BACKGROUND AND JUSTIFICATION:
The diverseness of fresh water mussels ( household Unionidae ) in the United States is unmatched in the remainder of the universe, yet this biodiversity is extremely vulnerable, with more than 70 % of recognized species considered to be either endangered, threatened, or of particular concern ( Williams et al. 1993 ) Most species inhabit limited scopes and have little ( and/or frequently unknown ) dispersion abilities due to reliance on specific host fish for reproduction, doing them easy fragmented and peculiarly susceptible to the activities of worlds, such as habitat alteration, pollution, and over-harvesting ( Neves 1993, Williams et Al. 1993 ) Recent debuts of foreign molluscs such as the zebra mussel ( Dreissena polymorpha ) pose an extra, big menace to this already destitute mussel zoology.
Despite the realisation that native mussel zoologies are threatened, cognition of basic biological science, ecology, and taxonomy of most unionids remains missing and bounds our ability to take immediate preservation action Further perplexing our cognition of unionid species is the deficiency of distinct morphological characters of usage in accurately naming species or finding evolutionary line of descents Phenotypic malleability has frequently been observed in both mussel shells ( shell collecting ) and in soft-tissue anatomy ( Kat 1983 ) , and as a consequence, our ability to specify species is compromised and deep species are frequently concealed At higher taxonomic degrees, convergence on similar sets of shell features by distantly related line of descents may confuse our ability to put species into higher categorization strategies that accurately reflect lineage.
Effective preservation and Restoration programs require clearly definable units of direction. Presently many unionid species are randomly defined and managed as metapopulations, in which the principal processes commanding kineticss are extinction, migration, and colonisation to set up new local populations ( Hanski and Gilpin 1997 ) The cardinal inquiries are how these processes jointly affect the kineticss and development of local populations and the full metapopulation Environmental patchiness forces species to be structured into systems of local populations within which conspecifics are more likely to interact with each other than with conspecifics from other populations Isolation, nevertheless, is normally non complete and since most beings have some power of dispersion, members of a local population have a low but positive chance of interaction with persons from other vicinities ( Wiens 1997 ) Depending on the rate of migration, demographic and familial kineticss will be influenced by this migration every bit good as by local birth and decease rates None of these parametric quantities are known for most unionids.
Despite a deficiency of cognition of the procedures at work within and between local populations for most unionid species, Restoration and supplementation attempts in unionid preservation progressively rely on relocating mussels to other sites or into hatchery installations for unreal extension An illustration of ongoing supplementation attempts is the province of the art hatchery installation at Virginia Tech, which has been let go ofing juvenile mussels into the Upper Tennessee River system since 1997 ( Neves et al. 2003 ) Through this plan, the Clinch River in Southwestern Virginia has received hatchery-reared juveniles as portion of a recovery effort for 18 endangered mussel species populating the country ( Zimmerman and Neves, 2003 ) Such supplementation attempts have been described as taking advantage of, “advances in engineering and proactive recovery ‘ ( McGregor and Shepard 2003 ) , yet without including a familial constituent in such work, the ‘technology ‘ available to directors is non being to the full utilized, and as a consequence, supplementation attempts could potentially backlash due to hazards that are built-in with hatchery supplementation plans One possible hazard of unreal extension is the creative activity of loanblends either between populations of a species that are really genetically distinguishable, or between closely related deep species Without cognition of connection ( degrees of cistron flow ) among natural populations, the possibility exists for species that are genetically distinguishable yet non to the full reproductively isolated to crossbreed, ensuing in an unreal loanblend and loss of both wild species This scenario was probably prevented through a familial survey of Potamilis inflatus, which detected big species-level familial differences between the Black Water and Amite River populations that were about to be assorted ( Roe and Lydeard 1998 ) The menace of hybridisation between other closely related taxa that co-inhabit an country being supplemented exists every bit good Again, understanding the phyletic relationships between closely related species, and divergency between populations of one species, will be our most utile tool in measuring the hazard involved in preservation attempts ( Perry et al. 2002 )
Another possible hazard involved in unreal extension is a loss of familial diverseness relation to natural populations Genetic diverseness is of cardinal concern to preservation attempts as it is required for evolutionary alteration to happen within populations ( Frankel and Soule 1981 ) In other words, familial diverseness allows populations to germinate in response to environmental alteration, such as new diseases, parasites, rivals, alimentary degrees, pollutants, etc Genetic diverseness ( including polymorphism and heterozygosity ) has been shown through empirical observation to be lost at greater rates in little relation to big populations ( e.g. Montgomery et Al. 2000 ) Likewise, the importance of keeping big effectual population sizes ( Ne, or Numberss of effectual genteelness persons ) in hatcheries in order to avoid inbreeding depression ( and loss of familial fluctuation ) has been good documented ( Ryman and Laikre 1991 ; Waples and Doh 1994 ; Tringali and Bert 1998 ) A counterintuitive yet possible effect of supplementation is a lowering of Ne in wild populations even as Numberss of animate beings increase This can go on when hatchery rise uping unnaturally increases juvenile endurance in selected households relative to household size in the wild and can significantly increase the discrepancy in household size, taking to inbreeding ( Ryman and Laikre 1991 ) For most unionid taxa, cognition of Ne in wild populations is unknown, yet is likely low for many taxa given the rareness of animate beings Estimates of Ne in both wild populations and in hatchery stocks are imperative because they allow for impact anticipations to be made about effects carrying and resettlement plans will hold on familial variableness ( see Villella et Al. 1998 ) .
The clubshell ( Pleurobema clava ) is a fresh water pelecypod that was historically widespread in the Ohio River basin and feeders of western Lake Erie, but has experienced a 95 % scope decrease Despite it ‘s naming as an federally endangered species in 1993, small is known about the ecology of P. clava, or about the connectivity between the staying populations populating widely scattered watercourses of worsening suitableness across the eastern United States P. clava is soon known to happen in rivers in six provinces ( Table 1 ) , but reaches maximal copiousnesss in the Allegheny River in Pennsylvania Currently, no familial information exists on population construction, degrees of cistron flow, or relatedness among geographic populations of P. clava.
Recently, several research workers have found a 2nd morphologically distinguishable signifier of P. clava in the Allegheny River near Hunter ‘s Station and in Gallic Creek, a feeder of the Allegheny The freshly identified shell type superficially resembles a non-congener that besides occurs in this river, Fusconaia subrotunda ( the longsolid ) Interestingly, these two shell types may hold been present in this part for many decennaries, as two shell forms of P. clava were besides noted by Ortmann ( 1919 ) Whether these distinguishable shell forms represent ecophenotypic fluctuations of P. clava or different species is unknown, yet is progressively of import as programs for unreal extension and supplementation of P. clava in the Allegheny drainage continue to be formulated.
On a broader geographic graduated table, there is inquiry about whether P. clava populations that occur in Michigan, Ohio, Indiana, and West Virginia ( Table 1 ) are really the same species as that found in Pennsylvania Due to the discontinuity between these geographically disjunct populations, small is known about the grade of familial connectivity between them Given the legion findings of important familial divergency among unionid populations busying stray drainages ( discussed supra ) , significant sub-structuring may be present between these locations
Besides of involvement is the relationship between P. clava and a similar signifier, Pleurobema oviforme ( Tennessee clubshell ) that occurs in locations where P. clava has besides been found in the Cumberland River in Kentucky and Tennessee Whether or non these species are distinguishable genetically besides remains unknown Unlike P. clava, P. oviforme is non protected under the Endangered Species Act, and since P. oviforme besides appears to be worsening, finding the relationship between these taxa is of import for preservation attempts A similar state of affairs, with two similar signifiers of Pleurobema species, P. decisum ( federally endangered ) and P. chattanoogense ( species of concern ) , occurs in the Mobile basin These species are presently being examined in a phyletic survey being led by David Campbell at the University of Alabama ( pers. comm. ) Consequences from the survey proposed here will be complimentary to the phyletic analysis of the Pleurobemini of the Mobile basin of Campbell et al. , and will make a complete model of relationships among these closely related and worsening species.
Many species belonging to the folk Pleurobemini ( Bivalvia, Unionidae, Ambleminae ) exhibit high sums of fluctuation in shell morphology, which has lead to systematic confusion In fact, species belonging to the genus Pleurobema have been described the undermentioned manner, “the members of the genus Pleurobema are among the most hard to place in North America” NatureServe Explorer ( ver. 1.6, 2003 ) Trouble in designation of Pleurobema species is particularly unfortunate in that most of the species are threatened or endangered at either the province or national degree ( see Table 2 for a list of preservation position of several Pleurobema species included in the proposed survey )
The pink mucket, Lampsilis abrupta, is in diminution, holding been badly impacted by dam building, dredging, pollution, competition with nonindigenous species ( e.g. , Dreissena polymorpha ) , and inordinate harvest home L. abrupta occurs over a broad geographic country that includes the Mississippi, Tennessee, and Cumberland River systems L. abrupta has been under the protective protections of the Endangered Species Act ( ESA ) since 1976 There is an sanctioned recovery program ( 1985 ) and USFWS has created a watershed execution agenda for the recovery program ( 1989 ) To program and implement biologically sound recovery plans in the Ohio River basin, cognition of the sum of familial diverseness nowadays and a thorough apprehension of the evolutionary relationships ( e.g. , degrees of cistron exchange ) among geographic populations of L. abrupta are indispensable The intended usage of civilized unionids as a preservation tool underscores the demand to acknowledge the familial composing of natural and managed populations However, no information exists on the phylogeographic relatedness among wild populations or the degrees of familial fluctuation within the confined broodstock ( s )
There are two major aims of this comprehensive undertaking The first aim is to use both mitochondrial and atomic DNA cistron parts to find whether phylogeographic construction exists among extant populations Along with finding relatedness among populations, this work will assist clear up systematic uncertainnesss affecting both species
The 2nd aim is to use freshly developed microsatellite Deoxyribonucleic acid markers to assess degrees of familial diverseness and heterozygosity within selected geographic populations with the largest concentration of staying persons Microsatellite markers will besides let a fine-scale analysis of cistron flow between main-stem and feeders that contain populations of both species, which will let for designation of possible beginnings for enlisting Furthermore, the public-service corporation of the markers will be demonstrated for usage in placing and keeping familial diverseness during the development of confined broodstock, for taking natural populations to supplement, and for supervising effects on wild populations as supplementation returns This proposed research represents the first probe into the population familial construction of and will supply the baseline familial informations necessary in order to efficaciously pull off staying populations
Cardinal to attempts to protect mussel populations is the ability to right define between species and distinguishable direction units Molecular tools applied to systematic, phylogeographic and population familial surveies are needed to find the graduated table ( watercourse, drainage, biogeographic part ) at which preservation attempts should be directed for unionid taxa of concern In fact, the usage of systematic cognition of unionids for preservation intents was late described this manner, “Mussels may stand for the best aquatic illustration of how an increased apprehension of phyletic relationships will be built-in in forestalling the loss of biodiversity” ( Perry et al. 2002 ) The limited figure of molecular systematic surveies that have been done to day of the month on unionid taxa have shown that morphology-based designation and categorization may non reflect distinguishable evolutionary line of descents At the generic degree and supra, several surveies have shown that species belonging to a peculiar genus frequently do non constellate together on a phyletic tree ( Potamilus, Roe and Lydeard 1998 ; Lasmigona, King et Al. 1999 ; Fusconaia, Quincuncina, Obovaria, Lydeard et al. 2000 ; Alasmidonta, Morrison et Al. 2003 ) Conchological characteristics of unionids, which are what categorization strategies have been built around, may non reflect the ‘historical bequest ‘ of species ( Lydeard et al. 2000 ) .
At the species degree, zoogeographic ( i.e. phylogeographic ) forms have frequently been uncovered utilizing molecular techniques when species are examined throughout their scope For illustration, strong geographical structuring and/or designation of deep species have been detected in stray drainages ( e.g. Quadrula quadrula, Berg et Al. 1996 ; Amblema plicata/elliottii, Mulvey et al. 1997 ; P. inflatus, Roe and Lydeard 1998 ; Lasmigona subviridis, King et Al. 1999 ; several Lampsilis species, Roe et Al. 2001 ) The Mulvey et Al. survey has been criticized, nevertheless, for unequal sample sizes, and it has been pointed out that in order to know apart among species within a genus ( particularly when proposing systematic alterations affecting common and threatened species ) , one must quanitify the sum of fluctuation both among populations within a species every bit good as between species ( Berg and Berg 2000 ) With an equal sampling design along with recent promotions in statistical analyses of mitochondrial DNA sequence informations such as analysis of molecular discrepancy ( AMOVA, Excoffier et Al. 1992 ) and nested clade analysis ( Templeton et al. 1995 ) , geographic expedition into the forces determining intraspecies familial diverseness seen today is possible, such as historical isolation versus current ecological procedures For illustration, allopatric atomization was detected utilizing both AMOVA and nested clade analysis in Lampsilis hydiana populations from the Arkansas River drainage relation to other Eastern Highlands populations ( Turner et al. 2000 ) The absence of phylogeographic patterning has besides been found in some instances, where some species do non look to be distinguishable based on analysis of DNA sequence informations ( e.g. Epioblasma florentina walkeri and E. capsaeformis, Buhay et Al. 2002 ; three Megalonaias species, Mulvey et al. 1997 ) Again, cautiousness must be taken in construing these types of surveies based on little sample sizes ( e.g. Berg and Berg 2000 ) , as from a preservation point of view, it is better to mistake on the side of acknowledging distinguishable evolutionary units instead than neglecting to acknowledge them ( Daughterty et al. 1990 ; Lydeard et al. 2000 ) .
Deoxyribonucleic acid extraction
Genomic DNA will be isolated from preserved mantle tissue utilizing the Puregene DNA extraction kit ( Gentra Systems, Inc. , Minneapolis, MN ) , and will be resuspended in TE buffer ( 10 mM Tris-HCl, pH 8.0, 1 millimeter EDTA ) DNA concentrations will be determined by fluorescence check ( Labarca and Paigen 1980 ) and unity of the DNA will be visualized on 1 % agarose gels ( Sambrook et al. 1989 ) .
Mitochondrial DNA COI and 16S cistron parts
The bulk of phyletic and phylogeographic surveies of unionids to day of the month hold used either the mitochondrial 16S rRNA cistron ( Mulvey et al. 1997, plus allozymes ) ; Turner et Al. 2000 ; Krebs et Al. 2003 ) or the cytochrome C oxidase I ( COI ) cistron ( Roe and Lydeard 1998 ; King et Al. 1999, plus ITS ; Graf and O’Foighil 2000 ; Buhay et Al. 2002, plus ND1 ) or a combined analysis of both cistron parts ( Lydeard et al. 2000 ; Roe et Al. 2001 ) Most significantly to this survey, an on-going phyletic survey of the folk Pleurobemini is good underway ( D. Campbell, pers. comm. ) utilizing the 16S, COI, and ND1 cistron parts The survey by Campbell et Al. dressed ores on species found in the Mobile basin, and hence the proposed work ( concentrating on northeasterly species ) will congratulate that survey, and will shortly profit from the sequence informations that will be available for many species in the Pleurobemini as that undertaking concludes
A 710-bp fragment of the mtDNA COI cistron will be amplified from genomic DNA utilizing the polymerase concatenation reaction ( PCR ) , with the COIL 1490 and COIH 2198 primers developed by Folmer et Al. 1994, and PCR conditions as described in King et Al. 1999 Additional internal COI primers have besides been published ( Mulvey et al. 1998 ) , and may be utilized as necessary
A limited sum of COI informations exists from antecedently published work ( available in Genbank, accession Numberss shown under species names, Fig. 1 ) for several Pleurobema ( P. clava, P. decisum, and P. sintoxia ) , plus several Fusconaia species ( F. flava, F. escambia, and F. ebena ) The multiple alliance of these sequences, one P. clava sequence generated from the King lab ( unpublished ) , was 680 bp in length, and included 203 variable and 171 phylogenetically enlightening sites Consequences of a parsimony analysis of the COI informations are shown in Figure 1 This preliminary analysis suggests that P. decisum is more closely related to P. clava than is P. sintoxia We can besides see that the two P. clava sequences are reasonably distinguishable from each other by the long subdivisions that lead to each single relation to the subdivisions linking the two persons of both P. sintoxia and F. flava In fact, the sequence divergency between the two P. clava sequences from the Allegheny ( Hunter ‘s Station ) and Michigan ( Hillsdale County, 1.75 % ) is approximately 10 times the familial distance between the two P. sintoxia sequences ( 0.15 % ) Divergence between species ranged from 5 % between P. clava and P. decisum to 8 % between P. clava and Fusconaia flava, and upwards of 20 % between Pleurobemini taxa ( clique ) and Amblema or Quadrula ( tribe Amblemini, data non shown ) One of the three sequences for Quadrula quadrula is about 10 % divergent from the other two sequences for this taxon, a familial distance seen between genera in other comparings in this information set, which could be declarative of a deep species It is notable that when multiple persons have been sampled for a individual taxon, these persons cluster together and have high bootstrap support The consequences of this preliminary COI analysis suggest several things: foremost, it is likely that sub-structuring exists between geographically disjunct P. clava populations ( Pennsylvania and Michigan ) ; 2nd, that this mitochondrial cistron part is appropriate for casting visible radiation on population sub-structuring and phyletic relationships among closely related taxa ; and 3rd, that trying of persons should include as many populations as possible in order to adequately measure the familial fluctuation throughout the scope of a individual taxon.
PCR primers used to magnify the 16S cistron part will be the 16Sar-L-myt and 16Sbr-L-myt described in Lydeard et Al. 1996, which amplify about 500 bp at the 3 ‘ terminal of the mtDNA cistron ( see Mulvey et Al. 1998 for conventional ) Two internal PCR primers are besides available for sequencing ( 16Sint2-H and 16Sint1-H ; Lydeard et al. 1996 ) To day of the month, there are merely two 16S sequences in Genbank for Pleurobema ( P. decisum and P. pyriforme ) In add-on, we will study the cytochrome B and/or ND1 parts to seek for polymorphisms and phylogeographic signal.
Nuclear ITS cistron part
It has become standard pattern to compare phyletic hypotheses from both atomic and mitochondrial cistrons when interested in historical relationships among taxa ( see Nichols 2001for a reappraisal ) By analyzing the best hypothesis of relationships based on one cistron ( or several cistrons from the same genome, such as the mitochondrial genome that is inherited as a linked unit ) , the forms observed could stand for the cistron ‘s line of descent, or a “gene tree” , and may or may non fit the true historical relationships, or the ‘species tree” ( Nichols 2001 ) When phyletic hypotheses based upon cistrons from different genomes agree, a more robust hypothesis consequences Despite credence of the utilizing both mitochondrial and atomic DNA among taxonomists, really few systematic surveies of unionids have been done One ground for this is a deficiency of illustrations of atomic cistron parts that have been amplified in unionids that demonstrate appropriate rates of alteration to be enlightening at turn toing species-level phyletic hypotheses The merely published survey we are cognizant of trades with evolution of the genus Lasmigona and phylogeography of L. subviridis ( King et al. 1999 ) . This work utilizes the internal transcribed spacer ( ITS ) part of the tandemly repeated ribosomal RNA cistron bunch that separates the 5.8S and 18S structural ribosomal cistrons The ITS is under less functional restraint than the structural cistrons neighbouring it, ensuing in a faster rate of development, doing this part appropriate for species-level comparing Similar phyletic hypotheses for the genus Lasmigona were produced utilizing the mitochondrial COI cistron and ITS ( King et al. 1999 ) , showing that this atomic cistron part will probably be utile for spoting relationships among members of the Pleurobemini as good Furthermore, ITS fluctuation was seen between populations of L. subviridis that corresponded to differences seen in the mitochondrial COI cistron, showing the public-service corporation of ITS for intraspecies phylogeographic surveies.
A 640-bp fragment of the atomic ITS-1 cistron will be amplified from genomic DNA utilizing the polymerase concatenation reaction ( PCR ) , with conserved primers based in the flanking 18S and 5.8S structural RNA cistrons ( see King et Al. 1999, for PCR primers and conditions ) Additional ITS primers are listed in Mulvey et Al. 1998 In add-on to the internal transcribed spacer parts ( ITS-1 and ITS-2 ) , S7-1 and S7-2 ribosomal parts will be surveyed and tested for phylogeographic signal.
Deoxyribonucleic acid sequencing
PCR merchandises from both atomic and mtDNA cistrons will be purified with Exonuclease I and Shrimp Alkaline Phosphatase ( Promega Corp. ) and so used as templets in sequencing reactions with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit ( Applied Biosystems ) Sequencing reactions will be run utilizing a PTC-200 Thermal Cycler ( MJ Research, Watertown, MA, USA ) and electrophoresed on an ABI Prism 3100a Genetic Analyzer in Tim King ‘s lab at the Leetown Science Center Forward and change by reversal sequencing reactions will be assembled organizing a consensus sequence for each person and cistron utilizing Sequencher 4.0 ( GeneCodes Corporation, Ann Arbor, MI ) Multiple alliances will be performed for each cistron part utilizing Clustal X ver. 1.4b ( Thompson et al. 1994 ) .
The extent of DNA polymorphism within and divergency between populations of P. clava and L. abrupta for each cistron part sequenced will be estimated utilizing DnaSP version 3 ( Rozas and Rozas 1999 ) The mean figure of nucleotide differences per site ( p, Nei 1987, Eq. 10.5 or 10.6 ) will be calculated for persons within populations for each cistron part individually The figure of net nucleotide permutations per site with trying discrepancies ( Da, Nei 1987, Eq. 10.21-10.24 ) plus Jukes and Cantor rectification ( 1969 ) between populations of P. clava and L. abrupta will be estimated for each cistron part utilizing DnaSP.
Gene geneologies will be estimated for populations of P. clava and L. abrupta based upon mitochondrial COI and 16Sdata individually utilizing the TCS computing machine plan ( Clement et al. 2000 ) , which follows the parsimony-based attack of Templeton, Crandall and Sing ( 1992, ‘TCS ‘ method, after writers ) Nesting of haplotypes within subnetworks will be carried out following the nesting regulations given in Templeton et al. , ( 1987 ) and Templeton and Sing ( 1993 ) .
The familial construction among populations was examined by pairwise FST statistics ( Excoffier et al. 1992 ) utilizing Arlequin ver. 2.000 ( Schneider et al. 2000 ) The significance of pairwise FST values was calculated by commuting haplotypes between populations 1,000 times.
For each molecular informations divider ( species and cistron ) , maximal parsimoniousness ( MP ) , distance-based neighbor-joining, and maximal likeliness ( ML ) analyses will be run utilizing PAUP* 4.0b10 ( Swofford 2002 ) For parsimoniousness analyses, heuristic hunts will be run utilizing unweighted, parsimony-informative ( PI ) characters with the undermentioned scenes: starting trees for subdivision swapping obtained via stepwise add-on, 100 random add-ons of sequences per tally, and tree bisection-reconnection ( TBR ) subdivision trading on best trees For 16S and ITS information dividers, spreads will either be coded as losing informations or as a fifth base in parsimony-based analyses, and consequences from these scenes will be compared An estimation of support for each node on parsimoniousness trees will be assessed utilizing bootstrap resampling ( Felsenstein 1985 ) with 1,000 replicates and the full heuristic hunt algorithm Additionally, the Bremer support, or disintegrate index ( Bremer 1988, 1994 ) will be calculated for nodes happening in rigorous consensus trees utilizing TreeRot ( Sorenson 1999 ) .
We will find the most appropriate theoretical account of DNA permutation for each species and each DNA part independently utilizing hierarchal likeliness ratio trials ( Posada and Crandall 2001 ) in Modeltest 3.06 ( Posada and Crandall 1998 ) ML analyses were run in PAUP* with the best-fit theoretical account from Modeltest and heuristic hunts with scenes as in parsimoniousness except the ‘as-is ‘ option for the add-on of sequences. ML bootstrap analyses will be run with the “fast” stepwise add-on algorithm and 500 replicates.
Population construction and affinity
Observed genotype frequences will be tested for consistence with Hardy-Weinberg and linkage equilibrium outlooks utilizing randomisation trials available in genepop 3.1 ( Raymond and Roussett 1995 ) Pairwise familial distances between sampled populations will be described by Da of Nei et Al. 1983 Statistical significance of familial differences will be tested utilizing the familial distinction randomisation trial available in genepop Da distances will be visualized in a neighbor-joining tree utilizing TREEVIEW ( Page 1996 ) Isolation by distance will be examined by comparing values between samples and their geographic river distance utilizing a Mantel randomisation trial available in Arlequin Assignment trials will be used to find the likeliness of an person ‘s genotype being found at the sample site from which it was collected Paetkau 1995 ; Cornuet et Al. 1999 ) The computing machine plan GENECLASS ( ver. 1.0.02 ) will be used for assignment trials ( hypertext transfer protocol: //www.montpellier.inra.fr/URLB/geneclass/geneclass.html ) .