fishes Article Spawning Locations of Pallid Sturgeon in the Missouri River Corroborate the Mechanism for Recruitment Failure Tanner L. Cox 1,*,†,‡, Christopher S. Guy 2 , Luke M. Holmquist 3 and Molly A. H. Webb 4 1 Montana Cooperative Fishery Research Unit, Department of Ecology, Montana State University, P.O. Box 173460, Bozeman, MT 59717, USA 2 U.S. Geological Survey, Montana Cooperative Fishery Research Unit, Department of Ecology, Montana State University, P.O. Box 173460, Bozeman, MT 59717, USA 3 Montana Fish, Wildlife and Parks, 205 W. Aztec Drive, Lewistown, MT 59457, USA 4 United States Fish and Wildlife Service, Bozeman Fish Technology Center, 4050 Bridger Canyon, Bozeman, MT 59715, USA * Correspondence: tanner.cox@tu.org † Current Address: Trout Unlimited, Western Water and Habitat Program, 215 N 200 E, Brigham City, UT 84302, USA. ‡ This work was part of the Master of Science thesis of Tanner Lewis Cox. Master of Science program at Montana State University, Bozeman, MT, USA. Abstract: Conservation propagation of pallid sturgeon (Scaphirhynchus albus) upstream of Fort Peck Reservoir, MT, USA, has successfully recruited a new generation of spawning-capable pallid sturgeon where there would otherwise be fewer than 30 remaining wild reproductively mature pallid sturgeon. Successful recovery of pallid sturgeon will now rely on the behavior of pallid sturgeon (e.g., successful spawning in locations that provide adequate drift distance for larvae to recruit). We used location data of pallid sturgeon during four putative spawning seasons to answer the following questions: Where do pallid sturgeon spawn? Are spawning locations related to discharge? Are substrate characteristics at the spawning locations similar to other river reaches? Do spawning-capable females, spawning-capable males, and female pallid sturgeon undergoing mass ovarian follicular atresia use the river similarly? Additionally, we considered if spawning locations are far enough from the Citation: Cox, T.L.; Guy, C.S.; river–reservoir transition zone to provide adequate drift distance for larvae to recruit. Spawning- Holmquist, L.M.; Webb, M.A.H. capable pallid sturgeon did explore upstream locations, and four spawning-capable pallid sturgeon Spawning Locations of Pallid were located in the Marias River during the spawning season in 2018 when discharge was at an Sturgeon in the Missouri River Corroborate the Mechanism for unprecedented high. Pallid sturgeon exited the Marias River and moved downstream prior to Recruitment Failure. Fishes 2023, 8, spawning, and when spawning occurred, it was not far enough upstream to prevent larvae from 243. https://doi.org/10.3390/ entering the transition zone of Fort Peck Reservoir. Thus, management of discharge and water fishes8050243 temperature to mimic 2018 conditions may increase use of the Marias River by pallid sturgeon during the spawning season, which would increase drift distance available to larvae and increase the Academic Editor: Manuel O. Nevarez Martinez probability of successful recruitment. Received: 3 April 2023 Keywords: pallid sturgeon; recruitment failure; spawning substrate; spawning location; spawn- Revised: 24 April 2023 ing movement Accepted: 28 April 2023 Published: 6 May 2023 Key Contribution: Spawning locations of pallid sturgeon in the Missouri River upstream of Fort Peck Reservoir do not provide adequate drift distance for free embryos. This finding corroborates the mechanism for recruitment failure and anoxic conditions at the river–reservoir transition zone. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and conditions of the Creative Commons Conservation propagation of endangered species has been used to prevent the extir- Attribution (CC BY) license (https:// pation or extinction of many taxon, such as plants [1], insects [2], birds [3], mammals [4], creativecommons.org/licenses/by/ amphibians [5], and fish [6]. However, individuals from a conservation propagation pro- 4.0/). gram may not perform well (i.e., survive and reproduce such that the population can Fishes 2023, 8, 243. https://doi.org/10.3390/fishes8050243 https://www.mdpi.com/journal/fishes Fishes 2023, 8, 243 2 of 22 self-sustain) in the wild such that recovery of the population is never achieved. For ex- ample, the Trumpeter Swan (Cygnus buccinator [Richardson]) conservation propagation resulted in increased abundance; however, young swans from the captive breeding program also had to be trained to effectively migrate to wintering areas or they would have perished during the winter [7]. Therefore, it is imperative to study the behavior of individuals from conservation propagation programs after they are placed in the natural environment to assess performance as it relates to recovery objectives, where performance is defined as reproductive output and survival that result in population persistence. Conservation propagation of fishes has increased abundance and prevented the extir- pation of several species (e.g., cutthroat trout or Oncorhynchus clarkii (Richardson) [8], lake sturgeon or Acipenser fulvescens (Rafinesque) [9], white sturgeon or Acipenser transmontanus (Richardson) [10], and razorback sucker or Xyrauchen texanus (Abbott) [11]). However, not all conservation propagation of fishes has been successful, and an analysis of case studies on endangered fish reintroductions (i.e., conservation propagation and translocations from existing populations) found 42% were unsuccessful [12]. Unforeseen behavioral deficits associated with hatchery-origin fish, such as a lack of predator avoidance [13,14], limited migratory behavior [15,16] and reduced foraging ability [17], may result in poor perfor- mance of an augmented population (e.g., in stocked populations). Therefore, the success of conservation propagation programs for species recovery requires an understanding of the behavior of the augmented population to ensure that behavior results in adequate performance as it relates to achieving recovery objectives. Conservation propagation is being used to conserve pallid sturgeon (Scaphirhynchus albus [Forbes & Richardson]) because recruitment failure has reduced the abundance of wild pallid sturgeon in the Missouri River upstream of Fort Peck Reservoir [18], such that there are probably fewer than 30 individual wild pallid sturgeon [19]. Pallid sturgeon were first stocked upstream of Fort Peck Reservoir in 1998 with pallid sturgeon hatched in 1997 [18]. As the abundance of wild pallid sturgeon continues to decline, recovery of pallid sturgeon upstream of Fort Peck Reservoir has begun with hatchery-origin pallid sturgeon becoming reproductively mature, successfully spawning, and contributing recruits to the population. Recruitment failure of pallid sturgeon in the upper basin of the Missouri River is related to the spawning location juxtaposed with the location of the river–reservoir transi- tion zone [20,21]. Thus, the behavior of hatchery-origin pallid sturgeon upstream of Fort Peck Reservoir regarding spawning movements and location will determine if successful recruitment is plausible. In the past, a lack of successfully ovulating hatchery-origin female pallid sturgeon has prevented spawning locations and spawning-related movements of successfully spawning females from being characterized [22]. If hatchery-origin pallid sturgeon spawn in upstream locations of the upper Missouri River or Marias River, the drift distance available to free embryos (i.e., pallid sturgeon in the developmental stage between hatching and exogenous feeding) will be optimized. Otherwise, spawning in downstream locations will result in inadequate drift distance, which could necessitate management actions for successful recruitment to occur. Increasing discharge has been suggested as a potential method to prompt upstream migration prior to spawning. However, it is unknown if discharge is correlated with the spawning location of pallid sturgeon. Discharge in the Marias River was shown to be associated with spawning of shovelnose sturgeon (Scaphirhynchus platorynchus [Rafinesque])—spawning occurred when discharge was greater than 28 m3/s [23]. Furthermore, spawning location could be limited by substrate composition, which has been associated with the spawning location of other sturgeon species [24]. If discharge, substrate composition, or other abiotic factors affect spawning location, management actions that improve drift distance available to free embryos could be implemented. Recovery of pallid sturgeon upstream of Fort Peck Reservoir was initiated by success- fully augmenting the population using conservation propagation. However, successful recovery of endangered species using conservation propagation requires augmented pop- ulations to perform such that recovery criteria are eventually met. For pallid sturgeon Fishes 2023, 8, x FOR PEFEisRh eRs E2V02IE3,W 8, x FOR PEER REVIEW 3 of 23 3 of 24 Recovery of pallidR esctouvregreyo no fu pastllrieda mst uorfg eFoonr t uPpesctkre aRmes eorfv oFior rtw Pase ckin iRtieasteedrv obiyr was initiated by successfully augmseunctciensgs futhlley paoupgumlaetniotin gu sthineg pcoopnusleartvioanti onu sipnrgo pcaognasteiorvna. tiHono wpervoepra, gation. However, successful recoversyu cocfe sesnfudla nregceorveder ys poefc ieesn duasningger ecdo nsspeervciaetsio nu sipnrgo pcaognasteiorvna trieoqnu ipreros pagation requires Fishes 2023, 8, 2a4u3 gmented populaatuiognmse tnot epde rpfooprmul astuiocnhs t htoa tp reercfoovrmer ys uccrhit etrhiaa ta rreec oevveenrytu carliltye rmiae at.r eF oerv entually me3t.o fF2o2r pallid sturgeon inp altlhide Mstuisrsgoeuorni Rini vethr e uMpsitsrseoaumri oRf ivFeorr tu pPsetcrke amRe soefr vFoiorr, t rPeaecchk inRge servoir, reaching reproductive maturreitpyr oadnduc stpivaew mniantug riinty l oacnadti ospnasw thnaitn pgr ionv liodcea atidoenqsu tahtaet dprrioftv didiset aadnceeq ufoart e drift distance for free embryos are thfree en eecmesbsrayroys naerxet t shtee pnse cife stshaer yp onpeuxlta sttieopns i sif gtohien pg otpo ublea tsioelnf- issu going to be self-sustaining in the Missouri River upstream of Fort Peck Reservoir, reachinstgairneipnrgo ductive maturity as outlined in the as aRnedvoiusetlspdin Reawnedc ininogv ethrye PRleavnised Recovery Plan for Pallid Sturgeon [18]. We d in locati ofonrs Pthalaltidp Srotuvrigdeeoand [e1q8u].a te drift distance for free embryos We designed nthecise ssstuardyy entsoei xgatnsestdwe pethrs itsihf sett hufoedlylpo otwop iuannlgas tqwioueners tithsioegn foso ilanlbogowtuoitn tbghe qe suheealsftt-cishouensrsty aa-iobnroiinuggti ntah se ohuattlcihneerdy a-rio e nrithe tghien pallid sturgeon: (1Rp)e aDvllioisd es dpstaRuwercgnoeinvogenr-:yc (a1Pp)l aaDbnolef o sfpreamPwaalnlleiidsn,g Ss-tpcuaarpwgaenboilnneg [f1-ec8ma].paalebsl,e smpaawlens,i nagn-dc afpemaballee males, and female pallid sturgeon unpdaellriWgdo eisntduger sgmigeaonsnes d uotnvhdaisreirsagtnuo difnoygllt iomcuaalnasssrw oaetvrraetrshiiaean f uoflsoleol lwitchiuenl garriq vaueterr sestsiiioman isulasarebly ot?uh te(2 thr) ievehra tscihmeirlya-rolyr?ig (in2) Where do pallid sptWuarlhlgiederoesn tdu sorpg apewaolnnli:?d ( (1s3)t)u DWroghesaoptna a wsrpena iswnugnb-s?ct a(r3pa)ta eWb clehaafert amacrateel ersisus,tbsicsptsar aawtte nt hicnehg as-rpcaaacpwteanrbiilsnetgimc sa alet st,haen sdpafewmnainleg locations? Additiopnlaoalcllaliydti, oswtnuesr ?gc Aoeondnsdiuditneiordenedarg lilofy it,nh wge ems pcaoasnwssonivdinaegrrie aldon cifafo ttlihloiecn usslp aaarrwea tfnraeirns eigan luoosuceagtthiho efnrrosi mvaer reth sfieam r ielnaroluy?gh(2 f)rWomh etrhee river–reservoir tradnroisvipetiaro–lnlrie dzsosetnruvero gsieuro ctnrha sntphsaaiwtti otnhn?e z(r3eo) nwWeo shualctdha b rteeh saautd btehsqteruraaett eewc dohurairlfdat c dbteiesr tiasadtnieccsqeu aftaottreh f edresrepif at wdnisitnagncloec faotiro fnrse?e embryos to survivAeedm adbnirtdyio ornesa ctlroluy i,stu,w raevnidvco ewn aseni ddceo rrneescdirduiefirtet, hdae nifds p dwaiswec hncaionrnggseild oiescr aretedilo aintfe sdai srtcoeh fsaaprrgawee nnisoi nurggel hatferdo mto tshpeawrivneinr–g locations. Answerrilneosgce arttvhiooesinres t.qr Aaunnesssitwtiiooennrsinz wogn itlehl esinusefco hqrmuthe asmttitaohneasrg ewewmilole uinnltdf odbreemcia sdmioeanqnsua asgtueecmdhr eainfstt dhdoieswctai sntiocoe nsfo srufcrhee aesm hborwyo tso manage discharget moanasdun rarvegsieve dreviasocnihrd awrragetceer aru-nistdu, rrafenasdceerw vleoevirec lowsn atsotie dpre-rsoruemdrfoaitfceed s liuesccvcheealssrs gtfoue pli srreorcmerluaoittteemd seutnoctc seopsfs afwuln riencgruloitcmateinotn os.f progeny from haAtpcnhrosewgryee-nroyirn igfrionthm ep sahellaqitduc hesestrtuiyor-gnoesroigwni nila lnpidna floltihrdme rsetmbuyrag nieanogcnre emaanseend t tthdheee crlieiskbioeyln ihisnoscourdecah soeaf sthheo wliktoelimhoaonda goef recovery. driesccohvaerrgye. and reservoir water-surface levels to promote successful recruitment of progeny from hatchery-origin pallid sturgeon and thereby increase the likelihood of recovery. 2. Materials and M2e. tMhoadterials and Method 2.1. Study Area 22..1M. Satteurdiya lAsraena d Method The study ar2e.a1 .iSs tTluohdceya tsAetruded aiyn athreea Gisr elaotc aPtleadin isn M thaen aGgreemate nPtl aUinnsi tM daenscargiebmede nbty U thneit described by the United States Fish UanidtTe Whde iSlsdttaultidfeesy SFaeirsrehva iiacsenl do(Uc WaStFeildWdliSinf,e t2 hS0ee1Gr4v)r iec[a1et8 (P]U laaSniFndWs cMSo,na 2nsi0as1gt4se )mo [fe1 nt8h]t eUa nMndiit scdsooensucsrriisi btse dofb tyhteh eMUisnsioteudri River from the upsSRttraievtaeemsr Ff erinoshdm ao tnfh dFeo Wurpti lsPdterlciefkae mRSee ersnvedricv eoof(i UrF otSorF tMW PoeScr,ko2n 0Ry1e 4Ds)ea[r1mv8o ](irrai ntvode Mrc kooinlrosoimnstyes tDoerfa mt[hrk e(mrMiv] iesrs okuilroimReivteerr [frrkom] 3010 to rkm 3388)t 3ha0en1du0 ptthsote rr eMkamma r3iea3ns8 d8R)oi vafenFrdo f rrtthoPem eM ctkhaerR iaceoss neRrflviuvoeeirnr cftreoo wMmio tthrho etnh cyeo DnMflaimusseon(urcirevi ewRriiktvhiel rot hmtoee Mteris[srokumri] R30iv1e0r ttoo Tiber Dam (rkm 0rT ktimob er3rk3 mD88a )1ma2n 6(d,r kFtmhige u0Mr eta o1r i)ra.k sTmRh ie1v 2es6rtu,f rdFoyimg uatrrheeae 1 cd)o. enTsflchrueieb snetcdue dhwye iratehr ertaeh pedrMeessicesrnsiobtsue drthi hRe eivree rrteopTreibseenr tDs atmhe furthest upstream( frdukirmstthr0iebtsuot turiokpnms tor1ef2 ap6m,aFl ldiigdisu strrteiub1ru)g.teiToohne iosnft utphdaeyl lMiadri essatsuodruegrseic orRinbiv einedr t hbheaers eMinrei s[p1sro8e]us. erin Rtsivtheer fbuarstihne [s1t8u]p. stream distribution of pallid sturgeon in the Missouri River basin [18]. Figure 1. Map of theF FMiigguisursreoeu1 1.r.i MRaiavppe oro fffr tthohmee Mthiisess srooiuvuerriri– RRrieivvseeerrr vffrorooirm tr ttahhnees rriitivivoeenrr– –zrroeesnseeerr vavtoo iFirro trtrrta aPnnessciitktiio oRnne zszeoornnveeo aiartt, FFoorrtt PPeecckk RReesseerrvvooiirr,, Montana, USA, to MMoTor,onUntaSynA Da,,a tUomSM, AMo, rotoonn tyManDoara,o mUn,ySMA D T(a,rmiUv,Se MrA ko(irnliotvamenreatk,e iUrlo S[mrAke mt(er]ir v3[er0rk1 m0k–i]lrok3m01e 0t3e–3r8 k[8mr)k, m3a3n]8 d38 0)t,h1a0en– drktmhe 3M38a8r)i,a asnRdiv tehre Marias River from tfhMroeam croiatnhsfl eRucievonencrfle fu rwoemnitch et htwhe eict ohMntflihsuseoeMnucrieis sRwoiuivtrheir t Rhtoiev MTeribisteosro TuDirbaie mRri,Dv Mearmo tno,t MTanibTae,, rU DSSAam ((,rr kMkmon 00t–a–rnkam, U1S2A6) .(rDkmam 0s–rakrme rkm 126). Dams are d1e2n6o).tt eDeda bmys ▬are ,a adnneddn popotoeinidnt sbts yoo f▬ frre,e faefenrreden npcceoe ianartrese dodefe nrneoofteteerdde nbbcyye ○ #a.r .e denoted by ○. The population oTfT hhpeeap llpoidopp usultalutairtogionenoo nfo pfi anpl latidhlleisd tMu sritgsuesroognueroiin R thiinve eMtrh ieus spMosutirsresiaoRmuivr ieo rfR uiFvpoesrrtt r euPapemsctkro efaFmo rtoPf eFcokrRt ePseecrk- Reservoir is bounvRdoe irsbeiysr vbaooniutrh nirdso pboyoguaenntdihc r bouypp osagtnretenhairmcoup paonsgtderne adicmo wuapnnssdttrrdeeaoamw n asbntardrera imderosbw. anUrrspitersretsar.emUa mpbs atrreiaemrs.m Uopvestmreeanmt movement beyonbdme yorovinvedmerre invktei rlobkmeilyeootmenred t e3r3i38v38e8 r8 isiks ilpporrmeevveeetnenrtte edd3 3b8yb8yM ioMsr oopnrryoenvDyea nmtDe.adPm rib.o yrP troMiocoro rnotsnotr yu cDtioanmo. f PMroiorro ntyo Dam, upstream movement was naturally prevented by the Great Falls of the Missouri River fewer than 7 km upstream of Morony Dam [18]. Downstream movement of pallid sturgeon is limited by the transition to lacustrine conditions at the headwaters of Fort Peck Reservoir near river kilometer 3010. The location of the Fort Peck Reservoir river–reservoir transition zone varies depending on reservoir elevation. Fishes 2023, 8, 243 4 of 22 Discharge in the Missouri River upstream of Fort Peck Reservoir is influenced by unregulated tributaries (i.e., the Smith, Teton, and Judith rivers; and the Belt, Arrow, Dog, and Cow creeks [25]), impounded tributaries, and mainstem impoundments. Upstream of Fort Peck Reservoir, the mainstem Missouri River contains nine dams. Eight of the nine dams have negligible influence on the hydrograph because outflows are maintained roughly equal to inflows [26,27]. Canyon Ferry Dam (rkm 3626) is the exception and is used to store water and regulate discharge in the Missouri River [28]. Tiber Dam on the Marias River and Gibson Dam on the Sun River are also used to store water and regulate discharge, which can reduce discharge in the Missouri River during the spring and summer [28]. Peak discharge typically occurs between late May and late June in the Missouri River. The first five mainstem impoundments upstream of Fort Peck Reservoir have little effect on the downstream water temperature other than reducing the daily variation in water temperature [29]. The effects of the Canyon Ferry, Hauser, and Holter reservoirs have not been thoroughly evaluated. Water temperature in the Marias River can be decreased by hypolimnetic water releases from Tiber Dam [30]. However, use of the surface spillway, an auxiliary water outlet completed at Tiber Dam in 1969, or a combination of the two could counteract cold-water temperatures from the hypolimnetic release. Substrate within the Missouri River upstream of Fort Peck Reservoir transitions from larger substrate in the upstream reaches to smaller substrate in the downstream reaches. Substrate is primarily cobble from river kilometer 3340 to 3130 [31]. At river kilometer 3,130, composition shifts to mostly gravel for several kilometers before transitioning into fine and sandy substrate [31]. Turbidity and substrate are likely influenced by the upstream impoundments, as sediment trapping at impoundments is a ubiquitous occurrence, and mitigation of sediment trapping is typically not focused on returning downstream sediment loading to preimpoundment conditions [32]. 2.2. Sampling Pallid sturgeon were sampled between early May and late July of 2018 and 2019. Pallid sturgeon targeted for sampling had been previously radio telemetered by Montana Fish, Wildlife & Parks as part of a concurrent study [22]. Pallid sturgeon were captured during the prespawning season (i.e., prior to the peak of the hydrograph) to determine sex and stage of maturity, and spawning-capable females were recaptured at the end of the spawning season (i.e., when water temperatures neared 24◦C) to determine ovulatory outcome. Discharge and water temperature were used to define the spawning season because spawning of pallid sturgeon typically occurs in late spring to early summer on the descending limb of the hydrograph [33,34], and water temperatures during spawning are estimated to be 12–24 ◦C based on embryo survival [35]. Prespawning-season sampling was prioritized using reproductive assessment data from 2011 through 2017. Pallid sturgeon known to be female that experienced reproductive activity in the past were considered high priority and were targeted for recapture first. After sampling high-priority pallid sturgeon, other known females were targeted. Male pallid sturgeon and pallid sturgeon of unknown sex were assigned lower priority and were sampled opportunistically. Pallid sturgeon locations were estimated using radio telemetry, and trammel nets 45.7 m long and 1.8 m deep with a 10.16 cm inner bar mesh and a 25.4 cm or 20.32 cm outer bar mesh were used to capture relocated pallid sturgeon. Smaller mesh trammel nets 45.7 m long and 1.8 m deep with a 5.08 cm inner bar mesh and a 25.4 cm outer bar mesh were occasionally used if the larger mesh trammel nets were ineffective at capturing an individual. Biological samples were collected from all captured pallid sturgeon. Handling and sampling procedures conformed to protocols developed for pallid sturgeon [36]. Blood was sampled from the caudal vasculature of each pallid sturgeon using a 3 mL syringe. Blood samples were immediately transferred to a 7 ml lithium heparinized vacutainer, stored in a cool environment, and transported to the field station the same day. Once at the field station, blood samples were centrifuged at 1228× g (relative centrifugal force) Fishes 2023, 8, 243 5 of 22 for 5 min to separate blood plasma from red and white blood cells. Blood plasma was transferred to 1.5 mL vials and stored at −20 to −80 ◦C until analyzed at the USFWS Bozeman Fish Technology Center. Gonadal tissue was sampled from pallid sturgeon of unknown sex, and ovarian follicles were sampled from females that were known or expected to be spawning capable. A small abdominal incision (1–2 cm) was made anterior to the urogenital pore between the midline and the ventral scutes. An otoscope was used to identify the gonad, and gonadal tissue samples were taken through the otoscope specula using a Miltex biopsy cup [37]. The otoscope speculum was angled to collect gonadal tissue from three different locations to account for gonadal heterogeneity. All tools used for the collection of gonadal tissue were disinfected with 70% isopropyl alcohol and were rinsed with sterile saline prior to use. Incisions were closed with 1–3 evenly spaced single interrupted sutures using size 0 absorbable suture material attached to a CP-1 suture needle (Ethicon PDS*II). Ovarian follicles and gonadal tissue were preserved in 10% phosphate-buffered formalin. 2.3. Sex and Stage Determination Blood, ovarian follicles, and gonadal tissue were analyzed at the USFWS Bozeman Fish Technology Center. Sex steroids (testosterone [T] and estradiol-17β [E2]) were extracted from blood plasma using methods described in Fitzpatrick et al. (1987) [38]. An extraction solvent (2 mL of diethyl ether, extracted twice) was added to tubes with 100 µL of the plasma and vortexed. The aqueous phase was removed by snap-freezing with liquid nitrogen, and ether was allowed to evaporate overnight in a chemical hood. The extract was reconstituted in 1 mL of phosphate-buffered saline with gelatin (PBSG). Following this, 10 or 50 µL of reconstituted steroid extract was analyzed using radioimmunoassay as described in Fitzpatrick et al. (1986) [39] and modified by Feist et al. (1990) [40]. A slightly more concentrated charcoal solution (6.25 g charcoal and 4.0 g dextran/L PBSG) was used for all assays. Testosterone and E2 concentrations were validated by verifying that serial dilutions were parallel to standard curves. Recovery efficiency was determined by adding tritiated steroids to tubes containing plasma (n = 4), which were extracted as described above. Recovery efficiencies were 91–95% for T and 83–93% for E2. All steroid assay results were corrected for recovery. Nondetectable plasma sex steroid concentrations (i.e., not quantifiable) were assigned half of the minimum quantifiable concentration for statistical purposes (0.10 ng/mL for T and 0.05 ng/mL for E2) [41]. The intra- and interassay coefficients of variation for all assays were less than 5% and 10%, respectively. Plasma sex-steroid concentrations were used to assign sex and stage of maturity to pal- lid sturgeon prior to spawning. Concentrations of T greater than 38 ng/mL and E2 less than 0.3 ng/mL were used to assign a reproductively active male pallid sturgeon, with reproduc- tively active male pallid sturgeon considered to be spawning capable. A spawning-capable male pallid sturgeon based on steroid concentrations would have testicular cysts with germ cells that were meiotic (spermatocytes, spermatids, and/or spermatozoa). Concentrations of T greater than 10 ng/mL and E2 greater than 0.3 ng/mL were used to assign a repro- ductively active female pallid sturgeon. Reproductively active females were vitellogenic or spawning capable (Figure 2, Level 4) and were differentiated via collection of ovarian follicles that were examined under a microscope, processed histologically, or both. Vitel- logenic females would not be able to spawn during the year they were sampled and were not included in this study. Reproductively active females classified as spawning-capable females were capable of spawning during the year they were sampled; however, not all spawning-capable females successfully spawned. Therefore, an additional subclassification was added (Figure 2, Level 5), and during analyses females that underwent mass ovarian follicular atresia (hereafter referred to as atretic females) were grouped separately from females that successfully ovulated (hereafter referred to as spawning females). Histolog- ical analysis of gonadal tissue collected postspawning season was used to differentiate spawning females from atretic females. During histological analysis, postovulatory ovarian follicles indicated successful spawning (spawning females), and mass ovarian follicular Fishes 2023, 8, x FOR PEER REVIEW 6 of 24 classified as spawning-capable females were capable of spawning during the year they were sampled; however, not all spawning-capable females successfully spawned. Therefore, an additional subclassification was added (Figure 2, Level 5), and during analyses females that underwent mass ovarian follicular atresia (hereafter referred to as atretic females) were grouped separately from females that successfully ovulated (hereafter referred to as spawning females). Histological analysis of gonadal tissue Fishes 2023, 8, 243 collected postspawning season was used to differentiate spawning females from a6torfe2t2ic females. During histological analysis, postovulatory ovarian follicles indicated successful spawning (spawning females), and mass ovarian follicular atresia (atretic females) was aitnredsiicaat(eadtr betyi c>5fe0m%a olef sth) ew oavsairniadnic faotleldiclbeys u>n5d0%ergoofinthge aotrveasriaia [n42fo] lalnicdle isnduincdaeterdg osipnagwantrine-g sfiaai[l4u2r]ea. Ondnein hdaictcahteedrys-poarwigninin mgafaleil puarell.idO nsteuhrgatecohne rcyla-ossriifigiendm asa lreeppraolldiducsttuivregleyo ancctilvases uifiseindg asstereropirdo dcuocntciveenltyraatciotinvse ubsuitn gwsittehroouidt ccoonrrceesnptorantdioinngs bguotnwaditahlo utitsscuoerr ewspaso nrdeminogvgeodn afrdoaml tiasnsualeywsisa sdrueem toov ae dtoftraol mlacakn aolfy smisodveume teonta dtoutrailnlga cskpaowf mnionvge smeaesnotnd u(sreien gHsoplamwqnuiinstg este aa-l. s2o0n19(s e[e22H])o. lmPaqluliidst settuarlg.eo2n01 9th[a2t2 ])h.adP alcloidmsptluertgeedo nputhbaetrthya—dacso mdeptleertmedinpeudb ebryty —praiosr dseatmerpmliinnged—bwyepreri ocor nsasimdperliendg r—epwreordeuccotnivseidlye rmedatruerper o(Fdiugcutrive e2ly, Lmevateul r1e) (iFrrigeuspreec2t,ivLee voef lt1h)e irrreepsrpoedcuticvteivoef ctlhaessriefipcraotdiounc taivt eancyla gssivifiecna ttiimone.a t any given time. FFigiguurere2 2. .H Hieierararcrchhicicaal lc lcalasssisfiificacatitoionno of fr erpeprordoduuctcitvievleylym matautruerep aplalildlids tsutrugregoeno.n. 22.4.4. .T Trarcakcikningg PPaalllildids stuturrggeeoonnw weerreet rtraacckkeedd foforrt htheed duurraatitoionno offt hthees pspaawwnnininggs seeaassoonnu ussininggr araddioio tetelelemmeetrtyrye eqquuipipmmenent.t.T Trarcakckininggb begegananin int htheefi fiftfhthw weeekeko of fM Maayyi nin2 2001188a annddt htheef ofouurrththw weeeekk of May in 2019 before peak discharge. Tracking ended in the first week of July in 2018 and the second week of July in 2019 after all spawning-capable female pallid sturgeon had been determined to have experienced ovulatory success or failure. Tracking equipment included boat-mounted, handheld, and land-based receiver systems with three- or four-element Yagi antennas. Each system used Lotek SRX 400 telemetry receivers (Lotek Wireless, Inc.; Newmarket ON, Canada) to receive and decode radio transmitter signals. Boat-mounted and handheld systems were used manually and provided real-time information about transmitters in range. Land-based systems were autonomous and recorded data for each Fishes 2023, 8, 243 7 of 22 transmitter that passed through the field of detection. Transmitters had a unique code allowing each pallid sturgeon to be individually identified. Signal directionality and signal strength were used to maximize accuracy when lo- cating a pallid sturgeon. Direction of a pallid sturgeon was determined by rotating the handheld antenna until finding the bearing that resulted in maximum signal strength. The boat was then moved in the direction of the pallid sturgeon until passing over the fish, which was indicated by a sudden decrease in signal strength. When the boat was positioned over a pallid sturgeon, a GPS location was recorded. Pallid sturgeon were systematically tracked based on priority given the sex and stage of maturity. The relocation of spawning-capable female pallid sturgeon was attempted every two days. Relocation of reproductively mature male pallid sturgeon was attempted once per week. Land-based telemetry stations were used to aid in searching for undetected pallid sturgeon by documenting if a fish left the last known location. In addition to this study, location data collected by Holmquist et al. (2019) [22] were combined with these data to produce larger sample sizes and better answer the questions posed. Holmquist et al. (2019) [22] used similar methods to this study and located pallid sturgeon weekly. Individual pallid sturgeon located for fewer than half the weeks tracked in a given year were excluded from analyses because minimal data on individual fish may not accurately represent movement or location of the individual. 2.5. Data Analysis Locations of pallid sturgeon collected during this study and by Holmquist et al. (2019) [22] were used to estimate movement rates of pallid sturgeon in the Missouri River upstream of Fort Peck Reservoir. Movement rates were quantified for each relocation of individual pallid sturgeon as net movement per day (km/day). Then, the median net- movement rate per day was calculated for each individual within specified reproductive classifications (i.e., atretic female, spawning female, or spawning-capable male). A negative median net movement rate indicated a downstream movement, and a positive median net movement rate indicated an upstream movement. Total movement by individual pallid sturgeon was calculated as the sum of distances between locations (i.e., river kilometers) throughout a putative spawning season. Total movement for pallid sturgeon tracked during two putative spawning seasons was determined by calculating total movement for each season and averaging the values. The median net movement rate and total movement were calculated using the minimum distance moved between relocations because pallid sturgeon might have made undetected movements between relocations. The median net movement rates and total movements of pallid sturgeon were summarized using reproductive classifications (i.e., atretic female, spawning female, and spawning-capable male). Summarized data were visualized in box plots for comparison, and the interquartile range (IQR) was used to describe variation within classifications. Interquartile ranges were calculated as the difference between the 25th and 75th quantiles (i.e., the difference between the lower and upper quartiles). Location data collected in this study and by Holmquist et al. (2019) [22] were used to summarize and compare locations among reproductive classifications. The median location was calculated as the median river kilometer among all recorded locations of an individual pallid sturgeon during specified reproductive classifications (i.e., atretic female, spawning female, and spawning-capable male). The maximum upstream location was calculated as the most upstream location among all recorded locations of an individual pallid sturgeon during specified reproductive classifications (i.e., atretic female, spawning female, and spawning-capable male). Median locations and maximum upstream locations of pallid sturgeon were summarized for atretic females, spawning females, and spawning-capable males. Summarized data were visualized in box plots for comparison, and the IQR was used to describe variation within classifications. Putative spawning reaches of pallid sturgeon that successfully ovulated were esti- mated using kernel densities. Using ArcMap 10.5.1 (ESRI, Redlands, CA, USA), a kernel- Fishes 2023, 8, x FOR PEER REVIEW 8 of 24 was calculated as the most upstream location among all recorded locations of an individual pallid sturgeon during specified reproductive classifications (i.e., atretic female, spawning female, and spawning-capable male). Median locations and maximum upstream locations of pallid sturgeon were summarized for atretic females, spawning females, and spawning-capable males. Summarized data were visualized in box plots for Fishes 2023, 8, 243 comparison, and the IQR was used to describe variation within classifications. 8 of 22 Putative spawning reaches of pallid sturgeon that successfully ovulated were estimated using kernel densities. Using ArcMap 10.5.1 (ESRI, Redlands, California, USA), dae knesritnyeel-sdtiemnsaitteym esatpimwaates mcreaapt ewdafsr cormealtoecda ftrioonms loofciantdioivnisd oufa ilnsdpiavwidnuianlg spfeamwanleinsga ffteemr athlees ianfitteiarl tpheea kinoitfiatlh epehaykd roofg trhaep hh.yTdhroegcrealpl hv.a lTuhees wceellr evascluaelesd wtoerree pscreasleedn tttoh erecpornetsiennuto tuhse rceolanttiivneudouens srietlyatbiveetw deeennsiotyn beeatnwdeezne roon,ew ahnedr ezearov,a wluheeoref ao nvealwuea os ft honeem waaxsi mthuem marexliamtiuvme dreenlastiitvyeo dfelnocsiattyio onfs loocfatthioenins doifv tihdeu ainldainvdidzuearlo arnedp rzeesreon treedprneoselnotceadti onnos l.oRcaeaticohness. Rsceoarcihnegs rsecloatriivneg dreelnastiitvye vdaelnuseistya pvpalruoeasc haipnpgroonacehainngd othnee aarneda btheetw aereean breeatwcheeesn srceoarcinhgesr esclaotriivneg dreenlastiitvyev adleunessitayp pvraolaucehsi nagpopnreoawcehriengin coluned edweinret hienecslutidmeadt edinp uthtaet ivesetismpaawtendi npgurteaatcivhe. Lsopcaawtionninsgo fremaachtu. rLeomcaatlieopnasl loidf mstuatrugreeo nmwaleer epoavlleidrl asitdurognetohne wkeerrnee lodveenrlsaiitdy mona pthteo vkeerrinfyel ifdmenastiutyre mmaapl etos hvaedrifoyc cifu mpiaetdurteh emdaelleisn heaatde docscpuapwiendin tgher edaeclhinese.ated spawning reaches. SSuubbssttrraattee imimaaggeerryy( F(Figiguurere3 )3)w wasacs oclloelcletecdtedin i2n0 12901in9 tihnr etherseues psuecstpeedctsepda wspnaiwngnilnog- claotciaotniosnosf opfa lplidallsidtu rsgtueorgne—ono—neo2n0e1 820s1u8s pseucstpeedctsepda wspnainwgnilnogca tlioocnataionnd atwndo 2tw01o9 2su01s9- pseucstpedectsepda wspnaiwngnilnogc aloticoantsio. nLs.o Lcaotciaotniosnws ewrerceo cnosnidsiedrerdeds ussupsepcetcetdeds pspaawwnnininggl oloccaatitoionnss wwhheenns sppaawwnniningg--ccaappaabblelef feemmaalelep paallildids sttuurrggeeoonnw weerreeo obbsseerrvveeddw wiitthhiinn0 0.2.255k km o offm maattuurree maalelep paallildids sttuurrggeeoonnl oloccaatitoionnss. .A llls suussppeecctteedds sppaawnniinnggl looccaattiioonnsst thhaattw eerreem aappppeeddw weerree wwitithhininp puutatatitvivees sppaawwnnininggr reeaacchheessd deelilnineeaateteddb byyk keernrneel-ld-deennssitiytya annaalylysseess. .S Suubbssttrraatteew waass maappppeeddf foorrt thheef fuulllw wididththo offt htheer riviveerra anndd~ ~00..55k km a abboovvee aannddb beelloow s suussppeecctteedds sppaawwnniningg llooccaattiioonnss.. SSubssttrratte maappiing wass ccoomplletted bbyy aaddaappttiinngg meetthhooddss ddeessccrriibbeedd iinn KKaaeesseerr LLititttss( (22001100))[ [4433]].. A ssoonaarr aand GPSS uniitt ((Hummiinbiirrd HELIIX 77 CHIIRPP MEEGA SSII GPPSS G33)) wwaass uusseedd ttoo rreeccoorrdds sididee--ssccaann ssoonnaarr iimaaggeess ooff tthhee rriivveerrbbeed aand rreeccorrd GPSS ccoooorrdiinattes.. IImaaggee rerecocordrdininggssw wereereg egoeroerfefrernecnecdedu suinsignSgo SnoanrTarRTXR(XS o(nSaornTaRrTXR, XH oHnonluollu,luH IH, UawSaAii), aUnSdAim) apnodrt eidmtpooArtercdM taop A1r0c.5M.1a.pS u1b0.s5t.r1a.t eSutybpsetrsawtee rtyepmesa nwuearlley mdealninueaalltyed daeslipnoealytegdo nass rpeporlyesgeonntsin rgesparnedse,ngtrianvge ls, and, cgorbabvlel(,F aignudr eco3)b.bDlee l(iFniegauterde s3u).b sDtrealtienetyapteeds wsuebrestvraerteifi teydpaets owpeproer tvuenriisfiteicdl oact aotpiopnosrbtuynciostlilce cltoincagtisounbss tbryat ceoslalemctpilnegs .sTuhbestsruartefa scaemarpelaeso.f Tthee psuorlyfagcoen asraeta eoafc hthsei tpeowlyagsocnasl cautl aetaecdh, sainted wthaes rcealalctuivlaetpedro, paonrdti othneo rfeelaatcihves upbrostproatretiotynp oefw eaasche sstuimbsattreadte. Tthyepec ewnatrsa elstteimndaetnedcy. Tahned cveanrtiraatli otennodfetnhcey parnodp voartriioantiaolns oufb tshtrea pterotyppoertsiownearle scuhbasrtaracttee rtiyzpedes uwsienrge tchhearmacetdeiraizneadn udsIiQngR t.he median and IQR. FFiigguurree3 3.. EExxaammppllee ooff ssuubbssttrraattee iimmaaggeerryy aanndd ddeelliinneeaattiioonn ooff ssuubbssttrraattee ttyyppeess ((ssaanndd,, ggrraavveell,, aanndd ccoobbbbllee)).. SSuubbsstrtraatetei mimaaggeerryyw waassc coollleleccteteddi nint htheeM MisisssoouurriiR Riviveerrn neeaarrr riviveerrk kiliolommeeteterr3 3111177. .S Suubbssttrraatteei mimaaggeerryy wwaassc coolllelecctetedda annddp poolylyggoonnssw weerreed deelilnineeaateteddu ussininggm meeththooddssa addaappteteddf rforommK Kaaeeseserr& &L Lititttss( 2(2001100))[ 4[433].]. Mean daily discharge data from the U.S. Geological Survey stream gages were used to characterize discharge in the Missouri and Marias rivers from April through July [44]. The mean daily discharge in the Missouri River near Landusky, MT, USA (stream gage 06115200), from 1956 through 2019 was summarized as the median monthly discharge by calculating the median of the daily mean discharge values for each month of each year. The mean daily discharge in the Marias River near Loma, MT, USA (stream gage 06102050), from 1960 through 2019, excluding 1973 through 2001 when discharge data were Fishes 2023, 8, 243 9 of 22 not recorded, was summarized as historical median monthly discharge by calculating the median of daily discharge values for each month of each year. Historical monthly discharge values were summarized for each river by calculating the 10th, 25th, median, 75th, and 90th quantiles for each month, and the summary of historical discharge data were compared with the discharge when tracking occurred. The mean daily temperature data from the U.S. Geological Survey stream gages were used to characterize the temperature in the Missouri and Maris rivers from April through July during years that tracking of pallid sturgeon occurred. The mean daily temperature data for the Missouri River near Landusky, MT, USA (stream gage 06115200), and the Marias River near Loma, MT, USA (stream gage 06102050), were summarized as the median monthly temperature from April through July of 2015, 2016, 2018, and 2019 by calculating the median of daily temperature values for each month of each year. 3. Results In general, the median discharge of the Missouri River declined from the mid-1960s to 2019, and discharge during the spring was particularly low during the early 2000s (Figure 4). The median monthly discharge in the Missouri River in 2015 and 2016 was below the historical median from April through July and was below the historical 25th quantile in May 2015, June 2016, and July 2016 (Table 1). Conversely, the median monthly discharge in the Missouri River in 2018 exceeded the historical 90th quantile in April and May, exceeded the historical 75th quantile in June, and exceeded the historical median in July (Table 1). In 2019, the median monthly discharge in the Missouri River exceeded the historical 75th quantile in April and May and was between the 25th and 75th quantiles in June and July (Table 1). During April, May, and June, the median monthly water temperature in the Missouri River was slightly warmer in 2015 and 2016 than in 2018 and 2019, but in July, the median monthly water temperature was coldest in 2015 and 2019 and warmest in 2016 and 2018 (Table 2). Table 1. The 10th, 25th, median, 75th, and 90th quantiles of monthly median discharge (m3/s) in the Missouri River from 1956 through 2019 from April through July, and the median discharge from April through July for 2015, 2016, 2018, and 2019. Data from the U.S. Geological Survey stream gage station near Landusky, MT, USA (stream gage 06115200). 1956–2019 2015 2016 2018 2019 Month 10th 25th Median 75th 90th Median April 152.3 196.4 246.2 293.1 408.3 210.7 205.3 511.1 406.3 May 187.3 253.2 351.1 441.0 610.2 209.5 280.9 869.3 487.0 June 194.3 299.5 407.1 653.1 868.8 301.6 238.7 831.1 375.2 July 139.8 173.5 248.2 332.0 512.0 184.3 171.3 322.8 303.0 Table 2. Median water temperature (◦C) in the Missouri River from April through July of 2015, 2016, 2018, and 2019 excluding April of 2015 and 2018 when the water temperature data were not collected and June of 2018 when the stream gage was inoperable due to flooding. Data from the U.S. Geological Survey stream gage station near Landusky, MT, USA (stream gage 06115200). Median Water Temperature (◦C) Month 2015 2016 2018 2019 April – 11.4 – 10.4 May 14.0 14.8 13.8 12.0 June 19.9 20.7 – 19.0 July 21.4 23.8 23.5 22.7 Fishes 2023, 8, x FOR PEER REVIEW 10 of 24 F ishes 2023, 8, 243 10 of 22 Figure 4. Median discharge (m3/s) in the Missouri River by month from 1956 through 2019. Ad- dFitiigounrael ly4,. tMheemdieadn iadnisocfhamrgoen t(hmly3/ms) eidni atnhed iMschisasroguerif rRomivepr reb-yC amnoynotnh Fferrormy D1a9m56 (1th9r3o4u–g1h95 32)01is9. inAcdludditeido.nDalalyta, tfhroem mtehdeiaUn. So.fG meoonlotghilcya lmSeudrivaeny dsitsrechamarggea gfreosmta tpioren-CneaanryLoann Fdeursrkyy D, MamT, (U19S3A4–(s1t9r5e3a)m is giangcelu0d6e1d15. 2D0a0t)a. from the U.S. Geological Survey stream gage station near Landusky, Montana, USA (stream gage 06115200). In general, the spring discharge in the Marias River has been relatively low throughout thTeab2l0e0 10. sTahse c1o0tmh,p 2a5rtehd, mtoedthiaend, 7is5cthh,a arngde 9d0uthri qnugatnhteile1s9 o6f0 ms aonntdhl1y9 m70esd(iaFnig duisrceh5a)r.gTe h(me 3m/se) dini athne mMoinsstohulyri dRiisvcehra frrgoemi n19t5h6e tMhroaurigahs R20i1v9e rfrwomas Abpertiwl tehernouthghe hJuislyto, raincda lth25e tmh eadnidan7 d5tishchqauragne tfirloesm frAopmrilA thprroiul gthhr Jouulyg hfoJru 2l0y15o,f 2200116,5 2a0n18d, awnads 2a0t19o.r Dbaetlao fwromth eth2e5 Uth.Sq. Guaeonltoilgeicfarlo SmurAvepyr siltrteharmou ggahge station near Landusky, Montana, USA (stream gage 06115200). June of 2016 (Table 3). Conversely, the median monthly discharge in the Marias River reached an unprecedented h1i9g5h6i–n2021091 8 exceeding the hist2o0ri1c5a l 90t2h0q1u6a ntile20d1u8r ing A20p1r9i l anMdoMntahy an1d0tehx ceed2i5ntgh theMmedeidainan i7n5Jtuhn e an90dthJu ly (Table 3). TMheemdieadni an monthly disAchparrilg e in15t2h.e3 Ma1ri9a6s.4R ive2r4i6n.22 01929r3e.m1 aine4d08b.3e twe2e1n0.t7h e h2is0t5o.r3i cal 52151th.1 and47056t.3h quaMntaiyle s fro1m87.A3 pril2t5h3r.o2u gh3J5u1n.1e of 2404119.0b ut r6o1s0e.2t o ab2o0v9e.5 the 92080th.9q uan86ti9le.3i n Ju4l8y7.o0f 201J9u(nTea ble 31)9.4T.3h e me2d99ia.5n mo4n0th7l.1y tem6p5e3r.a1t ure8in68t.h8e Ma3r0ia1s.6R ive2r3in8.72 015 8w3a1s.1w ithi3n75th.2e range of the temperature in other years of the study. However, from April through July of 2016Ju, ltyh e me1d3i9a.n8 mon1t7h3ly.5 temp24e8ra.2tu re w33a2s.0h ighe5r1t2h.0a n in128041.53, 201187,1a.n3d 2031292(.T8a ble340).3.I0n 2018 , the median monthly temperature in the Marias River was lower from April through June than it was during the other years of the study but reached temperatures similar to those of other years during July (Table 4). In 2019, the median monthly temperature was Fishes 2023, 8, 243 11 of 22 Fishes 2023, 8, x FOR PEER REVIEW 12 of 24 within the range of other years from April through June but was colder than in the other years of the study during July (Table 4). Figure 5. Median discharge (m3/s) in the Marias River by month from 1960 through 2019, ex cluding Figure 5. Median di1s9c7h3atrhgroeu (gmh32/0s0) 1inw thheen Mdisacrhiaarsg eRdivatear wbeyr emnoont rtehc ofrrdoemd. 1D9a6t0a ftrhormouthgehU 2.S0.1G9e, oelxocgliucadl iSnugrv ey 1973 through 2001 wstrheeamn dgaisgcehsatartgioen dnaetaar wLoemrea ,nMotT ,rUecSoArd(setrde.a mDagtaag efr0o6m10 2t0h5e0 )U. .AS.d dGiteioonloagllyic, athl eSmurevdeiayn of stream gage station mnoenatrh Llyommead,i aMnodnistcahnaarg, eUfSroAm (pstrere-Taimbe rgDaagme 0(16912022–0195506);. dAadtadrietcioorndaeldlya,t tUh.eS .mtheedGiaenol oogfi cal Survey stream gage 06102000) is included. monthly median discharge from pre-Tiber Dam (1922–1956; data recorded at U.S. the Geological Survey stream gage 06102000) is included. Fishes 2023, 8, 243 12 of 22 Table 3. The 10th, 25th, median, 75th, and 90th quantiles of the monthly median discharge (m3/s) in the Marias River from 1960 through 2019, excluding 1973 through 2001 when discharge data were not recorded, summarized by month, and the median discharge from April through July for 2015, 2016, 2018, and 2019. Data from the U.S. Geological Survey stream gage station near Loma, MT, USA (stream gage 06102050). 1960–1972 and 2001–2019 2015 2016 2018 2019 Month 10th 25th Median 75th 90th Median April 11.5 14.0 15.2 27.1 37.1 15.7 14.0 81.8 18.9 May 13.3 14.9 30.9 40.2 56.6 15.1 13.9 90.3 20.4 June 13.7 18.0 34.5 54.4 79.2 18.3 13.6 50.7 20.4 July 10.0 15.2 19.4 39.7 57.6 18.2 14.9 21.5 75.3 Table 4. Median water temperature (◦C) in the Marias River for April through July of 2015, 2016, 2018, and 2019. Data from the U.S. Geological Survey stream gage station near Loma, MT, USA (stream gage 06102050). Median Water Temperature (◦C) Month 2015 2016 2018 2019 April 10.7 11.0 6.0 9.6 May 14.7 15.6 11.9 12.3 June 20.3 20.4 16.5 18.9 July 20.8 22.8 20.0 19.9 Twenty spawning or atretic hatchery-origin pallid sturgeon were tracked during this study. Of the 20 pallid sturgeon tracked, two females were tracked during years that culminated in two different reproductive classifications (i.e., atretic and spawning) resulting in 22 individual classifications with tracking data—12 were classified as spawning-capable males, five as spawning females, and five as atretic females. The medians of median net movement rates were similar among the reproductive classifications and consisted of upstream movements less than 1 km/day (Figure 6). The median net movement rates had little variation within classifications, and all movements were between −3.3 and 4.1 km/day (Figure 6). The largest variation in median net move- ment rate was for spawning-capable males, with an IQR of 1.7 km/day. In contrast to the median net movement rates, the medians of total movement rates varied considerably among the reproductive classifications. Spawning female pallid sturgeon had the highest median total movement of 269.9 km, which was 176.3 km more than that of spawning- capable males (Figure 7). Atretic females had a median total movement of 142.3 km—less than that of spawning females but 48.8 km more than that of spawning-capable males (Figure 7). The total movement of atretic females had large variation with the 25th and 75th quantiles varying from 101.3 km to 221.6 km (IQR = 120.3; Figure 7). The median of the median locations of spawning females was within one km of the median of the median locations of spawning-capable males (Figure 8). The median locations of atretic females were highly variable (IQR = 203.0) compared to those of the spawning females (IQR 13.4) and spawning-capable males (IQR = 19.7). Furthermore, the median locations of atretic females overlapped the other classifications (Figure 8). The median locations tended to be in the lower reaches of the study area, with 19 of the 22 median locations downstream of Judith Landing (rkm 3194); however, three individual pallid sturgeon had median locations upstream of Judith Landing including one atretic female in the Missouri River, one atretic female in the Marias River, and one spawning-capable male in the Marias River (Figure 8)—all locations in the Marias River occurred in 2018 when discharge was high (Table 3; Figure 5). Interestingly, no pallid sturgeon had median locations between rkm 3126 and rkm 3293. Fishes 2023, 8, x FOR PEER REVIEW 15 of 24 Fishes 2023, 8, x FOR PEER REVIEW 14 of 24 Fishes 2023, 8, 243 13 of 22 Figure 6. Median net movement rate (km/day, circles represent individuals) for pallid sturgeon by Friegpurroed6u.cMtiveed icalansnsiefitcmatoiovne mduenritnrga tteh(ek pmu/tadtaivy,ec sirpcalewsnrienpgr esseeanstoinnsd oivf i2d0u1a5l,s )2f0o1r6,p 2a0ll1 id8, satunrdg e2o01n9b iyn rteFhpiegr uoMdreius 6scot. iuMvreei dcRliaiavsnes irnfi euctap mtsitoornevaedmu eroinnft g Froathrtete P(pkeumckt/a dtRiaveyse,e scrpivraocwilre.sn Iirnedgpirvseeisdaesunoatn lissn wdofiev2rie0d 1uc5laa,lss2s)0i 1fifo6er,d 2p a0as1ll 8iad,tra senttudicr 2gf0ee1om9na ilbney, tshrpeepaMwronidsisunocgut irfveiemR cialvaleesr,s iaufinpcdast trisoepnaam wdunorifninFggo-c rtathpPeae bpclkuet Ramteiasveleer .vs poBaiorwx. Ineninnddgivs sidreeaupsaorlenssswe noetfr e2th0ce1l a52s, 5s2ti0fih1 e6ad,n a2ds0 17a85t,rt heat niqcduf ae2nm0t1ia9lle eisn,, shtphoaerw izMnoiinnstsgaoluf leirmni eRasli aev,reera tnuhdpe smstpreeadawmiann ion, ftg hF-eco aurptp aPpbeelcrek wm Rhaeilsseke.ervrB oeoixxrt.e eInnndddsis vtoride tpuhraee llssa erwngteersteht oecbl2as5sestrhivfiaaetdnio dans 7n 5aott hrfueqtrituch afenermt tihlaealsen,, 1spawning female, and spawning-capable male. Box ends represent the 25th and 75th quantiles, hho .5ri ×z oinnttearlqliunaerstile range (IQR) from the 75th quantile, and the lower whisker extends to the smallest obosreizrvoanttiaoln li nnoesa areret htheem meeddiaiann, ,t htheeu uppppeerrw whhisiskkeerre exxtetennds to the largest observation no further than 1.5 × interquart ifluerrtahnegre th(IaQnR 1).5fr ×o mIQtRh efr7o5mth tqhue a2n5ttihle q, aunadn dtsi lteo. the largest observation no further than 1.5 × interquartile range (IQR) from the 75th quantile, andt thheel loowweerrw whhisiskkeerre exxtetennddsst otot hthees smmaalllelesstt oobbsseerrvvaatitoionnn noof ufurrththeerrt hthaann1 1.5.5× × IIQQRR ffrroom tthhee 2255tthh qquuaannttiillee.. Figure 7. Total movement (km; circles represent individuals) for pallid sturgeon b y reproductive cFlaigssuirfiec a7t. ioTnotdalu mrinogvetmheenptu (tkatmiv; ecisrpcalews nrienpgresseeansto innsdoivf i2d0u1a5l,s)2 0fo16r ,p2a0l1li8d, astnudrg2e0o1n9 biny trheepMrodisusoctuivrie Rcl Fiv aessification during the putative spawning seasons of 2015, 2016, 2018, and 2019 in the Missouri Riivgeu rruep 7st. rTeaomtalo mf FoovretmPeecnkt R(kemse;r vcioir. Individuals were classified as atretic female, spawning female, and srp auwpsntirnegam-ca opfa bFloermt Paelec.kB Roexsee rc nrvledossi rrr.e e pI pnrred es siev eindtntu ind thales iv2w ideruea lcsl)a sfosirfi pedal laids satturergtieco n by reproductive classification during the putative spawning seasons o5f t2h0a15n,d 27051t6h, 2q0u1a8n, tailneds, 2h0o freimzoanleta, lslpinaewsnairneg female, and spawning-capable male. Box ends represent the 25th and 75th quantile1s9, hin ×ortihzeo nMtaisl slionuersi tahRreievm etrhe deu ipamnst,ertdehaieamnu ,p otpfh eeFr owurtph piPseekrce krw eRhxetissekenredvrs oteiorx. ttehInneddlisav rigtdoeu satthloseb wsleaerrrvgeae tscitol anossbnisofieefrudvr atathisoe nra ttrhneaotni cf 1uf.e5rtmhaeirlne ,tt ehsrapqnau wa1rn.t5iil ne×g rifanentmegreaql(euI,Qa arRntid)le fs rproaamnwgtnehi ne(Ig7Q-5cRtah)p qfarbuolamen m ttihlaele,e a.7 Bn5odthxt heqneudalosn wrtieleper,r ewasnhednis tk tthehere el2ox5wttehen ard nswdth o7i5sttkhhee rqs umeaxanteltlnieldessts, o htboos retihrzveo ansttmiaolan llilneoests fouabretsh eerthvreat htimaonne d1n.io5a nf×u, rIttQhheRe r futrhpoapmne rt1h .5we ×2h 5iIstQhkReqr uf raeoxnmtei lntehd.es 2t5oth t qhue alnatriglee.s t observation no further than 1.5 × interquartile range (IQR) from the 75th quantile, and the lower whisker extends to the smallest observation no further than 1.5 × IQR from the 25th quantile. Fishes 2023, 8, x FOR PEER REVIEW 16 of 24 The median of the median locations of spawning females was within one km of the median of the median locations of spawning-capable males (Figure 8). The median locations of atretic females were highly variable (IQR = 203.0) compared to those of the spawning females (IQR 13.4) and spawning-capable males (IQR = 19.7). Furthermore, the median locations of atretic females overlapped the other classifications (Figure 8). The median locations tended to be in the lower reaches of the study area, with 19 of the 22 median locations downstream of Judith Landing (rkm 3194); however, three individual pallid sturgeon had median locations upstream of Judith Landing including one atretic female in the Missouri River, one atretic female in the Marias River, and one spawning- capable male in the Marias River (Figure 8)—all locations in the Marias River occurred in 2018 when discharge was high (Table 3; Figure 5). Interestingly, no pallid sturgeon had median locations between rkm 3126 and rkm 3293. Fishes 2023, 8, 243 14 of 22 Figure 8. Median location (rkm; circles represent locations in the Missouri River, and diamon ds Figure 8. Median locaretpiorense n(rtklomca;t iocnirscinletsh erMeparrieassRenivte rl)ofocraptiaollnidss tiunrg tehone bMy riespsrooduurcit iRveivcleasrs,i fiacnatdio nddiaurminognthdes represent locations in pthutea tMiveasrpiaaws nRinigvesera)s ofnosro pf a20l1li5d, 2 s0t1u6,r2g0e18o,nan bdy2 0r1e9purpostdreuacmtiovfeF ocrltaPsescikfiRceasetrivoonir .dIundriivnidgu tahlse were classified as atretic female, spawning female, and spawning-capable male. Box ends represent putative spawning sethaes2o5nthsa nodf7 52th01qu5a, nt2il0es1,6h,o ri2z0on1t8a,l lianensda re2t0h1e 9m eudipans,ttrheeaumpp eor fw hFisokrert exPteencdks toRtehseelarrvgoesitr. Individuals were clasosbifiseerdva taiosn antorefutricth efretmhaanle1.,5 s×pianwternqiunagrt ilfeermanaglee,( IQanR)df rsopmatwhen7i5nthgq-cuaapntailbe,lean md tahleel.o wBeorx ends represent the 25wthi sakenrde xt7e5ntdhs tqouthaenstmilaellse,s thoobsreirzvoantiotnaln olifnuertsh earrthea nth1e.5 ×mIeQdRiafrnom, tthe2 5uthppquearn twileh. isker extends to the largest observation no further than 1.5 × interquartile range (IQR) from the 75th Atretic females had the highest median maximum upstream location (median = 3292.6 rkm), quantile, and the lowesrp awwhniisnkge-rc aepxatbelnedmsa tleos thhaed stmheallolewsets ot b(mseerdviaanti=on3 1n6o2. 3furrktmh)e,ra tnhdasnp a1w.5n ×in IgQfeRm farloems the 25th quantile. were between the other classifications (median = 3207.0 rkm, Figure 9). In 2018, maximum locations were recorded for four pallid sturgeon in the Marias River—one spawning female, Atretic femalestw hoaadtre ttihc fee mhailgesh, eansdt omneesdpiaawnn inmg-acaxpimabluemma lue p(Fsigturerea9m). location (median = The putative spawning reaches for five spawning females were between rkm 3072 3292.6 rkm), spawannidngrk-mca3p1a41bl(eFi gmureasle10s anhdad11 ).thTeh e lloowweersbto u(nmd eodf sipaanw n=in g3r1e6a2ch.3es wrkemre )fu, rtahnerd spawning females wdeorwen bsteretawmeienn2 0t1h9et hoatnhienr2 c01la8.sIsnifi20c1a9t,itownosf r(eme emdbiaryno s=w 3e2re07ca.0pt urkremd d, oFwignustrea m9). In 2018, maximum loofctahteiopnutsa twiveersep arwecnoinrgdreedac hfoesr afnodurw peraellliindk esdtutorgaesopnaw inni ntghefe Mmaaleri(aIDs R8_i9v2e)rb—y one spawning femaglee,n etwticoa naatlryestisic[ 4f5e]m. Tahleess,u abnstdra toenwea sspsiamwilanrinamgo-cnagpthaebmleapped reaches and was predominantly composed of gravel and sand with a small amount mofacoleb b(lFei(gTaubrlee 59).)T. he furthest upstream mapped reach (rkm 3117.5–3119.0) contained the largest proportion of cobble (Table 5). Fishes 2023, 8, x FOR PEER REVIEW 17 of 24 Fishes 2023, 8, 243 15 of 22 Figure 9. Maximum upstream location (rkm; circles represent locations in the Missouri Ri ver Figure 9. Maximum uapndstdreiaammon ldoscraetpiroesne n(trklomcat;i ocnirscilnetsh ereMparreiasseRnitv elor)cfaotriopanllsi dinst utrhgee oMn bisysroeuprroid Rucitviveer calans-d diamonds represent loscifiactaitoionns diunr itnhget hMe apruitaasti vReivspearw) nfionrg pseaalsloidns sotuf r2g01e5o, n20 b16y, r2e01p8r,oadnduc2t0i1v9ei ncltahsesiMfiicssaotuiorin River upstream of Fort Peck Reservoir. Individuals were classified as atretic female, spawning during the putative spfeamwanlei,nagn dsespaaswonnisn go-fc a2p0a1b5le, m20al1e6. , B2o0x1e8n, dasnrdep 2re0s1en9t itnh eth25et hMainsdso75utrhi qRuiavnetirle us,phsotrrizeoanm- of Fort Peck Reservotailr.l inIensdarievitdheumalesd iawn,erthee ucplapsesriwfiehidsk earse xatetnrdestitco tfheemlaarglee,s t sopbsaewrvnatiinong nofefmurathleer, thaannd spawning-capable ma1l.e5 .× Binotxer qeunadrtisle rreanpgree(sIeQnRt) ftrhome t2h5et7h5t haqnuda n7ti5let,ha nqduthaenlotiwleesr ,w hioskreizr oexntetnadl sltiontehse samrael letshte median, the upper wohbissekrveart ieonxtneonfdursth teor ththane 1l.5ar×gIeQsRt forobmsethreva25ttihonqu annoti lefu. rther than 1.5 × interquartile range (IQR) from the 75th quantile, and the lower whisker extends to the smallest observation no further than 1.5 × IQRT farbolem5. tPhreop 2o5rttihon qouf asunbtsitlrea.t e types for three mapped reaches in the Missouri River upstream of Fort Peck Reservoir and the median of proportion of substrate type at locations with the interquartile The putative spraangwe n(IQinRg) inrepaarentheses. The river kilometer (rkm) of the upper and lower boundary of the mapped reaches iscdheenost efdo. rT hfievmea psppedarweanchiensgw efreemwiathlienst hwe peurtaet ivbeestpwaweneinng rrekamche s3(0se7e2 and rkm 3141 (FiguFrigeusr e1s 010 aandd1 11) a1n)d. wTehrees elloecwtederfo rbmoaupnpidng owfh esnpsapawwniinngg-c arpeabalechfeemsa lewpealrlied sfturrgtehoenr downstream in 2019w etrhe aobnse irvne d2i0n1te8ra.c tIinng 2w0it1h9m,a ttuwreom aflreepea lleidmstburrgyeoons. Twheemreap cpeadprteuacrheesdin dcluodwednas~t0r.5ekam of the putative spadwisntainncega broevaecanhdebse laowndw hwereeirnete rlaicntioknesdw etreoo bas esrvpeda.wning female (ID 8_92) by genetic analysis [45]M. aTppheed Rseuacbhs(trrkamt)e was similar amPoronpgor titohneS umbsatrpatpe Teydp ereaches and was predominantly composed of gravel and sandSa nwdith a small amGorauvenlt of cobble (TCaobbblele 5). The furthest upstream mapp30e8d1. 5r–e30a8c4.h0 (rkm 3117.05.4–33119.0) contain0e.5d6 the largest pro0.p01ortion of 3088.0–3090.0 0.41 0.53 0.06 cobble (Table 5). 3117.5–3119.0 a 0.38 0.49 0.13 Median (IQR) 0.41 (0.02) 0.53 (0.04) 0.06 (0.06) a Substrate imagery collected one year after spawning occurred. Fishes 2023, 8, x FOR PEER REVIEW 18 of 24 Fishes 2023, 8, 243 16 of 22 Figure 10. Kernel-deFnigsuitrye 1m0.aKpesr nsehl-odwen sthitye mrealpastisvheo wdethnesriteileatsi voef dleoncsaittiieosnosf lfoocra tiinondsivfoidr iunadli vfiedmuaallfeesm tahleast that successfully ovulatesdu c(csespsafuwllynionvgu lafteemd (aslpeasw) ndinugrfienmga ltehs)ed suprianwg tnhiensgp aswenaisnognse a2s0o1n82 0(1A8.( AID. I D99__116611,, BB. .I DID9_ 163, 9_163, and C. ID 9_a1n7d1C). . RIDel9a_t1i7v1e). dReelnastiivteiedse ncsliotiseesrc ltoos eor ntoeo innedinicdaictaet eaarreeaass wwhhereerefe mfeamlesalweesr ewmeorset fmreoquste ntly frequently located. Tlohceat elodw. Tehre alonwde ruapnpdeurp bpoerubnodusn dosf otfhteh eppuuttaattiivvee ssppaawwninnignrgea rcehaacrhe ianrdeic ianteddicbayttehde dbays hed the dashed lines, andlin tehs,ea rnidvethre kriilvoemr keiltoemr e(rtekrm(r)k mis) iins ipnapraernenththeesseess.. MMaattuurerem malealloec laoticoantsiodnusri ndgutrhine gsp tahwen ing spawning season arese iansodnicaarteeidnd bicya toedpebny ocpirecnlecisr.c les. Fishes 2023, 8, x FFOisRh ePsE2E0R23 R, 8E,V2I4E3W 19 of 24 17 of 22 Figure 11. KerFniegl-udreen1s1it.y Kmearpnes l-sdheonws itryelmatiavpes dsehnoswitireesl aotfi vleocdaetinosnitsi efsoro finlodcivaitdiounasl ffoermianldesiv tihdauta l females that successfully ovsuulactceedss (fsupllaywonvinugla tfeedm(aslpesa)w dnuirnigngfe tmhea lsepsa)wdunirningg stehaesosnp a2w01n9i n(Ag .s eIDas o8_n8260 a1n9d( AB.. IIDD 8_86 and B. ID 8_92). Relative densities closer to one indicate areas where females were most frequently located. The lower and u8_p9p2e)r. bRoeulantdivse odf ethnes iptiuetsactilvoes esrptaowonninegin rdeaiccaht earaer einads iwcahteedre bfye mthael edsawsheerde lminoesst, afrnedq uently located. the river kilomTetheer l(orkwmer) aisn idn uppapreenr tbhoeusensd. sMoaf ttuhreep mutaaleti vloecsaptiaownsn idnugrrineagc thhaer sepianwdinciantged sebaysothne adrea shed lines, and indicated by optehne criirvcelresk.i lometer (rkm) is in parentheses. Mature male locations during the spawning season are indicated by open circles. Table 5. Proportion of substrate types for three mapped reaches in the Missouri River upstream of Fort Peck Reser4v.oDir aisncdu tshsei omnedian of proportion of substrate type at locations with the interquartile range (IQR) in pareWntheecshesa.r Tachtee rriivzeerd kmiloomveemtere n(rtkamn)d oifd tehne tuifipepders panadw lnoiwnegr lobocautnidoanrsy ooff stuhcec essfully ovu- mapped reaches is denoted. The mapped reaches were within the putative spawning reaches (see Figures 10 and l1a1t)i nagndp walelirde ssetluercgteedo fnori nmtahpepMingis wsohuerni sRpiavwenriungp-sctarpeaabmle ofefmFoalret pPaelclikd Rsteusregrevoon ir for the first were observed tiinmteer.acIntingge nweitrha lm, saptuarwe nminalge fpeamlliadl esstumrgaedoen.l aTrhgee mmaopvpeemd reenatcshdesu irninclgudthede pa u~0ta.5t ive spawning km distance absoevaes aonnd bbueltohwa wd hmereed iinatnernacettiomnso vweemree onbtserarvteeds .t hat were less than 1 km/day and upstream. Spawning-capable males and atretic females had similar median movement rates as those of sp Proportion Substrate Type Mapped aRweancihng (rfkemma) les. However, the total movements by spawning-capable males and atretic females were fewer than thoSsaenodf spawning fGemraavleels . InterestinCgolby,baletr etic females had a 308l1ar.5g–e3v0a8r4i.a0t ion in total movem0.e4n3t rate where so0m.5e6i ndividuals mo0v.0e1d similar distances as 308s8p.a0w–3n0i9n0g.0f emales did, while0o.4t1h ers moved mu0c.5h3s horter distanc0e.0s.6 River locations used 311w7.5e–re31s1im9.i0l aar between spawni0n.3g8f emales and sp0a.4w9n ing-capable m0.a1l3e s, which is the result Meodfiabno t(hIQclRas) sifications bein0g.4lo1c (a0t.e0d2)n ear puta0t.i5v3e (s0p.0a4w) ning loc0a.t0io6n (s0..0H6o) wever, maximum a Substrate imagrievrye rcolollceactteiodn ondeif yfeeraer da,ftwerh sepraewsnpianwg oncicnugrrfeedm. ales had further upstream locations than did spawning-capable males. The disparity in maximum upstream location between spawning 4. Discussionf emales and spawning-capable males indicates that spawning-capable male pallid sturgeon do not move as far upstream as do spawning females during the putative spawning season. We characterWizeedd omcuomveemnteendt pauntda tiivdeesnptiafiwedn insgparweancihnegs olof csaptaiownnsi nogf fesmucacleesspfaulllliyd sturgeon up- ovulating palslitdre satmurogfeoFnor itnP tehcek MReissesrovuoriir Rfoivretrh eupfisrtsrtetaimme o. fD Fisotratn Pceecskf rRomesepruvtoaitri vfoers pthaew ning reaches first time. In togetnheertarla, nsspitaiwonniznogn efeamt athleesu mpsatdreea mlaregned moof vFeomrtePnetsc kdRuersinergv othire wpeurteatleivses than the esti- spawning seamsoante bdudt rhifatdd misteadnicaenr enqeut imreodvfeomr eonntt orgaetense ttihcadt ewveelroep lmesesn tthoafnp 1a lklimd/sdtuayrg aenodn free embryos. upstream. SpDawowninnsgt-rceaapmabolef FmoartlePse acnkdR eastreertvico ifre, mthaelems ihnaimd usmimrileaqru mireeddidanri fmt doivsetamnecnet has been esti- rates as those of spawning females. However, the total movements by spawning-capable Fishes 2023, 8, 243 18 of 22 mated to be 245 km and is positively correlated with water velocity [20]. Therefore, the required drift distance upstream of Fort Peck Reservoir may be further than 245 km. Pallid sturgeon putative spawning reaches described here were between 62 km and 131 km from the transition zone indicating that even the most upstream spawning reaches provided far less than the required drift distance. Substrate at suspected spawning locations within spawning reaches was a gravel–sand mosaic with little to no cobble or larger substrate. Substrate in the Missouri River transitions from smaller substrate at downstream reaches to larger substrate moving upstream [31]. Therefore, the substrate types we observed at spawning reaches are not commonly found at further upstream locations in the Missouri River. The Marias River may contain substrate compositions similar to what we docu- mented at suspected spawning locations, but the substrate in the Marias River has not been characterized. Additionally, the sediment input at tributary mouths may provide more heterogenous substrate favorable to spawning in upstream areas that are generally dominated by cobble. Pallid sturgeon were located in the Marias River and upstream of Judith Landing in the Missouri River. Upstream locations and locations in the Marias River occurred during the 2018 putative spawning season, which coincided with uncharacteristically high discharge. However, pallid sturgeon did not spawn while at upstream locations and instead spawned in lower portions of the Missouri River. A scarcity of spawning-capable male pallid sturgeon in the upstream portions of the Missouri River or in the Marias River may preclude upstream spawning opportunities even if female pallid sturgeon could spawn at those locations. Determining why spawning-capable male pallid sturgeon are apparently less likely to be located in upstream reaches during the putative spawning season could help elucidate ways to encourage spawning further upstream. Spawning in the Marias River or upstream of Judith Landing in the Missouri River would increase available drift distance, and given the apparent association between discharge and upstream location, future research and management efforts could consider discharge as a way to increase use of upstream reaches and tributaries. In closely related shovelnose sturgeon, discharge is linked to spawning in the Marias River [23]. When we observed spawning, female, atretic female, and spawning-capable male pallid sturgeon located in the Marias River in 2018, and the Marias River had a historically high discharge, which peaked at 157.2 m3/s on June 1, 2018. After peaking, the discharge was rapidly reduced to aid in flood control in the Missouri River and reached 20.3 m3/s by June 28, 2018. During the rapid decrease in discharge, all spawning-capable pallid sturgeon exited the Marias River. Pallid sturgeon in the Missouri River upstream of Fort Peck Reservoir spawned in locations where the substrate is a mosaic of sand and gravel, which is similar to other populations of pallid sturgeon [42,43]. Upstream of where we observed spawning, potential spawning substrate transitions to larger substrate types (e.g., cobble and gravel) [31]. If sand is an important substrate for spawning pallid sturgeon, the lack of sand further upstream could limit the likelihood of spawning in locations with adequate drift distance, regardless of discharge. However, the Marias River may contain preferable spawning substrate. If appropriate spawning substrate is present in the Marias River, discharge and temperature manipulation at Tiber Dam may be a viable management action to promote upstream spawning. Pallid sturgeon, like other sturgeon species (e.g., white sturgeon [46]), have adhesive eggs that probably evolved to adhere to hard substrates, and hard substrate, such as the large proportion of gravel observed in spawning reaches during this study, may be the preferred spawning habitat. However, why pallid sturgeon spawn in areas with high proportions of sand remains unknown. In the lower Missouri River, spawning reaches were dominated by sand, but selection coefficients were highest for hard substrates (i.e., gravel, cobble, boulder, or bedrock) and lowest for sand [47]. Spawning of pallid sturgeon in the Yellowstone River has occurred in reaches that are mostly sand [34], and white sturgeon have been documented spawning over sand; however, it was hypothesized that white sturgeon spawning over sand may be due to an absence of a preferred habitat [48]. Perhaps Fishes 2023, 8, 243 19 of 22 a gravel–sand mosaic of substrate the preferred habitat for reasons other than spawning. For example, in the lower Platte River, diet items of adult shovelnose sturgeon and age- 0 shovelnose sturgeon and pallid sturgeon are associated with sand substrate [49,50]. Furthermore, age-0 pallid sturgeon have been shown to use alluvial sand dunes as velocity refugia [51]. However, downstream drift of free embryos makes the link to spawning site selection and age-0 pallid sturgeon intangible. The variation of movement by atretic females is likely associated with the timing of ovarian follicular atresia. Ovarian follicular atresia is associated with a drop in sex steroid concentrations [52], and sex steroids (e.g., testosterone or estradiol) are closely associated with endocrine signaling that drives spawning-related behavior. For example, upstream migration can be induced in immature landlocked sockeye salmon (Oncorhynchus nerka [Walbaum in Artedi]) by implanting individuals with testosterone [53]. In pallid sturgeon, a decrease in sex steroids at the onset of follicular atresia would result in decreased drive to undergo spawning-related movements. Therefore, pallid sturgeon that initiate ovarian follicular atresia early in the spawning season would behave as though they were not reproductively active while pallid sturgeon that initiate atresia late in the spawning season would behave more similarly to spawning females. The movement of pallid sturgeon that are not reproductively active consists of slower movement and shorter distances compared to that of reproductively active pallid sturgeon (i.e., spawning females, atretic females, and spawning-capable males) [22]. The large variation in total movement by atretic females and the difference in total movement between atretic females and spawning females indicates that grouping atretic females with spawning females should be avoided when observing the behavior of pallid sturgeon. 5. Conclusions Here, we report the first observations of hatchery-origin pallid sturgeon spawning in the Missouri River upstream of Fort Peck Reservoir. In addition, we found that spawning- capable pallid sturgeon will use the Marias River, but we only observed this during an unprecedented discharge event. Recovery of pallid sturgeon as defined in the Recovery Plan will only be possible if pallid sturgeon spawn further upstream than the spawning locations identified here. Management actions such as modified water releases from upstream dams to encourage use of upstream locations, lowering Fort Peck Reservoir to provide more drift distance, or both may be necessary to promote successful recruitment as outlined in the Recovery Plan. Furthermore, we found that pallid sturgeon spawned in locations with a gravel–sand mosaic of substrate, which is negatively associated with distance upstream in the upper Missouri River. The Marias River may contain a more suitable spawning habitat provided that discharge and temperature regimes are managed to construct suitable spawning conditions. Management actions such as modifying discharge at Tiber Dam, altering reservoir levels at Fort Peck Reservoir, or constructing spawning habitat in desired spawning locations may be necessary to ensure spawning occurs in locations with an adequate drift distance for free embryos. Author Contributions: Conceptualization, T.L.C., C.S.G., L.M.H. and M.A.H.W.; methodology, T.L.C., C.S.G., L.M.H. and M.A.H.W.; software, T.L.C.; formal analysis, T.L.C. and C.S.G.; investigation, T.L.C., L.M.H. and M.A.H.W.; resources, C.S.G., L.M.H. and M.A.H.W.; data curation, T.L.C., C.S.G. and L.M.H.; writing—original draft preparation, T.L.C.; writing—review and editing, T.L.C., C.S.G., L.M.H. and M.A.H.W.; visualization, T.L.C. and C.S.G.; supervision, T.L.C. and C.S.G.; project administration, T.L.C., C.S.G., L.M.H. and M.A.H.W.; funding acquisition, C.S.G. and M.A.H.W. All authors have read and agreed to the published version of the manuscript. Funding: Funding and support for this work were provided by the Western Area Power Administra- tion and Montana Fish, Wildlife and Parks. The Montana Cooperative Fishery Research Unit is jointly sponsored by the U.S. Geological Survey; Montana Fish, Wildlife & Parks; Montana State University; and the U.S. Fish and Wildlife Service. Fishes 2023, 8, 243 20 of 22 Institutional Review Board Statement: This research was conducted under Montana State Univer- sity Animal Care and Use permit 2017-43 and U.S. Fish and Wildlife Service permit TE68706C-0. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors thank all of those who provided assistance in the field or laboratory. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the U.S. Fish and Wildlife Service. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. 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