Spring Chinook salmon from the upper Yakima River were placed into an artificial stream and allowed to spawn under quasi-natural conditions. Seven independent test groups were placed into the stream from 2001 through 2005. Four different types of males, large anadromous, jacks, yearling precocious, and sub-yearling precocious were placed into the test groups. Their breeding success or ability to produce offspring was evaluated by performing DNA-based pedigree assessments. These analyses disclosed that individual jacks and yearling precocious males produced approximately 26% as many fry as individual large anadromous males while the subyearling precocious males fathered 3% as many offspring as individual large anadromous males. The effects of four factors, body size (weight or length), spawning ground residency in hrs, tertiary sex ratios (absolute numbers of males to females) and mate number, on male breeding success were evaluated. There was a positive relationship between male breeding success and body weight in the large anadromous males that explained 26% of the variation in their breeding success. No relationship between fish length and breeding success was found in the other male life history types. A negative relationship between spawning ground residency and breeding success was found in the large anadromous males, however, none was found in the remaining male types. Individual breeding success in large anadromous males significantly decreased as tertiary sex ratios for females increased. Jacks and yearling precocious males experienced similar decreases in individual breeding success when tertiary sex ratios rose but these relationships were non-significant. The most important factor affecting individual breeding success in all four male types was the number of females they were able to spawn with. In the 2003 test group, this variable explained 93%, 91%, 64%, and 87% of the variation in breeding success in large anadromous males, jacks, yearling precocious, and sub-yearling precocious males, respectively. Significant differences existed among the male types in the acquisition of mates. Large anadromous males mated with more females than jacks and sub-yearling precocious males, and jacks obtained more mates than sub-yearling precocious males. Yearling precocious males and large anadromous males obtained similar number of mates. However, the number of fry produced per mate differed among the male types. Large anadromous males produced more fry per mate than did jacks and yearling precocious males. On the other hand, jacks and yearling precocious males produced similar numbers of fry per mate and mated with similar numbers of females. Overall in our test groups, large anadromous males produced 89%, jacks 3.2%, yearling precocious 7.4%, and sub-yearling precocious 0.8% of the fry. These percentages remained stable even though the proportion of large anadromous males in the test groups ranged from 48% to 87.5% and tertiary sex ratios varied from 1.4 to 2.4 males per female. Our data suggest that large anadromous males are able to produce most of the fry in natural settings when half or more of the males present on a spawning ground use this life history strategy.

Introduction

Four types of adult spring Chinook salmon males occur on upper Yakima River spawning grounds. Each type matures at a different age; two are anadromous while two others become mature without going to sea. In this report, males that have matured at the same modal age as females are referred to as large anadromous males. Most of them reach maturation at age 4 although a few are 5-yrs old at maturity [START_REF] Knudsen | Comparison of life-history traits between first-generation hatchery and wild upper Yakima River spring Chinook salmon[END_REF]. Anadromous males that mature a year earlier than the modal female age are referred to as jacks (after B. Berejikian, personal communication). For upper Yakima spring Chinook salmon, jacks are three-year olds. Males that mature after rearing 18 to 20 months in freshwater are called yearling precocious males while non-anadromous males maturing after just 7 to 8 months post emergence, are referred to as subyearling precocious males. The occurrence of such a diverse array of male life history types in upper Yakima River spring Chinook is not unusual. Precocious spring Chinook males have been recognized for over 100 years [START_REF] Rutter | Natural history of the quinnat salmon[END_REF] as cited by [START_REF] Pearsons | Abundance and distribution of precociously mature male spring Chinook salmon of hatchery and wild origin in the Yakima River[END_REF] and their presence in spawning populations can be quite variable, ranging from 1 to 12% or more [START_REF] Rich | Early history and seaward migration of chinook salmon in the Columbia and Sacramento rivers[END_REF] as cited by [START_REF] Larsen | Assessment of high rates of precocious male maturation in a spring Chinook salmon supplementation hatchery program[END_REF][START_REF] Gebhards | Biological notes on precocious male chinook salmon parr in the Salmon River drainage, Idaho[END_REF][START_REF] Mullan | Life histories and precocity of Chinook salmon in the mid-Columbia River[END_REF]). In the Upper Yakima River, yearling precocious males of hatchery and wild origin and wild sub yearling males have been observed [START_REF] Pearsons | Abundance and distribution of precociously mature male spring Chinook salmon of hatchery and wild origin in the Yakima River[END_REF]) and numerous jacks and large anadromous males may also be present.

In 1997 a spring Chinook supplementation program began in the upper Yakima River with the establishment of the Cle Elum Supplementation Research Facility (CESRF). This program has significantly increased the number of females and large anadromous male spring Chinook salmon returning to the upper Yakima River. However, hatchery conditions have also inadvertently increased the number of yearling precocious males in the upper Yakima. Analyses done by [START_REF] Larsen | Assessment of high rates of precocious male maturation in a spring Chinook salmon supplementation hatchery program[END_REF] indicated that 37 -49% of the males produced by the CESRF underwent precocious development and became sexually mature in the second year of life. Many of these fish emigrated downstream or perished after release but a portion of them either residualized in the upper Yakima River or migrated back upstream and became part of the spring Chinook spawning population [START_REF] Larsen | Assessment of high rates of precocious male maturation in a spring Chinook salmon supplementation hatchery program[END_REF][START_REF] Pearsons | Abundance and distribution of precociously mature male spring Chinook salmon of hatchery and wild origin in the Yakima River[END_REF]. The CESRF has also significantly elevated the number of jacks returning to the upper Yakima River [START_REF] Knudsen | Comparison of life-history traits between first-generation hatchery and wild upper Yakima River spring Chinook salmon[END_REF]. Increases in the abundance of precocious males and jacks due to artificial culture has been observed in other spring Chinook populations [START_REF] Gebhards | Biological notes on precocious male chinook salmon parr in the Salmon River drainage, Idaho[END_REF][START_REF] Mullan | Life histories and precocity of Chinook salmon in the mid-Columbia River[END_REF][START_REF] Unwin | Changes in life history parameters in a naturally spawning population of chinook salmon (Oncorhynchus tshawytscha) associated with releases of hatchery-reared fish[END_REF] and is likely caused by the growth patterns and energy stores salmon experience under artificial culture [START_REF] Shearer | The effect of whole body lipid on early sexual maturation of 1+ age male chinook salmon (Oncorhynchus tshawytscha)[END_REF].

The enhanced occurrence of jacks and yearling precocious males due to supplementation prompted several questions. First, even though large numbers of hatchery origin yearling precocious males were produced by the CESRF how many of them are present on upper Yakima River spawning grounds? Second, if they are present, how successful are they in producing offspring? Third, prior to the advent of the CESRF, wild sub yearling, yearling precocious males, and jacks were present in upper Yakima River spawning areas. In supportive breeding programs designed to recover depressed populations should males possessing these life history expressions be included in the broodstock and if so what should their genetic contributions be? [START_REF] Pearsons | Abundance and distribution of precociously mature male spring Chinook salmon of hatchery and wild origin in the Yakima River[END_REF] addressed the first question with their comprehensive assessment of the occurrence of hatchery-origin yearling precocious males in spring Chinook spawning areas in the upper Yakima River. The remaining two questions that deal with the relative breeding success of different male spring Chinook life history types have not yet been addressed.

In two previous papers we compared the breeding success of first-generation hatchery and wild female [START_REF] Schroder | Breeding success of wild and first generation hatchery female spring Chinook salmon spawning in an artificial stream[END_REF]) and male (Schroder et al. In Press) spring Chinook salmon spawning in an artificial stream. Because of the ubiquitous presence of precocious males and jacks in the upper Yakima, and the desire to mimic natural social conditions in the artificial stream, we included precocious males and jacks along with large anadromous males and females in seven test groups placed into an artificial stream. Their inclusion has given us an opportunity to compare the ability of four male spring Chinook salmon life history types to produce offspring while spawning under quasi-natural circumstances.

Here we present preliminary results of our efforts to compare the mean number of offspring produced by males possessing different life-histories by using DNA-based pedigree analyses. These analyses also allowed us to determine the breeding success of jacks, yearling precocious males and sub-yearling precocious males relative to that achieved by large anadromous males. Next, we compared the body lengths of males representing each life history type and examined the importance of body weight or length on breeding success within each male type. Longevity or the number of hrs males of each type resided in the artificial stream was compared and the effect of longevity on breeding success was also evaluated within each male type. The effect of different tertiary sex ratios (absolute number of males to females) in the test groups on the ability of individual males within each life history type to produce offspring was also investigated. When we compared the breeding success of large anadromous males of hatchery and wild origin the factor that accounted for the most variation in their breeding success was the number of mates they were able to spawn with (Schroder et al. In Press). Consequently, the mean number of mates each type of male spawned with was contrasted with one another. Additionally, we also determined the number of fry produced from each mating and ascertained whether that varied because of male type. Additionally, we assessed whether the paternal origin of fry produced from our test groups varied because of the composition of male types in a test group. Did for example, the proportion of fry produced by large anadromous males decline as their relative abundance decreased, or did it remain constant, or vary in some other fashion? This evaluation was done to provide insights into what genetic contributions should be made by various male life history types when supportive breeding programs are undertaken. Finally we examined how changes in tertiary sex ratios affected the overall paternity of fry in our test groups. This was done to determine if different levels of male intra-sexual competition, as estimated by the tertiary sex ratio, influenced which male types were able to produce offspring. Understanding the relationship between male competition for females and paternity may also help determine how different male types should be incorporated into artificial breeding programs.

Methods

Origin and Collection of Spring Chinook Salmon. Adult female, large anadromous males, jacks, and yearling precocious males were collected from the upper Yakima River from April through August at the Roza Adult Monitoring Facility. This facility is located at rkm 206 (as measured from the confluence with the Columbia River) and upper Yakima River spring Chinook must pass through it before reaching their spawning grounds. Methods described by [START_REF] Knudsen | Comparison of life-history traits between first-generation hatchery and wild upper Yakima River spring Chinook salmon[END_REF] were used to collect a representative sample of fish. Collected fish were transported 81 km to the CESRF and females, large anadromous males, and jacks were held in a 30.5 m long by 4.6 m wide x 3 m deep pond while yearling precocious males were placed into a 30.5 m long x 3.5 m wide by 1.1 m deep hatchery raceway. Sub yearling precocious males were collected off natural redds in the upper Yakima River by using hook and line or electrofishing gear. After capture they were transported to the CESRF and held in small circular tanks.

Fish selection and introduction into the artificial stream followed the protocols previously described in [START_REF] Schroder | Breeding success of wild and first generation hatchery female spring Chinook salmon spawning in an artificial stream[END_REF] and Schroder et al. (In Press). Briefly, beginning in early September, fish were examined weekly to determine their maturation status. Only ripe fish were selected for use in the artificial stream. Prior to being placed into the stream they were anesthetized in a 1:19,000 part solution of tricaine methanesulfonate [START_REF] Bell | A guide to the properties, characteristics, and uses of some general anaesthetics for fish[END_REF]. Once docile, the large anadromous males, females, and jacks were weighed to the nearest gram while yearling and sub yearling precocious males were weighed to the nearest tenth of a gram on electronic balances. Fork lengths to the nearest mm were taken on all fish. The large anadromous fish were tagged with Petersen disks but no external tags were applied to the precocious males due to their small size. A Tissue sample from the posterior edge of the dorsal fin was removed from each fish and placed into 100% ethanol for later DNA extraction and genetic analysis. The fish were then transported 200 m and released into the artificial stream. The origin, number, body weights, tertiary sex ratios, and the percentage of the males in each test group that were large anadromous fish are shown in Table 1.

Artificial Stream. A detailed description of the artificial stream can be found in [START_REF] Schroder | Breeding success of wild and first generation hatchery female spring Chinook salmon spawning in an artificial stream[END_REF]. It is located on the grounds of the CESRF and is 127 m long by 7.9 m wide. Concrete cross weirs subdivide the "U" shaped structure into six straight and one curved section. The straight sections are 15.2 long by 7.9 m wide while the curved section is 21 m long by 7.9 m. Each section has a level gradient and 30 cm falls separate one section from another. Gravel consisting of material ranging from 0.7 to 10 cm in diameter having a Fredle Index [START_REF] Lotspeich | A new method for reporting and interpreting textual composition of spawning gravel[END_REF] of 7 was used as spawning substrate. Water velocity (0.1 -2.0 m per second), discharge (0.37 m -3 •s -1 ) and depth (mean 0.4 m) were patterned after conditions that spawning Chinook salmon typically prefer [START_REF] Bjornn | Influences of forest and rangeland management on salmonid fishes and their habitats[END_REF][START_REF] Healey | Life history of Chinook salmon[END_REF]. A 2.1 m high wall of camofluage netting was installed on both banks with observation openings at eye level every m along its length.

Seven test groups of Chinook salmon were placed into the artificial stream from 2001 through 2005. In 2001 and 2002 the artificial stream was subdivided into an upper and lower part, each having three straight sections (total length equaled 45.6 m). In these two years, test groups were placed into each part of the stream. Single test groups were placed into the entire stream in 2003, 2004, and 2005. In all years, picket barriers were used to confine the fish to the portions of the stream set aside for their test group. Fish were introduced into a test group on the same day over a 2 to 3 hr period. They were always released into the uppermost section of the artificial stream allotted to their test group and could move freely within sections sequestered for their population. In those years where two test groups were placed into the stream, seven days elapsed between when fish were placed into the upper and lower parts of the artificial stream (Schroder et al. In Press).

Estimating Male Breeding Success. In 2001 and 2002 modified fyke nets with floating live boxes were installed just below each part of the stream that contained a test group. In 2003, 04, and 05 the traps were placed immediately below the last section of the artificial stream. The traps were used to capture fry as they emigrated from the stream. They were installed prior to fry emergence and were in continuous operation until fry migration ceased, then electrofishing gear and seines were used capture any remaining fry. Ten percent of each day's catch was preserved in 100% ethanol and used in microsatellite DNA pedigree assessments. The pedigree analyses indicated the number of fry each male had contributed to the ten percent sample collected from his test group. The number of progeny assigned to an individual male was used in the following formula from Schroder et al. (In Press) to estimate breeding success:

1) BrS 1 = (Fry 1 ) ⁄ (n)
Where BrS 1 = the breeding success of male 1

Fry 1 = The number of fry in the 10% sample that were fathered by male 1 n = The total number of fry in the 10% sample Pedigree Analyses. Methods used to perform the pedigree analyses were previously described in [START_REF] Schroder | Breeding success of wild and first generation hatchery female spring Chinook salmon spawning in an artificial stream[END_REF] and Schroder et al. (In Press). For test groups 1 and 2, genomic DNA was extracted from the fry and adult samples by digesting their tissues in a 5% chelex solution containing 0.4 proteinase K. Following digestion the samples were heated to denature proteins and DNA extracts were stored at 5 o C until all analyses were completed. Spin-column extraction kits from Machery-Nagel were used to purify genomic DNA from fish in test groups 3 -7. Adults and fry were genotyped at 10 or more loci (Table 2). The number of alleles per locus ranged from 5 in Ots-1 to 40 at Ots-100. Microsatellite DNA loci were amplified via the polymerase chain reaction (PCR) using fluorescent-labeled primers obtained from Applied Biosystems or Integrated DNA Technologies. Data were collected using an ABI-3100 Genetic Analyzer. Applied Biosystems Genemapper 3.0 software was used to collect, analyze and determine genotypes at each locus. Allele identification on sampled fry was attempted on all loci and fry had to be genotyped at six or more loci before they were assigned to a parent fish. A maximum likelihood procedure in Cervus 2.0 [START_REF] Marshall | Statistical confidence for likelihood-based paternity inference in natural populations[END_REF]) was used to infer parentoffspring relationships. The procedure uses allele frequency data to assign likelihoods to parentoffspring combinations and allows mismatching genotypic data to be evaluated concurrently with matching genotype data.

Data Analysis. Mean breeding success comparisons. Mean breeding success values for each male life history type spawning in a test group were calculated by using equation 2:

2

) x BrS A1 = (∑ BrS 1 + BrS 2 + . . . BrS n ) ⁄ (n)
Where x BrS A1 = the mean breeding success value in a test group for life history type A males BrS 1 = the breeding success of a single type A male in a test group n = the number of type A males in a test group These mean values represent the average percentage of fry in our 10% sample that individual males within a life history type had produced in a given test group. Three separate paired t-tests were conducted to see if mean individual breeding success of large anadromous males in the test groups differed from that achieved by jacks, yearling precocious, and sub yearling precocious males. Two additional paired t-tests compared the mean breeding success of jacks to yearling precocious and sub yearling precocious males. A final paired t-test compared the mean breeding success of yearling and sub yearling precocious males. Like all of our statistical analyses, these were two sided tests performed at an ∂ level of 0.05. The breeding success of jacks and the two precocious male life history types relative to large anadromous males was estimated by first determining an overall mean breeding success value for each male life history type in all the test groups which was calculated by using formula 3:

3) OBrS A = (∑ x BrS A1 + x BrS A2 + . . . x BrS An ) ⁄ (n A )
Where OBrS A = Overall mean breeding success for life history type A males,

x BrS A1 = Mean breeding success in test group 1 for life history type A males,

x BrS An = Mean breeding success in test group n for life history type A males Table 2.

Microsatellite loci and number of alleles scored for the pedigree analyses made on the Chinook salmon spawning in the artificial stream (From Schroder et al. 2008). n a = the number of test groups with life history type A males And then dividing that value by the overall mean breeding success value calculated for the large anadromous males, or:

4) Rel BrS A = (OBrS A ) ⁄ (OBrS LAM )
Where Rel BrS A = Breeding success of type A males relative to large anadromous males, OBrS A = Overall mean breeding success for life history type A males, and

OBrS LAM = Overall mean breeding success value for large anadromous males

Trait comparisons among the male life history types. Differences caused by male life history type on three traits linked to male breeding success, body length, spawning ground longevity and number of mates were examined. Body length comparisons were performed using a two-step process. First, Model I, 2-way ANOVAs were performed on the fork length data collected on males within a life history type. One fixed factor was test group origin while the other fixed factor was male origin, i.e. were the fish of wild or hatchery-origin. These analyses were performed to determine if all the length data obtained from males having the same life history could be pooled. A one-way ANOVA was then performed on the pooled data to assess whether the males with different life histories had different mean fork lengths at maturity. A post hoc Games-Howell Test was used to determine which male life history types had different fork lengths.

Longevity or the number of hrs a male resided in the artificial stream was calculated by subtracting the median time and date his test group entered the artificial stream from the time and date he was first observed dead. Mean longevity values for males representing each life history type within a test group were calculated by using the same approach shown in equation 2. That is, all the longevity data collected on a given male type were summed and then divided by the number of males of that type that were present in a test group. The resulting mean longevity values were used in three paired t-tests. In two of them, the longevity of large anadromous males was compared to that achieved by jacks or yearling precocious males. In the last paired t-test, longevity in jacks and yearling precocious males were compared. The longevity of sub yearling precocious males was not compared to the other male life history types because too few were recovered.

The pedigree analyses indicated the number of different females each male spawned with. The mean number of mates each male life history type spawned with was determined by using the method outlined in equation 2. In this instance, the number of females individual males having the same life history spawned with were summed and then divided by the number of those males in a test group. Six paired t-test were used to see if male life history type had an effect on the ability of individuals to acquire mates. In three of these tests, large anadromous males were compared with jack, yearling precocious, or sub yearling precocious males. In two other tests the mean mate numbers obtained by jacks were contrasted to those originating from yearling precocious or sub-yearling precocious males. The final paired t-test compared the mean mate numbers from yearling and sub yearling precocious males.

Factors affecting breeding success within a male life history type. The effect of body length or weight on breeding success in all four types of males was examined by using regression analyses. Data from each test group were analyzed separately. Seven regressions, one for each test group, were performed on large anadromous males. In these regressions body weight (log 10) was the independent variable and breeding success, after it had been normalized by the arcsine transformation, was the dependent variable. In all the other analyses, fork length rather than body weight was used as the independent variable. Six of these regressions were conducted on yearling precocious males, and three each were run on jacks and sub yearling precocious males. The P values obtained from the regressions performed on each male type were used in meta analyses referred to as combined probability tests (Sokal and Rholf 1995). These analyses were conducted to discover if body weight or length affected breeding success within a male type across multiple test groups.

The effect of longevity or the number of hrs males of each type resided in the artificial stream on breeding success was evaluated by using non-parametric methods. Kendall's Tau correlation analyses plus a combined probability test were used to look at this relationship in large anadromous males (Schroder In Press). Spearman Rank Correlations were used to examine the influence of longevity on breeding success in jacks and yearling precocious males. Not enough information on the longevity of sub yearling precocious males was gathered to assess the importance of longevity on their breeding success.

Regression analyses were used to ascertain the amount of variation in male breeding success that could be explained by the number of different females they spawned with. Data from the 2003 test group were used to produce four such regressions, one for each male type. Information from the 2003 test group was analyzed in this manner because it had the largest number of jacks, yearling and sub yearling precocious males of any our test groups (e.g. see Table 1). The slopes of the four regression lines were compared by using ANCOVA methods described in [START_REF] Zar | Biostatistical analysis[END_REF].

Results from the pedigree analyses were used to determine the number of fry produced by every female a male mated with. These raw count values were normalized by using the square root transformation and employed in One-Way ANOVAs that assessed whether male type affected the number of fry produced per mate. Seven of these ANOVAs were performed, one for each test group. When the overall ANOVA was found to be significant then a post hoc Tukey's Honestly-Significant-Difference Test was performed. The P values obtained from these post hoc tests were used in three combined probability tests that assessed whether there was an overall difference in fry produced per mate between large anadromous males and jacks, between large anadromous males and yearling precocious males, and between jacks and yearling precocious males.

Each test group had a different tertiary sex ratio (absolute number of males to females). Because all the fish placed into each test group were ripe at the time of entry and no new recruits were allowed to enter any of the test groups, the tertiary sex ratio was used to estimate male competition for females. Regression analyses were performed to determine how much variation in male breeding success (the dependent variable) could be explained by the tertiary sex ratios in the test groups (independent variable). Three regressions were performed; one for large anadromous males, another for jacks, and one for yearling precocious males.

Fry production by male life history types in the test groups. Four regression analyses, one for each male life history type, were performed to assess how much variation in fry paternity could be explained by how prevalent a male type was in a spawning population. In these analyses the percentage of the male spawning population representing a life history type was the independent variable. The dependent variable was the percentage of the fry population that had been fathered by that male life history. Prior to performing the regressions both percentages were normalized by using the arcsine transformation.

The effect of male intra-sexual competition for females on the proportion of fry produced by each male life history type in the test groups was also examined by regression. Four separate regressions were performed, one for each male life history type. The independent variable in these analyses was the tertiary sex ratio that was present in each test group. The dependent variable was the percentage of the fry population in a test group that a male life history type had produced. The arcsine transformation was used to normalize the percentage of the fry population in a test group that had been fathered by a male type. No data transformation was used on the tertiary sex ratios.

Results

Fry production from the test groups ranged from 18,960 in test group 2001 A, to 64,494 in test group 2003. A total of 30,683 fry were removed and set aside for DNA analysis and 13,779 were analyzed. Ninety-six percent or 13,216 of the analyzed fry were assigned to parental fish. The proportion of the total fry population originating from a test group that were assigned to parental fish averaged 4.3% and ranged from 1.8% in test group 2001 B, to 6.1% in test group 2004 (Schroder et al. In Press and Table 3).

Mean breeding success comparisons. Large anadromous males had greater mean breeding values than jacks (P <0.001), yearling precocious (P = 0.003), and sub yearling precocious males (P < 0.001). No difference was found in the breeding success values of jacks and yearling precocious males (P = 0.687) but jacks did have higher mean breeding success values than sub yearling precocious males (P = 0.011). Yearling, and sub yearling precocious males had similar mean breeding values (P = 0.152) (Table 4). The relative mean breeding success of jacks and yearling precocious males spawning in the test groups was about 26% of that achieved by the large Large anadromous males spawned with a greater number of females than jacks (P = 0.002) and sub yearling precocious males (P = 0.001) and jacks spawned with more females than sub yearling precocious males (P = 0.016). On the other hand, mate numbers for yearling On average, large anadromous males lived for shorter periods of time in the test groups than did jacks (P = 0.0275) and yearling precocious males (P = 0.026). No difference was observed between the longevity of yearling precocious males and jacks (P = 0.119). Large anadromous males had a mean longevity of 153 hrs; jacks had an overall longevity value of 173 hrs while yearling precocious males lived for an average of 221 hrs.

Trait comparisons among the male life history types.

Trait comparisons among the male life history types.

anadromous males. Sub yearling males had mean breeding values that were 2.85% of the large anadromous males (Figure 1). nadromous males. Sub yearling males had mean breeding values that were 2.85% of the large anadromous males (Figure 1).

Figure 1. Breeding success of jacks, yearling, and sub yearling precocious males spawning in the artificial stream relative to that achieved by large anadromous males.

Figure 1. Breeding success of jacks, yearling, and sub yearling precocious males spawning in the artificial stream relative to that achieved by large anadromous males.

The fixed factor two-way ANOVAs that examined whether male origin (first-generation hatchery or wild) or test group affected fork lengths in large anadromous males and jacks were non-significant and therefore length data within these two male types were pooled. Test group origin also had no effect on the fork lengths of yearling or sub yearling precocious males. However, hatchery origin yearling precocious males were significantly longer than wild yearling precocious males (P < 0.001). The one-way ANOVA and post hoc Games-Howell test that compared the fork lengths among males with different life histories indicated that there were dissimilarities in the mean fork lengths among all the life history types, with large anadromous males being the largest, followed by jacks, hatchery yearling precocious, wild yearling precocious, and wild sub yearling precocious males (Table 5). precocious males and large anadromous males (P = 0.098) were similar. And jacks and sub yearling precocious males also mated with a similar number of females (P = 0.540) as did yearling and sub yearling precocious males (P = 0.095) (Table 6).

Factors affecting breeding success within a male life history type. The effect of body size, weight for the large anadromous males, and fork length in the remaining life history types, on male breeding success was examined by using regression analyses. For the large anadromous males, regression coefficients ranged from 14 to 44% and averaged 26%. The regressions that examined the relationship between male breeding success and mate number in the 2003 test group showed that mate number had a significant positive effect on male breeding success in all four male life history types. For example, this factor explained 93%, 91%, 64%, and 87% of the variation in breeding success in large anadromous males, jacks, yearling precocious, and sub-yearling precocious males, respectively (Figure 3). The ANCOVA used to compare the slopes of these four regressions showed that large anadromous males obtained a greater gain in breeding success per mate than jacks (P = 0.035), yearling precocious (P <0.001) and sub yearling males (P = 0.029). The slope in the jack regression was greater than that

The regressions that examined the relationship between male breeding success and mate number in the 2003 test group showed that mate number had a significant positive effect on male breeding success in all four male life history types. For example, this factor explained 93%, 91%, 64%, and 87% of the variation in breeding success in large anadromous males, jacks, yearling precocious, and sub-yearling precocious males, respectively (Figure 3). The ANCOVA used to compare the slopes of these four regressions showed that large anadromous males obtained a greater gain in breeding success per mate than jacks (P = 0.035), yearling precocious (P <0.001) and sub yearling males (P = 0.029). The slope in the jack regression was greater than that

In three of the test groups, significant negative Kendall Tau correlations between spawning ground longevity and breeding success in large anadromous males were found. The combined probability test that examined the overall effect of longevity on breeding success showed that in our test groups, longevity in large anadromous males was negatively correlated to breeding success (P = 0.004) (Schroder et al. In Press). Conversely, both positive and negative relationships between these two variables were seen in jacks and yearling precocious males. And none of the Spearman Rank correlations that examined the relationship between male longevity in jacks or yearling precocious males and breeding success were significant.

In three of the test groups, significant negative Kendall Tau correlations between spawning ground longevity and breeding success in large anadromous males were found. The combined probability test that examined the overall effect of longevity on breeding success showed that in our test groups, longevity in large anadromous males was negatively correlated to breeding success (P = 0.004) (Schroder et al. In Press). Conversely, both positive and negative relationships between these two variables were seen in jacks and yearling precocious males. And none of the Spearman Rank correlations that examined the relationship between male longevity in jacks or yearling precocious males and breeding success were significant. probability test that examined the importance of this trait on breeding success in the large males showed that body weight was positively associated with breeding success (P < 0.001) (Schroder In Press). The combined probability tests that examined the importance of length on male breeding success in the other male life history types were all non-significant; the P values in these tests were 0.425 for jacks, 0.941 for yearling precocious, and 0.254 for sub yearling precocious males. The relationship observed between fork length and breeding success for yearling and sub yearling precocious males in the 2003 test group is shown in Figure 2.

robability test that examined the importance of this trait on breeding success in the large males showed that body weight was positively associated with breeding success (P < 0.001) (Schroder In Press). The combined probability tests that examined the importance of length on male breeding success in the other male life history types were all non-significant; the P values in these tests were 0.425 for jacks, 0.941 for yearling precocious, and 0.254 for sub yearling precocious males. The relationship observed between fork length and breeding success for yearling and sub yearling precocious males in the 2003 test group is shown in Figure 2. Relationship between male breeding success and number of mates in the four male life history types spawning in the 2003 test group.

observed for yearling precocious males (P = 0.021), however, no difference was detected between slopes in the jack and sub yearling regressions (P = 0.776). The slope of the sub yearling regression was steeper than the one observed for yearling precocious males (P = 0.049).

The effect that male life history had on mate number is shown in Figure 4, which depicts the average number of mates each male life history type obtained in the seven test groups. The mean number of mates each male life history type obtained in the seven test groups.

These above results, which were specific for test group 2003, prompted us to determine if the number of fry each mate provided to a male life history type varied in a similar manner across all the test groups. The three combined probability tests performed to evaluate whether there was an overall difference in fry produced per mate due to male life history type showed that large males gained more fry per mate than jacks (P = 0.008) and yearling precocious males (P <0.001). No difference, however, was observed in the number of fry produced per mate for jacks and yearling precocious males (P = 0.596). Although sub yearling precocious males were present in four of the test groups, too few of the fry they fathered were recovered to allow for similar comparisons.

As Tertiary sex ratios in the test groups increased mean breeding success in large anadromous males decreased (r 2 = 0.645, P = 0.029). Similar, but non-significant decreases in mean breeding values occurred in jacks (r 2 = 0.411, P = 0.121) and yearling precocious males (r 2 = 0.432, P = 0.156) (Figure 5).

Overall fry production by male life history type. If each male life history type were equally successful at producing offspring, then there should be a one-to-one relationship between the percentage of a male type present on a spawning ground and the percentage of the fry population 0.00 1.00 2.00 3.00 4.00 5.00 6.00

1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

Tertiary Sex Ratio Mean Arcsine Breeding Success

Lg Anadromous Jacks Yr Precocious Figure 5.

The effect of tertiary sex ratios on the mean breeding success values of large anadromous, jacks, and yearling precocious males spawning in the artificial stream.

originating from that type of male. The relationship between the occurrence of male life history types spawning in our test groups and the paternal origin of fry produced from those groups is shown in Figure 6. In every test group, large anadromous males produced about 89% of the fry regardless of their relative occurrence. No relationship was found between the percentage of large males in the test groups and the percentage of fry that life history type produced (r 2 = 0.334, P = 0.174). Similarly, the percentage of jacks, yearling and sub yearling precocious males in our test groups did not influence the percentage of fry they were able to produce. The regression analyses performed to examine the relationship between the abundance of a male type in the test groups and the corresponding proportion of fry they produced were all non-significant. The regression coefficients equaled 0.298 (P = 0.205) for jacks, 0.285 (P = 0.275) for yearling precocious, and 0.068 (P = 0.740) for sub yearling precocious males. The effect that tertiary sex ratios had on fry paternity in the test groups was examined in a similar fashion and we found that fry paternity was not affected by tertiary sex ratios in the test groups (Figure 7). The regressions that examined this relationship were all non-significant. In this case, regression coefficients equaled 0.002 (P = 0.919) for large males, 0.063 (P = 0.587) for jacks, 0.228 (P = 0.338) for yearling precocious, and 0.003 (P = 0.946) for sub yearling precocious males. .00 Figure 6.

The occurrence of male life history types in the test groups vs. the proportion of fry they fathered. The null line equals the proportion of fry each male type would be expected to produce if each life history strategy had an equal ability to produce offspring.

array of tertiary sex ratios and different mixtures of males extant in our test groups, large males consistently produced around 89% of the offspring and on average jacks produced 3.2%, yearling precocious 7.4% and sub yearling males 0.8%.

Discussion

Spring or stream type Chinook salmon [START_REF] Healey | Life history of Chinook salmon[END_REF] populations probably possess the most diverse array of male life history strategies in semelparous Oncorhynchids. Some males reach maturation after just 7 to 8 or 19 to 20 months of freshwater growth (sub yearling and yearling precocious males) while others utilize an anadromous life history strategy and reach maturation after spending one or more years in the ocean (jacks and large anadromous males) before returning to freshwater to spawn. In our experimental setting, large anadromous males achieved significantly higher mean individual breeding success values than the other three male life history phenotypes examined. Jacks and yearling precocious males had comparable mean breeding success values while sub yearling precocious males generally had the lowest breeding success values.

Significant differences existed among the male life history types in their body sizes, spawning ground longevity, and their ability to fertilize eggs from multiple females. Of these factors, body The effect of tertiary sex ratios in the test groups on the proportion of fry fathered by each male life history type.

size was probably the proximate factor largely responsible for the detected differences in breeding success. Large anadromous males were on average over 200 mm longer than jacks, the next largest male life history type, and obviously had even greater size advantages over the precocious males (Table 5). Their superior size allowed them to be socially dominant over the other three male life history types and provided them with a greater number of opportunities to spawn with females.

Although important, the ability to spawn with numerous females only partially explained why differences existed among the male life history types in their mean individual breeding success values. The number of fry produced per mate was another critical factor. In our test groups, for example, yearling precocious males and large anadromous males spawned with a similar number of females (see Table 6). Yet, the average number of fry produced per mate in these two life history types was significantly different (see Figure 3). In the 2003 test group, large anadromous males produced more than 600 fry per female they mated with however; yearling precocious males in this test group fathered one-sixth as many fry (101) per mate. The ability of large anadromous males to remain in close proximity to sexually active females, their success at fertilizing most of the eggs deposited by females either through sperm competition or by delaying sperm release by competitors, plus a proclivity for salmonid females to prefer relatively large mates [START_REF] Taborsky | Sperm competition in fish: 'bourgeois' males and parasitic spawning[END_REF][START_REF] Blanchfield | The cost of peripheral males in brook trout mating system[END_REF] were apparently responsible for their higher breeding success values.

The effects that body size, longevity, and different tertiary sex ratios had on individual breeding success values helped expose the selection pressures males in each life history type experienced while spawning. Large anadromous males mainly competed among themselves for access to females and in this life history type; body size was positively associated with social dominance and breeding success (Schroder et al. In Press). In our study, body size did not influence the breeding success of jacks and the two precocious male life history types. Males having these life histories established themselves next to courted females and were often exposed to aggressive attacks by the female and courting male. Yet, as male densities around a courting pair increase it becomes more difficult for any male to exclude all his potential rivals. In brook trout, for example, per capita aggressive interactions among males surrounding an active female decreased as male numbers increased [START_REF] Blanchfield | The cost of peripheral males in brook trout mating system[END_REF]. The inability to exclude multiple rivals leads to scramble competition among peripheral males and under this circumstance body size is not as important as being in close proximity when spawning events occur [START_REF] Jones | The influence of male parr size and mate competition on fertilization success and effective population size in Atlantic salmon[END_REF].

The negative relationship between longevity and male breeding success in large anadromous males was probably due to energy expenditures. Large anadromous males expend a considerable amount of effort on agonistic interactions and courtship. In our test groups, large anadromous males with high breeding success values engaged in more attacks and courting activities than those who had lower breeding success values (Schroder et al. In Press). Conversely, spring Chinook jacks and precocious males engage in less courtship and agonistic behavior (Schroder unpublished data); instead they spend time and energy avoiding attacks and on waiting behind pairs for spawning events. Because their breeding success is often determined by the outcomes of scramble competition, we speculate that energy expenditures expended by these males toward excluding rivals and staying in close proximity to a sexually active female don't affect their breeding success as strongly as they do in large anadromous males. Support for that speculation can be found in the relationship between tertiary sex ratios in the test groups and how this factor influenced individual breeding success values in the various male life history types. A negative relationship occurred between these two variables within each male life history type. However, it was only significant in large anadromous males; suggesting that intra-sexual competition among males possessing the same life history type is probably more important for large anadromous males than it is for the other male life history types.

Males typically mating at a disadvantage, such as jacks and precocious males are theoretically expected to employ behavioral, physiological or morphological strategies designed to offset this disadvantage [START_REF] Gage | Effects of alternative male mating strategies on characteristics of sperm production in the Atlantic salmon (Salmo salar): theoretical and empirical investigations[END_REF][START_REF] Taborsky | Sperm competition in fish: 'bourgeois' males and parasitic spawning[END_REF]. Precocious males and jacks did have longer spawning ground longevities than large anadromous males. Relatively long spawning ground residency may be a reproductive adaptation by jacks and precocious males to increase their likelihood of participating in spawning events. Additionally, [START_REF] Mjolnerod | Mate and sperm competition during multiple-male spawning of Atlantic salmon[END_REF] found that the spawning history of large anadromous Atlantic salmon had an effect on their ability to fertilize eggs. Individuals that had spawned extensively prior to a spawning event released fewer sperm. This meant that secondary males associated with such a spawning were able to achieve high breeding success values even though they had been socially dominated by a large anadromous male. Again, relatively long spawning ground residency may increase the probability of jacks and precocious males participating in a spawning of this type. The possible reproductive advantage associated with increased longevity was not fully evaluated in our experiment because additional males and females were not introduced into our test groups over time. Consequently, females were available to all the males over a shorter time period than they would have been under natural conditions.

Other reproductive adaptations observed in precocious males include higher gonadosomatic indices (GSI) than anadromous males [START_REF] Gage | Effects of alternative male mating strategies on characteristics of sperm production in the Atlantic salmon (Salmo salar): theoretical and empirical investigations[END_REF][START_REF] Vladic | Sperm quality in the alternative reproductive tactics of Atlantic salmon: the importance of the loaded raffle mechanism[END_REF][START_REF] Koseki | Differential energy allocation of alternative male tactics in masu salmon (Oncorhynchus masou)[END_REF]. And when body size is adjusted for, precocious males also produce greater quantities of sperm that are more motile and live longer (but see [START_REF] Mjolnerod | Mate and sperm competition during multiple-male spawning of Atlantic salmon[END_REF]) than those produced by anadromous males [START_REF] Gage | Effects of alternative male mating strategies on characteristics of sperm production in the Atlantic salmon (Salmo salar): theoretical and empirical investigations[END_REF][START_REF] Vladic | Sperm quality in the alternative reproductive tactics of Atlantic salmon: the importance of the loaded raffle mechanism[END_REF]. Conversely, when [START_REF] Hoysak | Raffles, roles, and outcome of sperm competition in sockeye salmon[END_REF] examined sperm competition between sockeye jacks (3-yr-old fish) and large anadromous males (5 yr-old fish) they found no differences in sperm quality. Instead they speculate that the relatively small size of jack sockeye allows them to gain closer access to females during spawning and thus enhances their fertilization success. Precocious spring Chinook have significantly higher GSI values than those found in large anadromous males (Knudsen and Schroder unpublished data). No evaluations, however, have been made on sperm quality in these fish to see if their sperm and milt possess traits similar to those found in other precocious salmonid males.

Even with greater investments in relative gonad size and possible enhancements in sperm quality large anadromous males consistently produced about 90% of the fry generated from each of our test groups. Similar results in other salmonid species have been found. In an 8 km long Scottish river containing 34 to 66 anadromous Atlantic salmon females 38 to 43 large anadromous males competed with an estimated two to three thousand precocious males for fertilization opportunities. In this population large anadromous males represented just 1 to 2% of the male population yet they still fertilized about 50% of the eggs that were deposited [START_REF] Taggart | Spawning success in Atlantic salmon (Salmo salar L.)[END_REF]. Additionally, [START_REF] Hutchings | Mating success of alternative maturation phenotypes in male Atlantic salmon, Salmo salar[END_REF] established populations in simulated streams where large anadromous male Atlantic salmon represented 5% of the spawning males. In their study, these individuals fertilized approximately 75% of the deposited eggs. In another study using Atlantic salmon spawning in an artificial stream, 16% of the males were large anadromous individuals while the remaining 84% were precocious parr; in this setting anadromous males fertilized between 63 and 77% of the deposited eggs [START_REF] Jones | Individual variation in Atlantic salmon fertilization success: implications for effective population size[END_REF]. In a natural spawning population of brook trout, [START_REF] Blanchfield | The cost of peripheral males in brook trout mating system[END_REF] and [START_REF] Blanchfield | Breeding success of male brook trout (Salvelinus fontinalis) in the wild[END_REF] found that male size and a female preference for large males meant that over 90% of the fry produced originated from large males even though there were numerous smaller "peripheral" males on the spawning grounds. In aggregate our study and those cited above indicate that large anadromous males produce a disproportionate number of fry when salmonids are allowed to spawn naturally.

In our introduction, we posed some questions related to the potential genetic effects that the occurrence of alternative male life history types may have on natural spawning populations of spring Chinook. In this report we provide preliminary information that can be used to address two of them, the relative success of each male history type and how that information plus ancillary data from other studies can be used to guide broodstock composition in salmonid recovery programs. The proportion of offspring produced by jacks and precocious males in spawning populations appears to be influenced by the relative abundance of large anadromous males. When large anadromous males are rare these life history types can achieve significant breeding success values. Although not specifically examined, we further speculate that if operational sex ratios (OSRs) are less than one, then breeding success values for jacks and precocious males will also be high. In most breeding populations of salmon, however, OSRs will be greater than one (i.e. more than one male will compete for each sexually active female) and male competition for females will therefore be intense. Under these conditions, large anadromous males appear to be able to fertilize a large majority of the eggs that are deposited. Consequently, if the goal of a spring Chinook recovery program is to mimic the genetic diversity present in a self-sustaining population then broodstock from all male life history types should be employed. What we found suggests that about 90% of the eggs should be fertilized by large anadromous males and the remaining 10% should be fertilized by jacks and precocious males.

Clearly refinements to these provisional suggestions will occur but given what we and others have observed they seem like a reasonable place to start.

Figure 2 .

 2 Figure 2. The effect of fork length on the breeding success values of precocious males spawning in the 2003 test group. Breeding success values have been normalized by using the arcsine transformation.

Figure 2 .

 2 Figure 2. The effect of fork length on the breeding success values of precocious males spawning in the 2003 test group. Breeding success values have been normalized by using the arcsine transformation.

  Figure 3.Relationship between male breeding success and number of mates in the four male life history types spawning in the 2003 test group.

  Figure 4.The mean number of mates each male life history type obtained in the seven test groups.

  Figure 7.The effect of tertiary sex ratios in the test groups on the proportion of fry fathered by each male life history type.

Table 1 .

 1 The number, weight, tertiary sex ratio, and percentage of the male population consisting of large anadromous fish present in the test groups placed into the artificial stream from2001 -2005.

	Test				Weight-	Mean	Tertiary	% Of Males In Test Group Using
	Group	Sex	Type	No.	Range (g)	Weight	Sex Ratio	The Large Anadromous Strategy
	2001 A Female	Large Anadromous a	16	2,100 -6,570	4,315
		Male	Large Anadromous	21	2,030 -7,460	4,234	1.5	87.5%
			Jack b	3	1,422 -1,527	1,467
			Yr-Precocious c	0
			Sub-Yr Precocious d	0
	2001 B	Female	Large Anadromous	17	2,870 -4,880	3,838
		Male	Large Anadromous	18	1,780 -5,250	3,552	1.76	60.0%
			Jack	2	1,109 -1,381	1,245
			Yr-Precocious	5	76 -155	120
			Sub-Yr Precocious	5	5 -9	7
	2002A	Female	Large Anadromous	22	2,930 -6,160	4,640
		Male	Large Anadromous	22	2,800 -5,560	3,840	1.36	73.3%
			Jack	2	849 -921	885
			Yr-Precocious	6	34 -96	65
			Sub-Yr Precocious	0
	2002B	Female	Large Anadromous	17	3,270 -5,100	4,055
		Male	Large Anadromous	16	2,720 -6,400	4,138	1.41	66.7%
			Jack	2	1,227 -1,300	1,264
			Yr-Precocious	6	35 -118	84
			Sub-Yr Precocious	0

Table 3 .

 3 Production of fry from each test group and the numbers sampled and assigned to parental fish via DNA-based pedigree analyses in each of the seven test groups spawning in the artificial stream(From Schroder et al. In Press).

						% Of Total Fry
						Population
					No. Of Fry Assigned	Assigned To
	Test Group	No. Of Fry Produced	No. Of Fry Sampled No. Of Fry Analyzed	To Parental Fish	Parents
	2001 A	18,960	1,936	1,014	991	5.2%
	2001 B	42,263	3,977	876	780	1.8%
	2002 A	45,889	4,635	1,581	1,566	3.4%
	2002 B	37,141	3,897	1,308	1,264	3.4%
	2003	64,494	6,522	3,000	2,750	4.3%
	2004	47,140	4,784	3,000	2,892	6.1%
	2005	49,552	4,932	3,000	2,973	6.0%
	Totals	305,439	30,683	13,779	13,216	4.3%

Table 4 .

 4 Results of the paired t-tests used to compare the mean breeding success values of large anadromous males, jacks, yearling and sub yearling precocious males spawning in the test groups.

		x Breeding
	Comparison	Value a	N b	P value	Conclusion
	Large Anadromous vs. Jacks	2.83% vs. 0.77%	7	0.00006 Reject H o : Large Anadromous > Jacks
	Large Anadromous vs. Yr Precocious	2.93% vs. 0.66	6	0.00256 Reject H o: Large Anadromous > Yr Precocious
	Large Anadromous vs. Sub Yr	2.61% vs. 0.02%	4	0.00071 Reject H o : Large Anadromous > Sub Yr Precocious
	Precocious
	Jacks vs. Yr Precocious	0.81% vs. 0.66%	6	0.68673 Fail to reject H o
	Jacks vs. Sub Yearling Precocious	0.61% vs. 0.02	4	0.01055 Reject H o : Jacks > Sub Yr Precocious
	Yr Precocious vs. Sub Yr Precocious 0.44% vs. 0.02%	4	0.15240 Fail to Reject H o
	a Mean breeding values in the test groups where both types of males being compared were present
	b Number of mean values compared in the paired t-tests

Table 5 .

 5 Results of the post hoc Games-Howell Test that compared the fork lengths of spring Chinook salmon males having different life histories that were placed into the artificial stream.

	Breeding Success Relative	To Large Anadromous Males	0% 25% 50% 75% 100%
			Lg Anadromous	Jack	Yr P Male	Sub Yr P Male
				Male Life History Type

Table 6 .

 6 Results of the paired t-tests that compared the mean number of mates each male life history type obtained in the test groups. Mean number of mates in the test groups where both types of males being compared were present b Number of mean values compared in the paired t-tests

	The combined

a

Of Fry Production Arcsine % Of Population Made Up Of A Male Type

	90.00
	80.00
	70.00
	60.00
	20.00 30.00 40.00 50.00 Arcsine % Null Lg Anadromous Jac Yr k P's Sub Yr P's
	10.00
	0
	0.00	10.00	20.00	30.00	40.00	50.00	60.00	70.00	80.00
								Thus, under the

Acknowledgements

This project would not have been possible without considerable assistance from many individuals. We thank Charles Strom, Vernon Bogar, DJ Brownlee, Greg Strom, Simon Goudy, Quinn Jones, Annie Jo Parrish, Jason Rau, and Dan Barrett for their help at the Cle Elum Supplementation Research Facility. Mark Johnston, Joe Hoptowit, Gerry Lewis, Ray Decoteau, and Antoine Marek collected and transported all the fish used in our experiments from the Roza Adult Monitoring Facility to the CESRF. Other Yakama Nation staff, Bill Bosch, David Lind, and Paul Huffman also supported us in numerous ways. Jen Scott, Mike Hamlin, Anthony Fritts, Gene Sanborn, Jordan Vandal, Kurt Fresh, Eric Volk, Gabriel Temple, Timothy Webster, Charity Davidson, Chris Johnson and Molly Kelly all from the Washington Department of Fish and Wildlife helped make observations, assisted us while we cleaned and repaired the artificial stream, or were involved in the capture and sampling of fry. Personnel in WDFW's Molecular Genetics Laboratory, Jim Shaklee, Alice Frye, Jennifer Von Bargen, Norm Switzler, Cherril Bowman, Mo Small, Janet Loxterman, and Denise Hawkins helped with pedigree analyses. We extend our appreciation to the Bonneville Power Administration (BPA) for funding this work as part of the Yakima/Klickitat Fisheries Project (project 1995-063-25). David Byrnes and Patty Smith of BPA provided administrative support for the project.

This report covers one of the many topics under the Yakima/Klickitat Fisheries Project's Monitoring and Evaluation Program (YKFPME). The YKFPME is funded under two BPA contracts, one for the Yakama Nation and the other for the Washington Department of Fish and Wildlife (Contract number 00037649, Project Number 1995-063-25). A comprehensive summary report for all of the monitoring and evaluation topics will be submitted after all of the topical reports are completed. This approach to reporting enhances the ability of people to get the information they want, enhances timely reporting of results, and provides a condensed synthesis of the whole YKFPME. The current report was completed by the Washington Department of Fish and Wildlife.


D Gálvez-López

B Quéméner

M F Devaux

D Bertrand

F Laurens

M Lahaye

).

Introduction

Apple texture relies on turgor, histology and cell wall chemistry. As a prerequisite in a study aiming at identifying genetic markers controlling these components, the variability of mechanical, histological and cell wall chemical characteristics was quantified in an apple progeny of 85 individuals.

Materials and methods

A progeny of 85 individuals was harvested in 2007 and stored for 2 months at 1.5 °C then analyzed.

-Sensory analyses parameters were scaled from 1 to 5 for firmness, crispness, juiciness, melting in the mouth, grains, fibers and mealiness, following the methods set up in the frame of the EU-Hidras project (Kouassi et al, 2009).

-Penetrometry and compression mechanical analyses were realized in order to collect the force, linear distance, dispersion standard, stress and others force data's (Camps et al., 2005).

-Histological analysis was done using macrovision acquisition system and image analysis for estimate Mean cell size, cellular spaces and size distribution (Devaux et al., 2008).

-Cell wall polysaccharides sugar composition was quantified by gas chromatography and by colorimetric methods uronic acid (Quemener et al., 2007)

Conclusion and Perspectives:

Cell wall pheno-/chemotyping data and instrumental measures of texture were compared to sensory evaluation of an apple progeny. All parameters measured varied in the progeny. Compression and histology data were related to crunchiness and most likely reflected a dominant contribution of cellular turgor pressure on texture. Further work will look for more specific cell wall contributions to apple texture. The identification of genetic markers controlling texture components (QTL mapping) is underway. Variability of cell wall determinants of texture in an apple progeny Biochemical results showed that cell wall was slightly richer in uronic acids than neutral sugars (Fig. 2a). High variation was observed for uronic acids in the progeny while other major sugar contents were homogenously distributed (Fig. 2b). The enzymatic fingerprints by mass spectrometry (Fig. 2c) showed a high variation in hemicelluloses fine structure for xyloglucans fragments (Fig. 2d).

Principal component analysis of all data (Fig. 3) showed a particular relationship between histological data, compression data and crunchy (¨croquant¨) perception.

  with previous contaminating starch measures by amylolysis (McCleary et al., 1997). -Hemicelluloses fine structure was determinate by combining g l u c a n a s e d e g r a d a t i o n a n d M A L D I -T O F M S o f oligosaccharides produced (Quemener et al., 2007). -Statistical analyses of the results were performed on MATLAB and R softwares.

Fig. 1 .Fig. 3 .

 13 Fig. 1. Variability in sensory perception (a), instrumental texture measures (b & c) and histology (d).

Acknowledgements

• This publication was carried out with the financial support from the Commission of the European Communities (IsaFruit contract N° FP6-FOOD 016279-2), Directorate-General Research -Food Quality and Safety Programme. It does not necessarily reflect the Commission's views and in no way anticipates its future policy in this area. Its content is the sole responsibility of the publishers. • CONACYT-MEXICO (Scholarship for PhD student: Gálvez-López D). • INRA Angers-Nantes people: R Robic, M Lormeau, Ph Guerif, R Looten, S Daniel, MJ Crépeau, J Vigouroux, C Surlève, D Ropartz.


Colette Grillet

Dominique Sionneau

Emmanuel Albina

Review on ring trials organised within EPIZONE and perspectives for standardisation

I -RECOMMENDED SET OF SAMPLES:

► Number of samples: the risk of not detecting an anomaly is directly determined by the number of samples included in the set. The table shows the risk interval according to the number of samples in the set (confidence interval of 95 %):

A compromise must be found between a lower risk and a reasonable number of samples: our suggestion would be 20-25 samples.

► Sample status : since it is impossible to assess the specificity of a participant using a low number of negative samples, we recommend to include more positive and positive limits in the set. Only few negative samples are to be included.

► Codification: the samples should be randomly codified.

► Volume :

The volume of samples should be limited to three repetition of the test.

Summary:

Theme 4 of EPIZONE is dedicated to the development and standardisation of the diagnostic tools for epizootic diseases. To achieve this objective, regular ring trials are organised within the network. However, to our knowledge, they are designed and organised by different laboratories with no established standards. In agreement with a recent report on the Euroepan network of excellence ( 1

III-REPORTING

The final report should include an overall analysis of the ring trial with an anonymous result reporting and a statement on conforming and non conforming results to individual laboratory participant.

Number of samples

  ), we believe that EPIZONE could somehow play a role in improving quality, particularly in the present case, for the standardisation of ring trials designed for the comparison and validation of diagnostic methods. As a starting point, we decided to review three European ring trials into which we took part (AIV/RRT-PCR, BT/ELISA & PCR(2), ASF/ELISA & PCR), one that we are currently organising for EPIZONE (PPR/ELISA & PCR) and three others that we have organised in 2008 either at the international, European or national level (AIV/RRT-PCR, Culicoides/PCR, BT/ELISA). From this evaluation, it is possible to make suggestions for harmonisation. EPIZONE could promote the definition of guidelines for the organisation of such ring trials, taking into account the international standards available (3, 4). Key-words: ring trials, quality assurance, standards, diagnostics. According to the OIE and ISO guidelines for Laboratory Proficiency Testing (3, 4), the criteria to be assessed should be: ► Sensitivity: capacity to detect a true positive sample. ► Specificity: capacity to score as negative, a true negative sample. ► Detectability: capacity to detect a minimum quantity of the target ► Dose/Effect response: for quantitative results only, capacity to generate a linear curve for serial dilutions.

►

  Repeatability: capacity to generate identical results on repetitions of the same sample.PROPOSITIONSStandardisation of the ring trials within EPIZONE could be based of a recommended set of samples, a list of criteria to be evaluated and a template for the final result reporting.