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INTRODUCTION AND BACKGROUND
Once again the National Marine Fisheries Service (NMFS) and the United States Fish and Wildlife Service(USFWS) (hereafter referred to as “the Services”) are under pressure from special interest groups to impose an “endangered” listing under the Endangered Species Act (ESA) on Atlantic salmon in a collection of rivers in Maine.  While previous listing attempts have failed, the political climate has once again brought the ESA to the forefront of the Atlantic salmon restoration debate.  The Services propose to list what they call “wild” Atlantic salmon in the following rivers within a proposed “Gulf of Maine Distinct Population Segment” collectively as endangered: the Sheepscot, the Ducktrap, the Narraguagus, the Pleasant, the Machias, the East Machias, the Dennys, and the Cove Brook tributary to the Penobscot River.  This rests on the assumption these rivers collectively represent a “distinct population segment” (DPS) as described by their “Policy Regarding the Recognition of Distinct Vertebrate Population” (FR 1996).  We feel this is an incorrect assumption that defies both common sense and the best scientific information available.  We also feel the Services should not succumb to political pressure from special interest groups seeking to further their political agendas and follow their own guidelines and criteria which do not support such a listing.

 The Endangered Species Act requires that “the best scientific information available” be used when determining if a species is endangered or threatened.  The 96th Congress instructed the Secretary of the Interior to use the authority to list a DPS “…sparingly and only when the biological evidence indicates that such action is warranted” (FR 1996).

 The NMFS developed a policy in 1991 to determine the existence of a “distinct population segment” (DPS) in pacific salmonids.  For a stock to qualify as a DPS, it had to be an “evolutionary significant unit” (ESU), and had to satisfy two criteria: 1) it must be substantially reproductively isolated from other conspecific populations, and 2) it must represent an important component in the evolutionary legacy of the species (FR 1996).

 In 1996, both the USFWS and NMFS adopted a more liberal joint interpretation of how to determine what constitutes a DPS, which continues to be in use today.  Three elements are considered when determining a DPS under the ESA, with the second only being considered if the first is proven, and the third being considered only if the first two are proven.  The three elements to be considered are (FR 1996):

1. Discreteness of the population segment in relation to the remainder of the species to which it belongs;

2. The significance of the population segment to the species to which it belongs; and

3. The population segment's conservation status in relation to the Act's standards for listing (i.e., is the                         population segment, when treated as if it were a species, endangered or threatened?).

In order to be considered discrete, a potential DPS must satisfy either of two conditions (FR 1996):

1. It is markedly separated from other populations of the same taxon as a consequence of physical, physiological, ecological, or behavioral factors. Quantitative measures of genetic or morphological discontinuity may provide evidence of this separation.

2. It is delimited by international governmental boundaries within which differences in control of exploitation, management of habitat, conservation status, or regulatory mechanisms exist that are significant in light of section 4(a)(1)(D) of the Act.

 If a potential DPS is determined to be discrete, its evolutionary significance is then considered.  Significance is defined as the subpopulation being ecologically persistent, having persisted outside of its historic range, or exhibiting unique genetic characteristics, which may include (but is not limited to) the following factors (FR 1996):

1. Persistence of the discrete population segment in an ecological setting unusual or unique for the taxon,

2. Evidence that loss of the discrete population segment would result in a significant gap in the range of a taxon,

3. Evidence that the discrete population segment represents the only surviving natural occurrence of a taxon that may be more abundant elsewhere as an introduced population outside its historic range, or

4. Evidence that the discrete population segment differs markedly from other populations of the species in its genetic characteristics.  Because precise circumstances are likely to vary considerably from case to case, it is not possible to describe prospectively all the classes of information that might bear on the biological and ecological importance of a discrete population segment.

 If a potential DPS is determined to be both discrete and significant, its conservation status is then considered under guidelines set forth by the ESA.

 In this paper, we first examine the geographical area in question to help determine if the proposed “Gulf of Maine DPS” is discrete using the second determining factor from above.  We then look at the first determining factor for discreteness, starting with a discussion on defining of a “wild” Atlantic salmon.  We then discuss the concept of “river-specific” stocks, while considering conceptual issues suggesting river-specificity to be virtually non-existent.  We then proceed to discuss the currently available molecular genetics data and how it relates to the discreteness of the proposed DPS in question.  Finally, we discuss other listing issues related to the genetic data available.
 
 

ASSESSING THE DISCRETENESS OF THE PROPOSED DPS

GEOGRAPHIC STRUCTURE
 One need only to look at a map to see that the supposed DPS is not geographically continuous or delimited by international governmental boundaries, and is separated by other salmon rivers not included, most notably the main stem of the Penobscot.  The following map, adapted from the “Maine Salmon Rivers” map on the Maine Atlantic Salmon Commission’s (ASC)  web page (http://www.state.me.us/asa/asrivers.html), illustrates this point very well.  The DPS rivers are in white, while the rest of the salmon rivers in Maine that are not being considered for listing are in black.

The proposed “Gulf of Maine DPS includes all of the salmon rivers “from the Kennebec River downstream of Edwards Dam northward to the mouth of the St.Croix River” (FR 1999), including the five Downeast rivers (Dennys, East Machias, Machias, Pleasant, and Narraguagus), and the Ducktrap, the Sheepscot, and Cove Brook – a tributary to the Penobscot.  However, it does not include the Kennebec, the main stem of the Penobscot, the Union, the St. Croix, and many other smaller rivers that had historical runs in the past because the salmon in these rivers are not considered to be “wild” by the Services, even though they are within the geographical boundaries.  This is important, because the documented returns in the DPS are low enough to be considered endangered only if the returns from the rest of the rivers within the proposed DPS are left out.

According to the Services, the “total documented natural (wild & stocked fry) Gulf of Maine DPS spawner returns to the rivers of the Gulf of Maine DPS range for the past 5 years are: 1995 (83); 1996 (74); 1997 (35); 1998 (23); 1999 (29)” (FR 1999).  Although this sounds as if only 29 salmon returned to the proposed DPS in 1999, the actual recorded number of returns to rivers within the geographical boundaries of the “Gulf of Maine DPS” as reported by the Atlantic Salmon Commission on their web page, “1999 TRAP/ROD CATCH STATISTICS” (http://www.state.me.us/asa/catchstats.html), is 1095 salmon.  The difference results from the inclusion of the returns from the Penobscot and other rivers within the geographical boundaries into the ASC’s tally.  This is a far cry from the 29 salmon the Services report, but they justify this number by saying that the salmon in the other rivers are not “wild” and are genetically different from “wild” salmon.

DEFINING A “WILD” ATLANTIC SALMON
 The next obvious question in this discussion is, “what exactly is a “wild” Atlantic salmon?”  The definition given to the authors by Ed Baum of the Atlantic Salmon Commission, came from the International Council for the Exploration of the Sea (ICES) and the North Atlantic Salmon Conservation Organization (NASCO).  A “wild” Atlantic salmon, according these respected organizations, is a salmon “that has spent its entire life cycle in the natural environment, with its parents having done the same (i.e., 2 generations in the wild without human interference)” (Ed Baum, pers. comm.).

This is an interesting definition, as its interpretation raises some interesting questions.  Using this definition, an aquaculture escapee of European origin could spawn in a Maine river, and after two generations, provided they didn’t run into any “human interference,” its progeny would be considered “wild.”  It’s hard to imagine that any “river-specific” genetic adaptations could evolve in two generations that would make the progeny more genetically fit than any other escapees’ progeny.  Yet, the progeny would now be considered a “wild” Atlantic salmon using the definition we have been given.

Clearly, using such an artificial definition of “wild” leads to all sorts of interpretation problems, including those encountered when considering which rivers in Maine contain “wild” Atlantic salmon.  It can hardly be said that any salmon in Maine rivers can be “wild” using this definition, taking into account the massive amount of past stocking and other human interference salmon have endured.  After all, using this definition, how can the “river-specific” salmon held in Federal hatcheries be considered “wild?”  Aren’t humans interfering them with?  In fact, using this definition, one could easily argue that a Maine “wild” salmon is no more “wild” than a Maine “wild” blueberry.  Thus, it must logically be concluded that calling salmon in one river more “wild” than those in another river is an artificial description, unless it is backed by scientific information that shows they are indeed “wild.”

In addition, no one can currently distinguish a fish of hatchery origin from a wild-spawned fish, making it impossible to claim a river population as a whole is “wild” since the Downeast rivers are included in the stock enhancement program.  To claim a river as a whole is “wild” makes the assumption that all hatchery-produced and subsequently stocked fish perish before reproducing.  If the Services claim this is the case, then a century’s worth of stock enhancement programs are a dismal failure.

Nonetheless, the Services claim to have information saying that “river-specific” adaptations have allowed the “wild” rivers in the proposed DPS to retain their genetic integrity, despite natural straying and past stocking efforts, while the other rivers within the geographical borders of the “Gulf of Maine DPS” have not.  As it will be shown, this claim on their part is nonsense.

“RIVER SPECIFIC” STOCKS
 Much discussion in the debate over listing the proposed DPS focuses on, and results from, the idea of “river-specific” stocks existing in Maine rivers.  Despite acknowledging that there is considerable debate over the “river-specific” stocking program in the 1999 Status Review, the 11-17-99 Federal Register notice calls it an “essential component of the strategy to rebuild salmon stocks in the DPS” (FR 1999).  While each river breeding and supplementation program is maintained assuming “river specificity”, several of these rivers have been pooled together on paper into one geographically inconsistent DPS.  River specificity in Atlantic salmon can occur if (and only if) sub-populations have been significantly reproductively isolated from each other for many generations and if the selective forces are strong enough to overcome the forces of genetic drift, migration, bottlenecks, etc.  The selective forces in a particular river must also favor significantly different phenotypes (and ultimately genotypes) from those in nearby rivers.

REPRODUCTIVE ISOLATION
 Anadromous Atlantic salmon populations in Maine are known to not be reproductively isolated (King and Smith 1994; May et al. 1994; Schill and Walker 1994; King et al. 1999) and have been extensively mixed due to past stocking practices (Baum 1995; Kornfield et al. 1995). These findings can be partially explained by examining the past stocking records and resulting stock introgression on each of these rivers.  Each of the seven rivers originally considered “wild” under the previous “threatened” status proposal by the Services are listed with the introduced stocks in parentheses: Sheepscot (various Maine and Canadian stocks); Ducktrap (Maine stocks); Narraguagus (Penobscot and Canadian stocks); Pleasant (Penobscot, Union, Narraguagus, Machias, and various Canadian stocks); Machias (Penobscot, Union, and Canadian stocks), East Machias (Penobscot and Union stocks); Dennys (Penobscot, Union, and Canadian stocks) (USFWS 1995b).  The use of the Penobscot “strain” alone has introduced genetic material from Downeast (Machias, Narraguagus, Orland) residual stocks and various Canadian stocks (Kornfield et al. 1995; USFWS 1995b).   The latest addition to the “Gulf of Maine DPS,” Cove Brook, is actually a tributary to the Penobscot River.

Although the main stem of the Penobscot River is not under consideration for listing, it represents the typical history of the Maine Atlantic salmon rivers. Regarding genetic distinctiveness of its stocks, the Penobscot represents the core population of the Maine / New Brunswick metapopulation model proposed by Kornfield et al. (1995). This core population itself is so well blended with other stocks that “if it were a dog, it would not be recognized by the American Kennel Club” (Kornfield et al. 1995, p7). This homogenization of stocks in other US Atlantic salmon rivers is what stopped the previously attempted “endangered” listing for all rivers, as USFWS Northeast Regional Director Ron Lambertson stated “We simply do not feel that salmon throughout the historic range warrants protection under the Act, because original stocks were lost from many rivers.” (USFWS 1995a). Considering the past stocking histories of Maine rivers “which facilitated introgression and eliminated local variability” (King 1995), it seems rational and consistent with previous policy that the eight rivers within the boundaries of the proposed DPS indeed not be considered a separate and unique entity. This would make them ineligible for an “endangered” status listing if the DPS / ESU is used to justify the listing.

Straying is also an important limiting factor to population subdivision in Atlantic salmon.  There are reported natural straying rates ranging from 1% to 5% (Kornfield et al. 1995 and references therein) to as high as 20% (Tallman and Healy 1994).  This would effectively eliminate any reproductive isolation between rivers by allowing significant gene flow into and out of the supposed “wild” rivers within the proposed DPS.  Of course, straying salmon must also reproduce to keep the populations from becoming isolated.  Some would argue strays would have little chance of interbreeding with "native" river fish because of phenotypic differences, but considering that the Atlantic Salmon Authority (now the Atlantic Salmon Commission) has acknowledged aquacultured fish can breed with "wild" salmon, that argument has little credibility.  Also, the fact that Atlantic salmon have been observed spawning with landlocked salmon that have dropped back from lakes into the rivers where they also spawn (Sgt. Francis Reynolds (ret.), Maine Warden Service, pers. comm.) furthers the idea that strays from one river to another can and do successfully breed.  In fact, given the historical accounts of high numbers of salmon that used to return to the rivers, and even using the conservative estimates if 1-5% straying rates cited by Kornfield et al. (1995), there would certainly be high gene flow between the rivers, quite likely high enough to prevent reproductive isolation and therefore river specificity.  This lends support to the idea of a Gulf of Maine DPS composed of rivers emptying into the Gulf of Maine and possibly even the Bay of Fundy.

 Further evidence against river specificity lies in the widely accepted metapopulation model, which has been proposed by many to explain the genetic make-up of Maine salmon.  A metapopulation is a collection of individual populations that appear to be physically separated but are genetically indistinguishable from one another.  These populations may be exchanging individuals or may have been exchanging individuals in evolutionarily recent times.  The ecological and evolutionary function of the metapopulation model is that it provides for recolonization of habitats when there are many subpopulations that are subject to frequent extinction events (i.e. glaciation, disease outbreaks, natural disasters, etc.) (Neigel 1997).  An example of this was seen when a dam on the Souadabscook Stream was removed and salmon started recolonizing the upstream portion of the river a short time later.   A direct result of this ability is a lack of genetic differentiation between the subpopulations since they are exchanging individuals at least at irregular intervals, if not each generation.

Maine populations and others in southern New England have been rebuilt from a variety of sources, including Penobscot fish and other Canadian stocks (Baum 1995; Kornfield et al. 1995: USFWS 1995a; USFWS 1995b).  Management officials claim existence of a “river-specific” breeding program will allow selection to lead to “river-specific” differences.  First, this hypothesis assumes that there are “river-specific” selection pressures, and that they are substantially different between rivers.  These selection pressures would have to be high enough to counteract the large amount of gene flow that is known to exist between Maine rivers.  Given the known phenotypic plasticity of Atlantic salmon, this hypothesis is unlikely and is not supported by the genetic data that will be discussed later in this paper.

PHENOTYPIC PLASTICITY AND “RIVER SPECIFICITY”
Phenotypic plasticity refers to a low or fluctuating correlation (relationship) between the phenotype (visible physical characteristics) and genotype (genetic make-up) of an organism.  Another term for the correlation of phenotype with genotype is heritability.  For a trait to respond to selection it has to be heritable, with the rate of genetic gain being proportional in part to the heritability.  The accrual of “river-specific” genetic differences (local adaptation) for various traits thus depends on the heritability of those traits.  It is widely known that those traits most important to gaining a selective advantage, such as reproductive traits, have the lowest heritabilities, a relationship that holds quite well across multiple vertebrate taxa.  Given the high phenotypic plasticity (low heritability) of many traits important to natural selection in Atlantic salmon, it is difficult to hypothesize there could be "river-specific" selection pressures high enough to overcome naturally high gene flow, much less the effects of stocking.  In fact, the idea of selection pressures being strong enough to have ever created "river-specific" stocks in Maine is highly questionable at best.

In practical terms, "heritability" is percent of phenotypic variation that is due to additive genetic effects, with the remainder being largely to due to environmental effects.  Thus, for a trait with a heritability of 8%, only 8% of the observed variation is due to differences in additive effects of genes among animals, while the other 92% is largely due to responses of fish to their environment.  Further, any observed morphometric trait differences such as growth rates, fecundity, and survivorship, cannot be extrapolated to claim the existence river-specificity when the heritability of these traits is low (i.e. <20%) or unknown altogether.  Hence, differences in means of morphometic traits between populations does not necessarily mean a true genetic change has taken place between populations.  For these types of traits to be of use in drawing conclusions about river-specificity, the observed differences must be consistent over many years.

The use of morphometric data (i.e. using physical characteristics) to draw conclusions regarding the evolutionary history of a salmonid is not an accurate assessment of their history (Allendorf 1988; Grant 1988; Pella and Milna 1988). Heritabilities of morphometric traits in fish are often on the low end (i.e. 8% for body weight in juveniles) of traits measured in other types of vertebrates.  Even Robin Waples, NMFS’s chief geneticist, acknowledges that differences in morphometric traits between populations are confounded by environmental variability (Waples 1991).  Waples (1991) also notes that visible or measurable differences between habitats do not necessarily mean local genetic adaptations have occurred.

In instances where morphometrics have been used to identify fish populations, “large phenotypic variation, coupled with the lack of information concerning the genetic basis for this variation, often results in oversplitting of stocks” (Allendorf 1988).  Alternatively, Allendorf (1988) claims accurate stock identification requires “analysis of distinct alleles at defined loci”.  For example, the Atlantic Salmon Authority (ASA) recently used morphometric identification to incorrectly identify the single salmon caught in its picket weir on the Pleasant River in 1997 (Horton et al. 1997, Ouelette, 1999).  The ASA thought the salmon was an aquaculture fish and sent it back out to sea only to later determine from a scale sample that it was "most likely of wild origin" (Horton et al. 1997).  And again in 1998, a salmon released upstream of a picket weir on the Dennys River was incorrectly identified as “a 2 sea-winter salmon of wild origin” (Ouelette, 1999), only to later be determined to have been of an aquaculture fish (Ouelette, 1999).

“RIVER-SPECIFIC” STOCKING:  EFFECTS ON POPULATION VIABILITY AND GENETIC VARIATION
 A serious problem with the “river-specific” stocking program is the remarkably small number of broodstock used to create each subsequent generation (“effective population size”).  One way of assessing the program’s merits is by examining its effects on population viability.  In addition to maintaining genetic variation, the ability of the species to adapt to a changing environment and persist over time is also related to the effective population size and the rate of population increase.

 The results in the May et al. (1994) study demonstrate the trend towards a loss of genetic variation in small populations.  This can result from inbreeding depression and genetic drift due to the low effective population size (Ryman and Laikre 1991; Verspoor 1988), known as the “hatchery effect” or the “Ryman-Laikre effect”.  This loss of genetic variation (heterozygosity) may manifest itself in the biological characteristics of future generations by limiting growth, survivorship, and fecundity (Avise 1994).  It is generally thought that genetic variation should be preserved and is essential in species facing a changing environment, and thus management efforts must be concerned with maintaining the genetic variation of “wild” stocks and preventing the loss of genetic variation in captive stocks used for stock enhancement (Frankham 1995; Lande 1995; Avise 1994; Ryman and Laikre 1991; Waples et al. 1990, Waples 1990, Lande 1988, Lacy 1987).

The reduction of genetic variation within a population, such as that which results from a population bottleneck, has been implicated in the extirpation of several wild populations by eliminating genetic variation in genes influencing disease resistance, leaving the population defenseless against new and existing strains of pathogens (Waples 1990).  In fact, it is recommended that fisheries managers swap between one and ten fish among subpopulations (rivers) each generation to slow the loss of genetic variation within a subpopulation by importing new genetic variation into finite populations (Mills and Allendorf 1996).  Whether this is accomplished via natural straying rates or not is currently unknown.  To continue on the course of promoting the loss of genetic variation under the guise of a conservative “river-specific” stocking program, whose laurels are not supported by existing data to begin with, does not make logical or scientific sense.

Effective population size, Ne, as derived in Waples (1990), is defined as the effective number of breeders (Nb) multiplied by the generation length of the fish, in this case 4 years.  For example, if the population size is growing by less than 0.1% per year or decreasing altogether, and has a broodstock size of 250 individuals (approximately 125 males and 125 females), its long term viability is severely compromised even if the environment remains constant (Lynch 1996)1.  Other recommendations for maintaining the long term viability of a population is a minimum of an effective broodstock size (Nb) of 100 fish per population per year, which translates to an Ne of about 400 given a 4-year generation interval (Waples 1990).  Given that this is the effective broodstock size, the actual broodstock size may have to be substantially larger than 100 fish.  At levels below an Nb of 100 fish, low frequency alleles are subject to rapid elimination from the population.  Lande (1988) suggests an absolute bare minimum Ne of 500 fish (mathematically equivalent to an Nb of 125 fish given a four-year generation interval).
Given the recommendations for broodstock sizes and the current returns in the DPS, the long-term viability of the proposed DPS is questionable at best.  This begs the question of whether or not an endangered species listing of the DPS can have a positive long term effect on the viability of the DPS, whose population size is already well below any of the recommended minimum viable population sizes.

The effective broodstock sizes for rivers in the “river-specific” program are well below 250 fish, often less than 50 fish per river (the Pleasant for example), despite attempts to expand the gene pool by breeding across year classes.  This relationship has important implications in Atlantic salmon management in that it provides more evidence that the “river-specific” stocking program is probably doing more harm than good for the long-term viability of the metapopulation.  Considering the broodstock sizes that have been and are currently being used, the term “river-specific inbreeding program” may be more accurate.
 The small broodstock sizes in the river-specific program have an additional downside which has a direct and large effect on the genetic data used to test for significant population subdivision.  This has been demonstrated in hatchery versus wild populations of Chinook salmon (Waples and Teel 1990).  In the Waples and Teel (1990) study, it was observed that significant differences in allele frequencies arose after 2-4 years in hatchery stocks but not in wild stocks.  The effective number of breeders (Nb) in the hatcheries was estimated at between 25-50 fish per year, while the actual number may have been much larger.  In fact, the number of loci accruing significant differences was highly correlated with, and inversely proportional to, the actual number of female broodstock spawned.  As expected, the smaller the population size, the greater the random fluctuations in allele frequencies, and the greater the likelihood of observing a significant change in allele frequencies.  To quote directly from the Waples and Teel (1990) study:

 These results have serious implications for the interpretation of population genetic data on the rivers in Maine.  The population sizes of each river, and even the proposed DPS as a unit, are comparable in size to, or even less than, the hatchery populations used in the Waples and Teel (1990) study.  It would follow suit that the allele frequencies in the individual river stocks will  be greatly influenced by random genetic drift and have a high probability of showing a statistically significant difference between rivers.  These differences could easily be misinterpreted to suggest that such statistically significant differences represent historical isolation and local adaptation.  In truth, the population sizes of the Downeast rivers and the DPS as a unit are not large enough to have maintained historical differences.  These differences have very likely been permanently obscured by the overriding factors of random genetic drift and inbreeding from the extremely low population sizes.

GENETIC DATA
 It is important to note that, while genetic information may not be required for listing, when available it is certainly “the best scientific information” and should be considered, especially when determining discreteness.  The purpose of using population-level genetic data is that it provides estimates of the degree of population subdivision, which results from reproductive isolation, be it absolute or nearly so.  While there is no official “gold standard” of what constitutes reproductive isolation from the standpoint of population genetics, the general rule of thumb is the presence of population-specific alleles (private alleles) and/or substantially statistically significant differences in allele frequencies between populations coupled with temporal stability of these differences.

ORIGINAL STUDIES
 Before the 1995 Federal Status Review on Atlantic salmon was conducted, several genetic studies were done on the salmon in Maine rivers.  When considering genetic data collected in these studies, it becomes clear that neither “river-specific” stocks nor a collective DPS exists. The data in May et al. (1994) show no population-specific alleles. In fact, 96.6% of the genetic variability was due to within population variation, meaning differences between rivers accounted for only 3.4% of genetic variation. This is strikingly similar to results presented in Jordan et al. (1992) who examined sea-run populations in Scotland. The data showed 96.4% of genetic variation was due to within population variability, with 1.6% of the variability attributable to different river populations.  While the May et al. (1994) study shows statistically significant differences in allele frequencies between rivers, the significance levels of each test were not corrected for multiple tests of the same hypothesis to maintain an experiment-wise Type I error rate of 5%.  Thus, these results should be interpreted with caution as the probability of  “false positives” of significant differences in allele frequencies is increased above the standard 5%.  In addition, the study did not sample more than generation of fish, thus it did not test for temporal stability or persistence of the observed differences, so the results could be the reflection of genetic drift.

 Schill and Walker (1994) used random amplified polymorphic DNA (RAPD) markers to test the hypothesis of significant population subdivision between Gulf of Maine rivers: Dennys, Ducktrap, East Machias, Narraguagus, Pleasant, Penobscot River broodstock, and St. Johns River broodstock.  Estimates of Nm ranged from 2.9 to 4.1, based on observed FST values, indicating considerable migration.  No population specific alleles were observed.  Interestingly, their cluster analyses showed the Penobscot to be as genetically similar to any of the DPS rivers as any of the DPS rivers were to one other.  In fact, the most distantly related river to the rivers sampled was the Sheepscot, not the St. Johns or the Penobscot.

 Kincaid et al (1994) analyzed morphometric data on Atlantic salmon collected from the Dennys, Ducktrap, Machias, Narraguagus, Penobscot, Pleasant, and Sheepscot Rivers, in addition to the Green Lake NFH and St. Johns aquaculture fish at the ASF Salmon Genetics Research Program at St Andrews NB.  While the data suggested stock differences in the traits measured, the authors did not account for the effects of environment on these traits.  The authors mention the stock differences found can result from both genetic and environmental factors and are, by themselves, inconclusive.  The problems associated with using morphometric data to assess population subdivision were noted above and discussed in Waples (1991) and Allendorf et al. (1987).

 King and Smith (1994) attempted to use allozymes and RFLP analysis of mitochondrial DNA to assess population subdivision.  Only one allozyme locus was consistently resolvable and showed no evidence of population subdivision.  No genetic variation was found in the mitochondrial DNA regions amplified (D-loop, NADH1, 12SrRNA) via PCR and was thus not useful for evaluation of population subdivision.  While mtDNA differences exist between North American and European Atlantic salmon, no variability was found within or between stocks of North American fish.

 The genetic data presented by May et al. (1994), Schill and Walker (1994), and Kincaid et al (1994) do not support the idea of the proposed “Gulf of Maine DPS” being distinct from any other river system in Maine or New England (i.e. Penobscot, Connecticut river systems).  In fact, the data questions the current existence of “river-specific” stocks altogether.  Regarding the temporal stability of putative statistical genetic differences between rivers, none of these studies sampled across multiple generations of fish.  In addition, the studies of May et al (1994) and Schill and Walker (1994) estimate an amount of gene flow between rivers in the proposed “Gulf of Maine DPS” that is significant and likely adequate to prevent reproductive isolation, a prerequisite for developing “river-specific” populations over evolutionary time.

MOST RECENT GENETIC DATA
Regarding the latest genetic data (King et al 1999), we do not entirely agree with some aspects of the conclusions reached in the report.  While Tim King is highly respected as a fisheries geneticist, we feel there are other possible explanations for the data he observed.  After reading the report, it almost seems it was written to be hyper-critical of aquaculture fish and the perceived threat to the "wild" gene pool.  In particular, we are referring to the authors’ comments in paragraph 1 page 3, and paragraph 2 page 31.  The authors use the presence of what they perceive as "European" alleles in the Downeast rivers to claim that aquaculture strays have introduced these alleles into Downeast rivers.  While this is one interpretation of the data, other alternative hypotheses can be postulated.

The data interpretation problem arises with the use of microsatellites to reach conclusions about population mixture.  One cannot distinguish alleles identical in state (iis) from those identical by descent (ibd), especially without pedigree information on the individual animals used in the study.  For instance, one could observe an allele 235bp in size in two different populations that arose by two independent mutation events (iis), or it could have resulted by a mutation occurring in one population and then migrants spread the allele into the other population (ibd).  Since microsatellites have the highest mutation rates of any nuclear marker system, and given both the number of fish sampled and the divergence time between North American and European Atlantic salmon populations, both scenarios are equally likely and should both be considered as possible underlying sources of variation.  Currently geneticists cannot tell whether two alleles scored by length in base pairs (i.e. 235bp) are identical in state or by descent without having pedigree information, and the assumption is often made that the 235bp allele arose once, and only once, during the course of evolution, as per the infinite alleles model of molecular evolution.  Since microsatellites have a finite number of scorable alleles, it is certainly possible to have two different mutational events creating alleles indistinguishable from one another because they are the same size in base pairs, creating alleles identical in state but not identical by descent.  In such cases, the two alleles could have different flanking base pair sequences.

 For example, in King et al (1999) the SSLEEN82 locus has a 228bp allele found only in the Pleasant but occurs in all the aquaculture stocks.  This asks the question of whether the 228 allele was introgressed into the Pleasant from an aquaculture escapee or occurs in both populations because of independent mutational events.  The answer is that we don't know which case is true, and the interpretation of the data depends on whether both 228 alleles are assumed to be iis or ibd.  Another possibility to explain this observation is the allele is or was a rare allele in either the Penobscot or St John sea-run populations used for stocking the Pleasant.  Allele frequencies tend to change dramatically in small populations due to genetic drift, and the Downeast populations are very prone to drift because of the small broodstock sizes.  Rare alleles at microsatellite loci could easily have been lost through drift or just missed when the populations were sampled (O’Connell and Wright 1997), thus absence of these “European” alleles in other North American populations should not be inferred to mean they are absent in those populations.

To use another example from King et al (1999), the existence of the “same” allele (Ssa85 – 106bp) in both the East Machias River and Sebago Lake landlocked fish would suggest introgression of Sebago Lake landlocked salmon into the East Machias River population, an unlikely situation.  However, if we extend King’s contention that the presence of the “same” alleles being found in river salmon and Landcatch origin aquaculture fish (which in this case the authors called “European” alleles) means introgression of Landcatch into Maine rivers, then we would have to logically conclude that a fish from Sebago Lake somehow swam across the state to the East Machias River and spawned successfully.  This inconsistency is not pointed out in the King et al. report.

The possible scenarios of multiple independent mutational events and rare alleles can be extended to any other case of supposed aquaculture contamination of "wild" fish in the study.  To reiterate, the existence of the same microsatellite allele in both a European population and a North American population does not provide conclusive evidence that the two populations have been mixed together.  That is only one of many possible interpretations of the data.

Regarding measures of population subdivision, the data of King et al (1999) do not support the DPS/ESU argument.  Coming from the gestalt of FST, 27% of observed genetic variation was attributable to differences between North America and Europe.  Differences between rivers in Maine accounted for 3.8% of observed variation, and the difference between Maine and Canada accounted for 6.2% of the observed variation, which is comparable to the previous genetic studies.  Regarding  AMOVA analysis of mtDNA haplotype frequencies, only 2% of observed variation was attributable to differences between rivers with 86% of observed variation being due to variation within rivers.

Interestingly, the DPS of the seven or eight rivers was not tested against Maine or Maine/New Brunswick by King et al (1999), so the use of the data as currently analyzed does not directly address the DPS question, but instead looks at the effect of river on FST and haplotype frequencies as analyzed through AMOVA.  Bearing in mind that the small population sizes can easily “create” significant differences between rivers due to genetic drift, homogenizing or pooling the rivers together into a DPS would be expected to reduce the amount of observed population subdivision when the DPS as a unit was compared to the rest of Maine or Maine / NB.  Considering that differences between rivers accounted for only 2% - 3.8% of observed genetic variability, pooling rivers into the DPS as a unit would be expected to reduce this amount even more if the DPS is tested against the Penobscot or a Maine / NB pool.

Using the information as analyzed, it seems more appropriate to consider, at bare minimum, a DPS comprised of all Maine rivers, and even perhaps those in Canada.  In fact, a DPS comprising the eight rivers in Maine was not justified or specifically recommended in the report, rather King et al. suggested an ESU based on the North American / European level of differentiation.

 In summation, the genetic data, which in the case of Atlantic salmon is “the best scientific information available,” clearly does not support the existence of the “Gulf of Maine DPS.”  In light of Congress’ directions to use the DPS designation sparingly and only when the evidence suggests it is warranted, designating a “Gulf of Maine DPS” of endangered Atlantic salmon as proposed, would violate both the Services’ own DPS policy and the directions given by the 96th Congress.

Though we have shown that the proposed “Gulf of Maine DPS” is not discrete as defined
in the 1996 Federal Register notice discussed on the introduction, we will briefly discuss two more issues for the sake of being thorough.  There has been much discussion of the proposed DPS containing the “last genetic remnants” of the “wild” populations and genetic effects of aquaculture escapees, which both deserve brief comment.
 
 

 ASSESSING SIGNIFICANCE TO LEGACY OF SPECIES AND GENETIC EFFECTS OF AQUACULTURE ESCAPPEES

ASSESSING SIGNIFICANCE
 Assessing the importance of the supposed DPS “strains” to the legacy of the species is difficult, if not entirely impossible, since there is no genetic data on these river populations prior to the time of extensive stocking and introgression between stocks.  It is also unknown if “river-specific” populations ever existed given the known natural straying rates and even a rudimentary understanding of population genetics.  To claim they are unique or evolutionarily important while observing proper scientific protocol would require quantitative comparison with stocks from other parts of their range (i.e. Connecticut, Miramichi, etc.).  The amount of historical genetic differentiation is unknown, although it can be easily assessed using current molecular genetic techniques in which DNA is extracted from fish scales preserved on decades-old scale cards.  This would permit one to track genetic changes that have occurred in these stocks over time and allow a robust comparison of current versus historical levels of genetic differentiation.  Claiming minute genetic differences between rivers seen today are “evolutionarily significant” is analogous to comparing apples and oranges when no one has ever seen an orange.

GENETIC EFFECTS OF AQUACULTURE ESCAPEES
 Atlantic salmon aquaculture is assumed by management officials to have deleterious genetic effects on the river “stocks.”  Using inferences from DNA markers, this is untrue when considering the St. John and Penobscot strains of aquaculture fish, but is relevant when considering the Scottish Landcatch  (Rodzen and Kindahl, in review).  The “hatchery effect”, which states hatchery reared fish have less genetic variation than, and are inferior to, their wild counterparts, was not supported by the data in that study.  This is probably because the "wild" fish in the eight rivers of the supposed DPS, as well as those in the Penobscot, are themselves hatchery reared.  Additionally, the aquaculture stocks are supplemented with gametes from the same pool of broodstock used to produce the sea-run stocks, which serves to limit divergence.  The Penobscot and St. John "strains" of aquaculture fish actually had a higher level of genetic variation than the "wild" fish which likely resulted from the much larger brood stock sizes used in commercial production in order to avoid inbreeding (Rodzen and Kindahl, in review).

 Peterson (1999), a quantitative geneticist and animal breeder, provides a viewpoint on the effects of introgression of farmed salmon that is quite different from the Services.  The author suggests that while we should avoid large scale intrusions of farm fish into wild populations, one should not overlook the new genetic material that small-scale intrusions introduce to the population and its potential for providing new genetic variation for selection to act upon, particularly when the wild populations have lost much of their variation.  Basically, he claims the new genetic material brought into the wild population may actually be of benefit to the long term viability of the wild population.  While this may seem radical to some, it does make logical sense from the standpoint of animal breeding and quantitative genetics.

 Peterson notes that small “wild” stocks have probably exhausted most genetic variation intrinsic to those populations due to drift and inbreeding.  This logic can be easily extended to the situation of small broodstock sizes in the DPS rivers.  In that type of scenario, Peterson recommends outcrossing the small population with other populations in an effort to restore genetic variability, which is consistent with the recommendations of Mills and Allendorf (1996).  This is analogous to acknowledging the river-specific program is not doing Maine’s Atlantic salmon any evolutionary favors, but rather fish should be pooled across all of Maine’s rivers to restore the genetic variability of the Gulf of Maine as a whole.
 

CONCLUSION

 In conclusion, we have presented information readily available from a variety of sources, including our own research concerning Atlantic salmon population genetics. The use of “the best scientific information available” is the backbone of the ESA. To disregard such existing and pertinent information is irresponsible to the point of constituting scientific negligence. It is readily apparent that the proposed listing of the eight Maine rivers within the proposed “Gulf of Maine DPS” is politically motivated and not based on the best available information; the best and current information does not warrant a listing when following the Services guidelines for the listing process and the directions of Congress.

Further, it is our opinion that the current "river-specific" stocking program, which the Services call “an essential component of the strategy to rebuild salmon stocks in the [proposed] DPS” (FR 1999), does not take into account the metapopulation concept and is not based on the best scientific information available.  It is apparent that there is either a general lack of knowledge or a complete disregard of population genetics concepts within the agencies involved in salmon restoration in Maine, which has allowed a current policy to develop which appears to be trying to artificially create "river-specific" populations where none currently exist and may never have existed.  In fact, the best scientific information available suggests that these "river-specific" populations most likely never did exist, and that the metapopulation model of genetically indiscreet subpopulations is the more scientifically realistic approach to understanding the genetic structure of Maine's salmon.  We feel continuing the current "river-specific" policies could have a severely detrimental and possibly unrecoverable impact on the genetic diversity of Maine salmon.

 Many people say that too much time is being spent debating the genetics issues in Atlantic salmon recovery.  The authors assert just the opposite is true - not enough time has been spent understanding the subject before making management decisions.  There are now extensive policies in place that are based on false presumptions of "river-specific" genetic differences. There are hundreds of thousands of dollars being spent on "river-specific" weir projects based on the idea that there are genetic differences between river and aquaculture salmon, even though there is evidence to the contrary (Rodzen and Kindahl, in review).  There are expensive "river-specific" hatcheries that are artificially creating (through inbreeding) "river-specific" stocks for a select few rivers, while other rivers receive no supplemental stocking at all due to lack of hatchery space and "river-specific" broodstock.  It seems as if everything to do with Atlantic salmon recovery is based on the same presumed "river-specific" genetic differences that don't actually exist.  With all the time, money, and energy that is being wasted because the agencies involved either don't understand or choose to ignore the basic concepts of population genetics, how can the genetics issues not be important?

 A fellow scientist recently commented to one of the authors that Maine Atlantic salmon are dying of "PCB" poisoning - not polychlorinated biphenyls, but rather Politically Correct Biology.  Unfortunately, there is an increasing trend in Maine and abroad for fisheries and wildlife management agencies to attempt to implement politically correct ideology under the guise of a hard science.  This is truly sad, as Atlantic salmon may end up paying the ultimate price if this trend continues.  Decisions based on political ideology and made by individuals with little or no regard to or knowledge of population genetics concepts can have a severe impact on the health of the remaining salmon and may help lead to their ultimate demise.  We can no longer sit idly by while Atlantic salmon are mismanaged out of existence.

We are simply requesting that the Services evaluate the proposed listing using both common sense and the best available scientific information. It is our sincere hope that the ultimate outcome of this process be based on such information and not political motives disguised as “conservative scientific philosophy”.
 
 

ABOUT THE AUTHORS
 Jeff Rodzen holds a B.S. in Marine Ecology from University of Maine at Machias and a Ph.D. in Animal Genetics from the University of California - Davis, where he worked in the Genomic Variation Laboratory under Dr. Bernie May.  His background is in fisheries / population genetics and animal breeding.  Alfred Moore, Jr. holds a B.A. in Biology from the University of Maine at Machias.  He studied population genetics of Atlantic salmon at UMM under Dr. Eric Kindahl, while remaining active in local conservation organizations and the Pleasant River Hatchery.
 
 

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Footnotes:
1. Ne= (4NmNf)/(Nm+Nf)
Long term population viability compromised if Ne < 1/(4rmax)  where rmax = rate of population increase  (p. 482, Lynch 1996)