88 0.22
5.00 4.45 0.60 0.61 0.02
9.00 8.58 0.92 0.82 −0.12
3.00 – 1.00 0.63 −0.60
3.00 – 1.00 0.63 −0.60
8.00 7.65 0.82 0.83 0.01
8.00 – 1.00 0.79 −0.27
6.00 9.58 0.91 0.74 −0.23
3.00 – 0.50 0.63 0.20
Zt22
NA AR HO HE FIS
4.00 2.57 0.07 0.17 0.57
3.00 2.00 0.09 0.09 −0.04
4.00 3.02 0.16 0.22 0.27
5.00 3.73 0.32 0.35 0.09
4.00 2.53 0.07 0.16 0.57
4.00 3.66 0.13 0.34 0.61
3.00 2.83 0.08 0.16 0.47
2.00 – 0.50 0.38 −0.33
1.00 – 0.00 0.00 N/A
2.00 1.65 0.06 0.06 −0.03
3.00 – 0.38 0.48 0.21
4.00 4.00 0.55 0.58 0.06
1.00 – 0.00 0.00 N/A
Zt23
NA AR HO HE FIS
7.00 5.27 0.61 0.63 0.03
7.00 5.69 0.73 0.69 −0.06
8.00 6.82 0.75 0.76 0.02
8.00 6.58 0.68 0.78 0.12
9.00 6.89 0.76 0.81 0.06
4.00 3.94 0.40 0.70 0.43
7.00 6.75 0.75 0.74 −0.02
3.00 – 1.00 0.63 −0.60
2.00 – 0.50 0.38 −0.33
7.00 5.94 0.71 0.77 0.08
4.00 – 0.38 0.60 −0.38
7.00 7.00 0.91 0.75 −0.21
4.00 – 1.00 0.75 −0.33
Mean
NA AR HO HE FIS PHWDeficit PHWExcess
6.50 4.96 0.53 0.61 0.20 P < 0.05 –
5.30 4.40 0.59 0.53 −0.08 – –
5.80 4.72 0.55 0.54 0.11 – –
7.70 6.31 0.64 0.65 0.01 – –
8.00 5.82 0.56 0.61 0.12 P < 0.05 –
4.70 4.35 0.46 0.57 0.26 P < 0.05 –
5.30 5.14 0.63 0.56 −0.06 – –
2.30 – 0.67 0.44 −0.49 – –
2.50 – 0.67 0.46 −0.44 – –
5.00 4.99 0.55 0.59 0.07 – –
5.00 – 0.63 0.61 −0.01 – –
5.00 5.00 0.73 0.62 −0.18 – –
2.80 – 0.67 0.52 −0.28 – –
demographic changes in segments E and J highlight the importance of having an established ex situ population that is representative of the in situ population. It also highlights how ex situ populations can protect populations and ultimately species; however, the ex situ population must be developed properly and have thorough planning, including regular monitoring to determine shifts in genetic structure and demographics. The second goal of the study was to evaluate temporal changes in population structure and diversity within the in situ San Marcos River population. The only other genetic evaluation of Z. texana within the San Marcos River was conducted by Richards et al. (2007) and although a comparison of the current study to their study is not possible for all metrics due to different sample collection methods, their raw data (allele nomenclature) when adjusted were comparable to the current study. A comparison, after adjustment, indicates that the genetic patterns of the in situ population is complex and dynamic on both temporal and spatial scales and indicates that tillers may move between segments, establishing new stands at a higher frequency as compared to Richards et al. (2007). That is, the number of identical multi-locus genotypes (clones) found had a wider distribution in the current study, occurred in multiple segments and grouped into a higher number of clusters (28 vs. 17). This suggests that movement and establishment of new stands within
the system is occurring more frequently; however, results may be biased due to varied sampling techniques. In terms of genetic diversity, the two studies were comparable in overall heterozygosity and total number of alleles per locus and similar to estimates within Zizania latifolia using microsatellites (Chen et al., 2012). The locus Zt13 did differ between the current study and Richards et al. (2007) as they reported 20 alleles at this locus, whereas the current study found 14. This is expected given that the number of samples genotyped was fewer in this study (245 vs. 471) and the number of alleles detected is correlated with sample size (Leberg, 2002). Correcting for sample size and estimating genetic diversity in terms of allelic richness (AR ), diversity within each segment has remained stable over time with estimates of AR slightly higher in 2012 compared to the combined 1998, 1999 and 2002 collections. For example, AR in segment F was 5.22 in the previous collection period and 5.82 in 2012; likewise, in segment C estimates were 4.21 and 4.72 respectively. Unlike genetic diversity estimates, the interpretation of inbreeding coefficients are more complex and linked to the varied reproductive strategies of Zizania. In general most in situ segments had negative FIS values indicating heterozygote excess between 1998 and 2002 (Richards et al., 2007) and were lower in the current study. The negative value of FIS in clonal organisms such as Z. texana is expected (Balloux et al., 2003) and is a strategy for an organism
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Fig. 3. Output of the Bayesian cluster analysis STRUCTURE from Richards et al., 2007 (right) and this study (left) from showing the probability (y-axis) of each of the sample (y-axis, individual bars) being part of the three genetic clusters (k = 3). Each cluster is represented by a different color (light grey, dark grey, and black).
to maintain high genetic diversity and is a pattern seen in other Zizania, such as Z. latifolia (Chen et al., 2012). If the clonal and pollination strategies of Z. texana (Power and Oxley, 2004) are taken into account, then the system may maintain high allelic diversity and negative FIS values through clonal reproduction and not random mating, but tillers do not seem to establish within stands (Richards et al., 2007) but may instead disperse and form new stands or become lodged within previously established stands. In contrast, segment F had positive FIS values in both studies which could be attributed to clonal reproduction of homozygous individuals. Significant excess of homozygotes as indicated by positive FIS was also found in segments A and H in the current study. These patterns could be explained by either of two hypotheses: (1) an increase in self-pollination or cross-pollination rates in certain segments or (2) a Wahlund effect; however, further research is needed to refute or confirm these hypotheses. The reproductive strategies of Z. texana are varied, with most plants being submerged most of their lifespan, which prevents flowering and pollination and only allows for clonal reproduction via tillers. At segment F it may be that when compared to other segments, flowering and pollination are more common overall due to ecological conditions (lower water levels), as the positive FIS values have been consistent temporally, indicating higher inbreeding. This is despite the
segment having more allelic diversity with the number of alleles ranging from 15 to 42 (1998–1999, Richards et al., 2007) and 48 (2012, current study). Although it is possible that increased flowering due to low water levels contributed to this pattern it is unlikely given that the plants in this reach did not show increased flowering in early 2000 when it was dewatered (J. Poole per. comm.). In addition, the backwater downstream from Cape’s Dam was enough to keep plants underwater (J. Poole per. comm.). It is more probable that the observed pattern in our study is due to a Wahlund effect. This occurs when two previously sub-divided populations (differing allele frequencies) come into contact skewing allele frequencies of all possible allele pairs (and thus HO ) indicating that there are fewer heterozygotes than would be expected (HE ) in a random mating situation. Given time and random breeding in a large population HO stabilizes and trends toward HE . There have been numerous plantings (sub-population mixing) in segments A, F and H both in the 1990’s and early 2000’s (J. Poole pers. comm.). These plantings along with a lack of random mating due to clonal reproduction may also explain the apparent excess of homozygotes as indicated by the positive FIS values. Analysis of population structures were also comparable between the two studies indicating that population structure has remained relatively stable over time. Based on the results of this
Please cite this article in press as: Wilson, W.D., et al., Genetic diversity assessment of in situ and ex situ Texas wild rice (Zizania texana) populations, an endangered plant. Aquat. Bot. (2015), http://dx.doi.org/10.1016/j.aquabot.2015.12.005
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Table 3 Zizania texana pairwise FST values (below diagonal) and associated P values (above diagonal) for each of the seven in situ and two ex situ (BR and FR) populations using the six microsatellite loci used by Richards et al., (2007). Significant P values, after Bonferroni correction for multiple comparisons, are in bold (P = 0.002).
A B BR C D F FR H K
A
B
BR
C
D
F
FR
H
K
– 0.079 0.033 0.101 0.071 0.086 0.100 0.087 0.054
0.000 – 0.040 0.019 0.031 0.061 0.039 0.026 0.069
0.016 0.003 – 0.042 0.038 0.034 0.046 0.033 0.005
0.000 0.018 0.001 – 0.036 0.048 0.027 0.043 0.038
0.000 0.001 0.002 0.000 – 0.015 0.018 0.034 0.037
0.000 0.000 0.004 0.000 0.043 – 0.057 0.037 0.024
0.000 0.004 0.008 0.037 0.072 0.001 – 0.050 0.059
0.000 0.026 0.019 0.003 0.007 0.003 0.014 – 0.037
0.002 0.000 0.299 0.004 0.003 0.023 0.005 0.021 –
study and Richards et al. (2007), it is important to continue genetically monitoring the in situ population of Z. texana in the San Marcos River to better understand temporal and spatial genetic patterns. Additional genetic analysis should lend support or refute the need to maintain ex situ samples from all segments or keep specimens that represent distinct genetic clusters. Plants should continue to be added to the ex situ population at the SMARC to match the relative proportions of genetic clusters and unique alleles found within the river. The formulation of a genetics management plan is warranted that includes methods and schedules for in situ and ex situ genetics monitoring, augmentation strategies, propagation methods, and a reintroduction protocol. Acknowledgements We thank Christopher Richards of the United States Department of Agriculture for providing us with data from the 2007 study. Doug Phillips, Daniel Huston, and Shannon Devine assisted with the collection of Texas wild rice samples from the San Marcos River. Meredith Bartron, Robert Doyle, Jackie Poole, and Christian Smith all provided helpful comments on earlier drafts. The conclusions in this article are those of the authors and do not necessarily represent the views of the U.S. Fish and Wildlife Service, and reference to trade names does not imply endorsement by the U.S. Government. This project was funded by the U.S. Fish and Wildlife Service. References Balloux, F., Lehmann, L., de Meeûs, T., 2003. The population genetics of clonal and partially clonal diploids. Genetics 164, 1635–1644. Chen, Y.Y., Chu, H.J., Liu, H., Liu, Y.L., 2012. Abundant genetic diversity of the wild rice Zizania latifolia in central China revealed by microsatellites. Ann. Appl. Biol. 161, 192–201. Christe, C., Kozlowski, G., Frey, D., Fazan, L., Bétrisey, S., Pirintsos, S., Gratzfeld, J., Naciri, Y., 2014. Do living ex situ collections capture the genetic variation of wild populations? A molecular analysis of two relict tree species, Zelkova abelica and Zelkova carpinifolia. Biodivers. Conserv. 23, 2945–2959. Christie, M.R., Marine, M.L., French, R.A., Waples, R.S., Blouin, M.S., 2012. Effective size of a wild salmonid population is greatly reduced by hatchery supplementation. Heredity 109, 254–260. Cibrian-Jaramillo, A., Hird, A., Oleas, N., Ma, H., Meerow, A.W., Francisco-Ortega, J., Griffith, M.P., 2013. What is the conservation value of a plant in a botanic garden? Using indicators to improve management of ex situ collections. Bot. Rev. 79, 559–577. Cohen, J.I., Williams, J.T., Plucknett, D.L., Shands, H., 1991. Ex situ conservation of plant genetic resources: global development and environmental concerns. Science 253, 866–872. Dobson, A., Lodge, D., Alder, J., Cummings, G.S., Keymer, J., McGlade, J., Mooney, H., Rusak, J.A., Sala, O., Wolters, V., Wall, D., Winfree, R., Xenopoulos, M.X., 2006. Habitat loss, trophic collapse, and the decline of ecosystem services. Ecology 87, 1915–1924.
Earl, D.A., vonHoldt, B.M., 2011. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the EVANNO method. Conserv. Genet. Resour. 4, 359–361. Evanno, G., Regnaut, S., Goudet, J., 2005. Selecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14, 2611–2620. Excoffier, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479–491. Falk, D.A., Holsinger, K.E., 1991. Genetics and Conservation of Rare Plants. Oxford University Press, UK. Fisch, K.M., Kozfkay, C.C., Ivy, J.A., Ryder, O.A., Waples, R.S., 2015. Fish hatchery genetic management techniques: integrating theory with implementation. N. Am. J. Aquacult. 77, 343–357. Goudet, J., 1995. FSTAT (Version 1: 2): a computer program to calculate F-statistics. J. Hered. 86, 485–486. Griffith, P., Husby, C., Lewis, C.E., Francisco-Ortega, J., 2011. Palm conservation at a botanical garden: a case for the Keys thatch palm. Palms 55, 93–101. Guerrant Jr., E.O., Havens, K., Mauder, M. (Eds.), 2004. Ex Situ Plant Conservation. Supporting Species Survival in the Wild. Island Press, Washington, D.C. Havens, K., Kramer, A.T., Guerrant Jr., E.O., 2014. Getting plant conservation right (or not): the case of the United States. Int. J. Plant Sci. 175, 3–10. Heywood, V.H., Iriondo, J.M., 2003. Plant conservation: old problems, new perspectives. Biol. Conserv. 113, 321–335. Kramer, A.T., Havens, K., 2009. Plant conservation genetics in a changing world. Trends Plant Sci. 14, 599–607. Leberg, P.L., 2002. Estimating allelic richness: effects of sample size and bottlenecks. Mol. Ecol. 11, 2445–2449. Maunder, M., Havens, K., Guerrant Jr., E.O., Falk, D.A., 2004. Ex situ methods: a vital but underused set of conservation resources. In: Guerrant Jr., E.O., Havens, K., Mauder, M. (Eds.), Ex Situ Plant Conservation. Supporting species survival in the wild. Island Press, Washington, D.C, pp. 3–20. Meirmans, P.G., van Tienderen, P.H., 2004. GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organisms. Mol. Ecol. Notes 4, 792–794. Michalakis, Y., Excoffier, L., 1996. A generic estimation of population subdivision using distances between alleles with special reference for microsatellite loci. Genetics 142, 1061–1064. Peakall, R., Smouse, P.E., 2006. GENALEX 6: genetic analysis in excel: population genetic software for teaching and research. Mol. Ecol. Notes 6, 288–295. Petit, R.J., El Mousadik, A., Pons, O., 1998. Identifying populations for conservation on the basis of genetic markers. Conserv. Biol. 12, 844–855. Poole, J.M., 2002. Historical distribution of Texas wild-rice (Zizania texana) from 1989 to 2001. Section 6 Final Report. Albuquerque, N.M.: U.S. Fish and Wildlife Service. Poole, J.M., Bowles, D.E., 1999. Habitat characterizations of Texas wild-rice (Zizania texana), an endangered aquatic macrophyte from the San Marcos River, Texas, USA. Aquat. Conserv. 9, 291–302. Poole, J.M., Carr, W.R., Price, D.M., Singhurst, J.R., 2007. Rare Plants of Texas. Texas A&M University Press College Station. Power, P., Oxley F.M., 2004. Assessment of factors influencing Texas wild-rice (Zizania texana) sexual and asexual reproduction. Final Report for Edwards Aquifer Authority, San Antonio, TX. Pritchard, J.K., Stephens, M., Donnelly, P., 2000. Inference of population structure using multilocus genotype data. Genetics 155, 945–959. Raymond, M., Rousset, F., 1995. GENEPOP (version 1: 2): population genetics software for exact tests and ecumenicism. J. Hered. 86, 248–249. Richards, C.M., Antolin, M.F., Reille, A., Pool, J., Walters, C., 2007. Capturing genetic diversity of wild populations for ex situ conservation: Texas wild rice (Zizania texana) as a model. Genet. Resour. Crop Evol. 54, 837–848. Rousset, F., 2008. Genepop’007: a complete reimplementation of the Genepop software for Windows and Linux. Mol. Ecol. Resour. 8, 103–106. U.S. Fish and Wildlife Service (USFWS), 1978, Determination that 11 plant taxa are endangered species and 2 plant taxa are threatened species. Federal Register 43, 81: 17910–17916. U.S. Fish and Wildlife Service (USFWS), 1995, San Marcos/Comal (Revised) Recovery Plan. Albuquerque, New Mexico. pp x + 93 with 28 pages of appendices. van Oosterhout, C., Hutchinson, W.F., Wills, D.P.M., Shipley, P., 2004. Micro-checker: software for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Notes 4, 535–538. Weir, B.S., 1996. Genetic Data Analysis II. Sinauer Associates Sunderland, Mass. Wilcove, D.S., Rothstein, D., Dubow, J., Phillips, A., Losos, E., 1998. Quantifying threats to imperiled species in the United States. Bioscience 45, 607–615.
Please cite this article in press as: Wilson, W.D., et al., Genetic diversity assessment of in situ and ex situ Texas wild rice (Zizania texana) populations, an endangered plant. Aquat. Bot. (2015), http://dx.doi.org/10.1016/j.aquabot.2015.12.005