Genetic Stock Assessment of Spawning Arctic Cisco (Coregonus Autumnalis) Populations by Flow Cytometric Determination of DNA Content

8 1991 Wiley-Liss, Inc. Cytometry 12:260-267 (1991)

Genetic Stock Assessment of Spawning Arctic Cisco (Coregonus autumnalis) Populations by Flow Cytometric Determination of DNA Content

Samuel F. Lockwood and John W. Bickham Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, Texas 77843 Received for publication January 24, 1990; accepted November 7, 1990

Intraspecific variation in cellular DNA tain, and Liard rivers, but was not ob- content was measured in five Coregonus served in kidney tissue preparations autumnalis spawning populations from from the Mountain or Carcajou rivers. the Mackenzie River drainage, Canada, The Liard River sample had significantly using flow cytometry. The rivers assayed elevated mean DNA content relative to were the Peel, Arctic Red, Mountain, the other four samples; all other samples Carcajou, and Liard rivers. DNA content were statistically indistinguishable. Sig- was determined from whole blood prep- nificant differences in mean DNA content arations of fish from all rivers except the among spawning stocks of a single spe- Carcajou, for which kidney tissue was cies reinforces the need for adequate used. DNA content measurements of kid- sample sizes of both individuals and pop- ney and blood preparations of the same ulations when reporting “C” values for a fish from the Mountain River revealed particular species. statistically indistinguishable results. Mosaicism was found in blood prepara- Key terms: DNA content, DAPI, flow cy- tions from the Peel, Arctic Red, Moun- tometry, fisheries stock assessment

Most studies of the biological significance of DNA report DNA content means, standard errors, and content have concentrated on defining a specific value, ranges for individual species, as well as means for iden- measured in picograms of DNA, for a particular species tified populations within that species. After a thorough or higher taxon (9). Such studies may prove to be pre- characterization of intraspecific variation in genome mature, because the biological significance of DNA size, the biological significance of that variation can be content variation (“genome size” variation) is difficult discussed relative to other taxa. to evaluate without knowledge of the amount of vari- Particularly well suited to an analysis of intraspe- ation within species and populations (20). The amount cific DNA content variation are members of the fish of DNA in an unreplicated haploid genome (the “C” family Salmonidae. A population structure based on value) was thought to remain constant within a species geographically distinct stocks is present in salmonids (38). Recently, however, significant differences were (6,39), which has accentuated chromosomal divergence found to exist among individuals within species in this group of tetraploid fishes (1).Population subdi- (7,8,18-20,30-32,37). Those results contrasted with vision is the result of the ability of these fishes to re- earlier views that intraspecific differences in genome turn to their natal streams at given times to spawn size either do not exist (12) or are insignificant or un- (11,34). Salmonids in general are plastic in morphol- important (3). A common theme of recent studies is ogy, growth rate, and spawning characteristics (2,13, that the extent of intraspecific variation in DNA con- 36,411, and many of these characteristics are geneti- tent must be well characterized as a first step towards understanding the biological properties of genome size, or before phylogenetic comparisons can be made. Mea- surements from single individuals, or even adequate FIG.1. Map of sampling area within the Mackenzie River, North- samples from a single population may be grossly insuf- west Territories, Canada, showing locations of the five tributaries ficient for determining the genome size of a species sampled: 1) Peel River, 2) Arctic Red River, 3) Mountain River, 4) (20). The trend in modern studies of genome size is to Carcajou River, 5) Liard River. t\

0 0 0 tv CD m r CD CD 262 LOCKWOOD AND BICKHAM cally based (35).In addition, intraspecific karyotypic Approximately 30 individuals were analyzed on a variability is well documented in this fish family (1, given day, including equal numbers from each river to 14,15). The tolerance of the salmonid karyotype to minimize any possible variation in DNA determination gains and losses of entire chromosomes could lead to by machine error. Samples were coded, randomized, chromosomal, and genome size, variation among pop- and run blind. Accuracy of DNA content measurement ulations (21). From these observations, it is inferred also was assessed each day by analyzing chicken and that variation within salmonid species might be tortoise (Geochelone elephantopus; Houston Zoo speci- greater than that normally encountered in diploid te- men HZG 5825) blood cells. leost fishes, and vertebrates, in general (20). Statistical analyses were performed using the BIO- A preliminary study of DNA content variation, as STAT Univariate Statistical Toolbox (29). DNA con- part of a larger analysis of population genetics, was tent measurements were ordered by specimen number, conducted in the Arctic cisco, Coregonus autumnalis corresponding to the order in which they were caught. (6). The results showed a high degree of DNA content One way analysis of variance was performed after Bart- variability within a coastal sample and indicated a lett’s test (Fmax test statistic) of the homogeneity of need for flow cytometric analysis of spawning stocks. sample variances. Student-Newman-Keuls multiple In the present study, modified techniques developed in range test, which controls for type I comparison-wise earlier studies (4-8,25) were used to quantify intraspe- error rate, was used to sort significantly different sam- cific variation in the Arctic cisco, Coregonus autumna- ple means. When sample sizes were unequal, a har- lis. This variation is considered relative to its potential monic mean was used in the analysis. Descriptive sta- physiological and zoogeographical implications. Flow tistics (means, standard errors, ranges, and 95% cytometry, a rapid and highly accurate procedure for confidence intervals) were compiled for all samples. estimating differences in DNA content within and The hypotheses that the data followed a normal distri- among populations (401, is assessed as a potential tool bution was tested by evaluating skewness (81 statistic) in fisheries stock assessment (6). and kurtosis (82 statistic), calculated separately for each sample. The original Kolmogorov test of goodness MATERIALS AND METHODS of fit (D test statistic) was also used to evaluate the Collections of Arctic cisco from spawning runs in the hypothesis that the data followed a normal distribu- tributaries of the Mackenzie River drainage, North- tion. The serial correlation test (C test statistic) was west Territories, Canada, were made between 15 July used to test the independence of successive samples 1988 and 23 September 1988. Samples were taken from (i.e.,whether the value of one sample is the function of the Peel, Arctic Red, Mountain, Carcajou, and Liard the value of the previous sample). All statistical tests rivers (Fig. 1).Gill nets were used to catch Arctic cisco were performed at the alpha = 0.05 level (27). at least 1 mile upstream from the mouth of each tributary to reduce the probability of sample contamina- RESULTS tion of fish from other stocks that might stray a short Multiple cell populations (mosaicism: Fig. 2F) were way up the wrong river. Whole blood and kidney sam- found in blood preparations in frequencies ranging ples were taken in the field from fish from all rivers from 48.4% in the Liard River to 32.1% in the Peel except the Carcajou River, from which only kidney River (Fig. 3).The increase in DNA content in the ad- samples were available. Whole blood was used to de- ditional peak (right hand peak) of the blood samples termine DNA content in all fish except those from the varied considerably among stocks: Liard (9.524), Carcajou River, for which kidney tissue was used. The Mountain (16.6%), Arctic Red (6.47%), and Peel DNA contents of blood and kidney samples taken from (7.69%).Mosaicism was not found in the kidney tissue the same fish from the Mountain River were compared preparations of fish from the Carcajou River. Tissue- to determine both the effect of tissue type on DNA con- specific mosaicism, in which certain tissues contained tent measurement, and the validity of comparing the additional “abnormal” cell populations, had been pre- kidney samples from Carcajou River fish with all other viously reported in turtles (4). Therefore, a duplicate blood samples. set of kidney samples from Mountain River fish was Methods for blood and kidney tissue preparation, analyzed to ascertain “normal” cell populations within staining, and flow cytometric analysis are described in individuals, as well as the effect of tissue type on DNA detail by Burton et al. (8) and Lockwood (23). Cells content measurement. were stained with DAPI (4‘,6-diamidino-2-phenylin- Duplicate samples of blood and kidney from 17 indi- dole) and analyzed on a Leitz MPV flow cytometer (8). viduals from the Mountain River were available for Chicken erythrocytes (2C = 2.54 pg; Rasch et al. [33]) comparison. DNA content measurements of “left hand” were used as an internal standard. Fluorescent micro- peaks of blood (mean = 6.34 pg) and the single peak spheres were used to align and focus the flow cytome- found in all kidney (mean = 6.38 pg) preparations from ter. Instrumental gain and the voltage to the photom- the same fish were compared statistically with the eter were adjusted so that the mean of the G, peak of paired Student’s t-test (27). Group variances (F = 1.38, the chicken red blood cells was at, or as near as possible df = 16,p = 0.26) and group means (T = -1.26, df = to, channel 50 on the pulse height analyzer (Fig. 2). 16, p = 1.00) did not differ significantly, hence the GENETIC STOCK ASSESSMENT OF SPAWNING ARCTIC CISCO 263 B X CRBC X CBC - CRBC I - _I x=49 X=126 W 15- -CBC d135- x-49 Z X=126 Z Z z 10- Coregonus autumnalis 2 9- Coregonus autumnalis c! 6 53 pg 6 53 PS (I) 45- _I 5- 2-1 1 W W 0 - 0 1 I I - I I I I I

N N 0 C 0 D r 7 X CRBC CBC X - - CBC 1 x=49 ' X-128 6 1 - W 9- W 9- x=122 Z Z Q 6- 6- Coregonus autumnalis I Coregonus autumnalis 2 CRBC 6 46 ps 3- 1 1 W 0 I I I I I I 50 100 150 200 250 50 100 150 200 250 CHANNEL NO + (DNA) CHANNEL NO -t (DNA)

N 0 EN, F 7 T

Geochelone elephantopus FIG.2. Representative DNA histograms for a member of each Arc- -,- tic cisco spawning population examined: A) Liard River, Bj Carcajou i.1 River, Cj Mountain River, D) Arctic Red River, Ej Peel River, F) 24 !l;'lmosaic individual from the Liard River. Gj tortoise used to calibrate 1 w the flow cytometer. The G1 peaks for chicken, Callus domesticus 0 (CRBC), and cisco blood cells (CBC) are labelled. DNA content is 50 100- 150 200 250 proportionate to relative channel ratios and the gain of the flow cytometer is adjusted so the G. domesticus G1 peak is as near channel 50 CHANNEL NO + (DNA) as possible (2.54 pgj. 264 LOCKWOOD AND BICKHAM

PERCENT MOSAICISM BY SAMPLE SITE

100

60 Liard 50

0 0 1 2 3 4 5 6

FIG.3. Percent mosaicism (numbers of individuals with multiple cell populations) in five Arctic cisco spawning populations.

larger Mountain River sample of 19 measurements lations sampled; no other significant differences were from blood was used in the ANOVA. These statistically found (Table 1). indistinguishable results justified the comparison of On the basis of the g, and g, statistics, the Peel, “normal” or “left-hand” peaks in mosaic individuals Mountain, and Carcajou river samples did not differ across all populations, as well as the comparison of significantly from the normal distribution (Table 1). mean DNA content in samples using different tissue The Arctic Red River sample was skewed slightly left. type in the analysis. Left-hand peaks of mosaic indi- The Liard River sample was skewed right and lep- viduals were the only cell populations used in the sta- tokurtotic with the outlier included; it did not differ tistical analyses. from the normal distribution with the outlier excluded. The average DNA content over all five spawning On the basis of the D statistic, no stock differed signif- stocks was 6.44 picograms (pg) and ranged from 5.56 pg icantly from the normal distribution. Lastly, the serial in one Peel River cisco to 8.14 in one Liard River cisco, correlation test was nonsignificant for all samples a variation of 2.58 pg (40.1%). One Liard River cisco tested except for the Liard sample with outlier ex- (AK 12372, 8.14 pg) was found to be an outlier; statis- cluded. In that sample, the data points tended to clump tical analyses were conducted with and without this near the extremes. Thus, for the five complete samples, outlier. The range of intra-population variation in ge- there was no evidence to suggest that the samples were nome size relative to the total range of variation was not independent (Table 1). considerable; 87.7% in the Peel River, 55.5% in the Arctic Red River, 54.8% in the Mountain River, 58.7% DISCUSSION in the Carcajou River, 58.1% in the Liard River with- The overall mean DNA content (6.44 pg, 128 indi- out the outlier, and 74.8% in the Liard River with the viduals) found in this study is well above the mean outlier included. (4.35 pg, 16 individuals from the Colville River, Bartlett’s test revealed that sample variances did not Alaska) found in a previous study (6) in the Arctic differ significantly; hence the variance assumptions of cisco. However, the trend toward lower DNA content the ANOVA were satisfied (F,,, = 3.46, df = 30, p > values and increasing variability in coastal samples .lo, outlier included; Fmax = 2.77, df = 28, p > .lo, relative to spawning stock samples is consistent with outlier excluded). One-way analysis of variance on results found by Lockwood (23). The overall mean stock DNA content means was significant (F = 9.88, df value of 6.44 pg of DNA in Coregonus autumnalis is = 4,p < 0.000001, outlier included; F = 8.89, df = 4, also higher than the overall mean of 5.0 pg for the p < 0.000001, outlier excluded). Student-Newman- three salmonid genera examined by Johnson et al. (20). Keuls multiple range test revealed that the Liard However, the value is consistent with the doubling of River sample (with and without the outlier) had signif- genome size by the salmonid tetraploid event (1) from icantly greater mean genome size than all other popu- the proposed primitive ancestral genome size of ap- GENETIC STOCK ASSESSMENT OF SPAWNING ARCTIC CISCO 265

Table 1 Descriptive Statistics for Arctic Cisco DNA Content Means (in pgi and Results of Normality and Independence Testing by Spawning Population (With and without Liard River sample outlier included) Sample Locality N Mean S.E. Range gl 82 D C Liard River 31 6.68 (.069) 6.21-8.14 1.76" 5.43" 0.12 -0.27 Liard River 30 6.64 (.051) 6.21-7.11 0.19 -1.22 0.13 0.38* Peel River 28 6.39 (.065) 5.56-6.92 -0.71 0.39 0.07 -0.02 Arctic Red River 29 6.35 (.039) 5.81-6.67 -0.98" 0.85 0.15 0.24 Mountain River 19 6.34 (.047) 5.79-6.64 -0.73 1.61 0.11 0.14 Carcajou River 21 6.20 (.051) 5.64-6.55 -0.70 0.28 0.12 0.23 *Indicates significance at the 0.05 level. proximately 3.0 pg (12). The trend towards reduction in This could be reflected in slightly higher genome size chromosome number and overall genome size may be in this population. Protein and suitable molecular data slowed in the coregonines relative to other salmonid are needed to establish the affinities of MacKenzie Arc- genera, or selection for higher genome size may be op- tic cisco populations relative to each other and also erating on this species. Bering cisco from tributaries in the Yukon River. This The range of DNA content variation within popula- would also provide a test of the historical biogeography tions of Arctic cisco relative to the overall range of the of the region already proposed (22). species was large. The Peel River population contained The range of intraspecific variation in genome size nearly as much within population variation (87.7%) as found in this study (2.58 pg) parallels that found in that found among all populations, and may reflect a other studies. This range is consistent with variation multi-stock composition of that tributary, as previously found in pocket gophers of the genera Thomomys (38) suggested based upon allozyme data (26). Statistical and Geomys (7), as well as variation in Neotropical bats analysis of the genome size data, however, does not (8).Variation is slightly higher in this study than that corroborate such a conclusion (the sample is not found in another study of salmonids (20). However, in- platykurtotic). In fact, deviations from normal distri- traspecific variation may have been underestimated by butions of samples from the Arctic Red and Liard rivers small sample size (10 individuals or less in 12 of 14 (outlier included) samples detected with tests of skew- species examined) in the previous study (20). In fact, ness and kurtosis were slight, and not detected with the greatest intraspecific variation found was in the the less powerful Kolmogorov test. These findings mir- second largest sample, Salmo clarki bouveri (15.4%). ror previous reports from fish, in which genome size Another possible confounding factor which could limit distribution was essentially continuous and normal variability in that study is the use of hatchery main- within populations (18,19,32). tained fish which may not reflect variation found in Significantly elevated mean DNA content in the natural populations of the same species. Reports of Liard River population, and the inability to distinguish large amounts of intraspecific variation in DNA con- statistically among the four northern populations on tent (6,30,32,37) reinforce the need for data from nat- the basis of DNA content may be the result of several ural populations which take into account both sample factors. Strong selection for elevated genome size may size and population structure. be acting on this population (10). Alternatively, the Variation in staining intensities of different tissue Liard River population may have originated from a types within individuals, and tissues stained on differ- stream capture event of high DNA content Bering cisco ent days, are common problems in cytometric studies from the headwaters of the Yukon River, Alaska (16). (J. Bickham, pers. obs.). Of critical importance is the A single Yukon River Bering cisco, with reported ge- selection of a fluorochrome, such as DAPI, with stoi- nome size of 7.0 pg (6), was within the range of varia- chiometric binding properties and a staining method- tion of the Liard River population but outside the ology that ensures consistent and complete saturation ranges of all other Arctic cisco spawning populations in of the DNA with the dye (24,28). Therefore, the non- this study. If such a capture event took place, genome significant difference in mean DNA content observed size variability may have been greatly reduced in the between left-hand peaks of blood and kidney prepara- Liard River founder population due to sampling error tions from 17 of the same individuals for which tissue (genetic drift). Yet the Liard River sample possessed was available was reassuring and substantiates the intra-sample variability (58.1 or 74.8%) intermediate staining protocols used in this study. between the other samples. Lastly, there is a possibil- The protocol was further reinforced by the observa- ity that the Liard River fish are not anadromous (R. tion that repeated staining of erythrocytes from the Dillinger, pers. comm.). If this were the case, and the tortoise Geochelone elephantopus on separate days re- physiological constraints of anadromy were absent in vealed a constancy of ratio (chicken channel 50, tor- this population, selection for reduced genome size and toise channel 119). This precision of DNA content mea- faster developmental rate might not be operative (17). surement may be a reflection of longer staining of 266 LOCKWOOI) AND BICKHAM cellular pellets (uniformly 72 h in stain at 4°C) in this Wildlife Service, US. Bureau of Land Management, Denver, Col- study relative to previous studies (4,7,8).Also, stained orado, 1979. 3. Bennett MD: DNA amount, latitude, and crop plant distribution. samples from one individual analyzed repeatedly over Environ Exp Bot 16:93-108, 1976. an eight month period yielded identical readings of 4. Bickham JW, Tucker PK, and Legler JM: Diploid-triploid mosa- DNA content. Although staining times less than 72 icism: an unusual phenomenon in side-necked turtles (Platemys hours might be adequate, previous studies in this lab- platycephala). Science 227:1591-1593, 1985. oratory corroborate the need for constancy in staining 5. Bickham JW, Hanks BG, Smolen MJ, Lamb T, Gibbons JW: Flow cytometric analysis of the effects of low-level radiation exposure time. The suggestion (40) that a second internal stan- on natural populations of slider turtles (Pseudemys scripts). Arch dard may increase resolution of DNA content determi- Environ Contam Toxic 17:837-841, 1988. nation by reducing error due to floating zero point was 6. Bickham JW, Carr SM, Hanks BG, Burton DW, Gallaway BJ: ignored, not without precedent; Johnson et al. (20) Genetic analysis of population variation in the Arctic cisco (Core- gonus autumnalis) using electrophoretic, flow cytometric, and found no improvement in genome size measurement mitochondria1 DNA restriction analyses. Biol Pap Univ Alaska using a second standard. 24:112-122, 1989. A large number of individuals assayed exhibited tis- 7. Burton DW, Bickham JW: Heterochromatin variation and DNA sue specific mosaicism (4). How this phenomenon content conservatism in Geomys attwateri and G. breuiceps (Ro- might be maintained in Coregonus autumnalis blood dentia: Geomydae). J Mamm 70580-591, 1989. 8. Burton DW, Bickham JW, Genoways HH: Flow cytometric anal- cells is unknown. The observation that blood samples ysis of nuclear DNA content in four families of neotropical bats. from coastal site fish contained very few mosaic indi- Evolution 43:756-765, 1989. viduals (23) suggests the possibility that such mosa- 9. Cavalier-Smith T: Introduction: the evolutionary significance of icism is not constitutional, but rather a physiological genome size. In: The Evolution of Genome Size, Cavalier-Smith T (ed).J. Wiley & Sons, New York, 1985, pp 1-36. response of some fish to the freshwater conditions en- 10. 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Ferguson A, Fleming CC: Evolutionary and taxonomic significance of protein variation in the brown trout (Salmo trutta L.)and was useful as a technique to differentiate genetic other salmonid fishes. In: Protein Polymorphism: Adaptive and stocks of salmonid fishes. It is likely that other tech- Taxonomic Significance, Oxford GS, Rollinson DE (eds). Aca- niques, such as protein electrophoresis and mitochon- demic Press, New York, 1983, pp 85-89. drial DNA analysis, have greater sensitivity. DNA 14. Frolov SV: Polymorphism and mosaicism for additional chromo- content analysis, however, has the distinct advantage somes in Coregonus surdinella. Tsitologia 28:740-746, 1986. 15. Frolov SV:The additional chromosome system in the karyotype of of being considerably less expensive than biochemical Coregonus nasus. Tsitologia 28:215-221, 1986. or molecular genetic studies and, under circumstances 16. 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