Population genetics of grotto sculpin (Cottus specus), a new cave-adapted fish species Julie L. Day & David E. Starkey & Ginny Adams & Shawn Brummett & Devon Keeney Received: 19 August 2013 /Accepted: 18 December 2013 # Springer Science+Business Media Dordrecht 2014 Abstract The grotto sculpin (Cottus specus) is a troglomorphic fish endemic to cave systems in Perry County, Missouri. These fish are state-threatened and were recently listed as federally endangered under the Endangered Species Act (ESA). Due to the unstable nature of the cave environment, grotto sculpins are highly susceptible to pollution via agricultural and waste runoff. Here we provide a summary of population ge- netic diversity within the range of the grotto sculpin. Samples from twenty-one cave, surface, and resurgence sites within and surrounding the Bois Brule drainage in Perry County, southeast Missouri, USA were collected and sequence data from the mitochondrial control were analyzed using analysis of molecular variance, maxi- mum likelihood, parsimony, and Bayesian techniques. We found a substantial degree of divergence from sur- face populations of Midlands and Black River banded sculpin races (7.7–8.0 %). Within the Bois Brule drain- age, two distinct lineages of grotto sculpin were identi- fied which correspond with northern and southern cave system boundaries and may represent independent cave invasions. Keywords Sculpin . Fish . Conservationbiology . Cave . Population genetics Introduction Population connectivity can be broadly defined as the degree to which populations are considered open or closed by gene flow and dispersal patterns (Hellberg et al. 2002), which is central to the evolution of diversi- ty. Measures of connectivity and diversity are critical considerations for species recovery and habitat restora- tion initiatives in already at-risk species, as well as conservation and management efforts for potentially threatened species and their associated habitats (Cook et al. 2007; Schick and Lindley 2007). These estimates are particularly important in complex karst systems, typified by sinkholes and complex cave systems, where endemism may be very high but problematic to sample and characterize (Culver et al. 2007). Questions of hab- itat relationships and connectance are particularly diffi- cult to ascertain but immensely imperative in under- standing the persistence of hypogean biota (Dunne et al. 2002). Understanding the complexity of the karst landscape is imperative to understanding the degree of intercon- nectedness and relatedness of cave fauna populations. Stygobitic species (aquatic, cave-adapted organisms) are restricted to underground environments, limiting Environ Biol Fish DOI 10.1007/s10641-013-0216-5 J. L. Day (*) : G. Adams : S. Brummett Department of Biology, University of Central Arkansas, 201 Donaghey Avenue, Conway, AR 72035, USA e-mail: [email protected]D. E. Starkey Department of Natural Sciences, College of Coastal Georgia, 1 College Drive, Brunswick, GA 31520, USA D. Keeney Department of Biological Sciences, Le Moyne College, 1419 Salt Springs Road, Syracuse, NY 13214, USA
Transcript
Population genetics of grotto sculpin (Cottus specus), a newcave-adapted fish species
Julie L. Day & David E. Starkey & Ginny Adams &
Shawn Brummett & Devon Keeney
Received: 19 August 2013 /Accepted: 18 December 2013# Springer Science+Business Media Dordrecht 2014
Abstract The grotto sculpin (Cottus specus) is atroglomorphic fish endemic to cave systems in PerryCounty, Missouri. These fish are state-threatened andwere recently listed as federally endangered under theEndangered Species Act (ESA). Due to the unstablenature of the cave environment, grotto sculpins arehighly susceptible to pollution via agricultural and wasterunoff. Here we provide a summary of population ge-netic diversity within the range of the grotto sculpin.Samples from twenty-one cave, surface, and resurgencesites within and surrounding the Bois Brule drainage inPerry County, southeast Missouri, USA were collectedand sequence data from the mitochondrial control wereanalyzed using analysis of molecular variance, maxi-mum likelihood, parsimony, and Bayesian techniques.We found a substantial degree of divergence from sur-face populations of Midlands and Black River bandedsculpin races (7.7–8.0 %). Within the Bois Brule drain-age, two distinct lineages of grotto sculpin were identi-fied which correspond with northern and southern cave
system boundaries and may represent independent caveinvasions.
Population connectivity can be broadly defined as thedegree to which populations are considered open orclosed by gene flow and dispersal patterns (Hellberget al. 2002), which is central to the evolution of diversi-ty. Measures of connectivity and diversity are criticalconsiderations for species recovery and habitat restora-tion initiatives in already at-risk species, as well asconservation and management efforts for potentiallythreatened species and their associated habitats (Cooket al. 2007; Schick and Lindley 2007). These estimatesare particularly important in complex karst systems,typified by sinkholes and complex cave systems, whereendemism may be very high but problematic to sampleand characterize (Culver et al. 2007). Questions of hab-itat relationships and connectance are particularly diffi-cult to ascertain but immensely imperative in under-standing the persistence of hypogean biota (Dunneet al. 2002).
Understanding the complexity of the karst landscapeis imperative to understanding the degree of intercon-nectedness and relatedness of cave fauna populations.Stygobitic species (aquatic, cave-adapted organisms)are restricted to underground environments, limiting
Environ Biol FishDOI 10.1007/s10641-013-0216-5
J. L. Day (*) :G. Adams : S. BrummettDepartment of Biology, University of Central Arkansas,201 Donaghey Avenue, Conway, AR 72035, USAe-mail: [email protected]
D. E. StarkeyDepartment of Natural Sciences, College of Coastal Georgia,1 College Drive, Brunswick, GA 31520, USA
D. KeeneyDepartment of Biological Sciences, Le Moyne College,1419 Salt Springs Road, Syracuse, NY 13214, USA
the movement of individuals to subterranean pathwaysand eliminating the use of surface conduits. Severalauthors have found that genetic population structure instygobites can be strongly influenced by the degree ofhydrologic connectivity and habitat availability(Verovnik et al. 2004; Buhay and Crandall 2005;Venarsky et al. 2009).
However, due to the dynamic, unstable nature ofkarst cave systems, connectivity between available hab-itats is often difficult to determine. Hydrological con-nectivity can vary from year to year and even week toweek from variations in rainfall, surface ponds collaps-ing into karst streams, and geological events that resultin the creation of new caves or restriction of existingcaves. Populations may be disproportionately sampledand misrepresented, or go undocumented entirely due tovariability in accessibility, geological stability, or hy-drology. This can impede or prevent comprehensivestudies of subterranean assemblages, which severelylimit our capacity to identify and preserve endemic
fauna. Species delineations, central to conservationmanagers, are often problematic and cannot always bemade using taxonomic methods typically employed forterrestrial organisms. Several studies have found thatwhile genetic divergence may exist among populations,morphological similarities may result from convergentadaptation to the cave environment (Humphreys andAdams 2001; Verovnik et al. 2004). If overlooked, thesedata may lead to inaccurate conclusions about related-ness and uniqueness. Studies of several cave speciesindicate that multiple invasions during separate, uniqueevents occur with some regularity (Borowsky andVidthayanon 2001; Trajano 2001; Strecker et al. 2003,2004), in which case traditional systematic, manage-ment, and conservation practices may not be applicable.
Grotto sculpins (Cottus specus) are found in five cavesystems and their corresponding resurgence streams inPerry County, southeast Missouri, USA (Fig. 1; Adamset al. 2013). A high degree of variability was detected ingrotto sculpin morphology and genetics and the
Fig. 1 a–c provide geographic reference within the interiorUnited States. d shows the geographic distribution of sam-pling localities and drainage relationship in Perry County,
Missouri. Shaded area denotes karst sinkhole plain bound-aries, and bolded streams indicate subterranean cavestreams. See Table 1 for site abbreviations
Environ Biol Fish
presence of an “intermediate” surface Cinque HommesCreek population add to the complexity of this species(Burr et al. 2001; Adams et al. 2013). Rather thanforming a discrete boundary, Cinque Hommes Creekwas found to contain fish belonging to two distinctlineages of grotto sculpin previously thought to be geo-graphically isolated. It remains unclear if similarity inmorphology among grotto sculpin populations is basedon convergence or if an adaptive event occurred within asingle cave with subsequent dispersal to nearby caves.
The extensive karst system underlying Perry Countymay have contributed to the occurrence of multiple caveinvasions. Separate invasions into the cave environmentwould result in a unique evolutionary lineage withineach cave, and an effective conservation plan wouldneed to be established to preserve the evolutionarydistinctiveness of each independent lineage identified.However, fewer invasions with subsequent movementof sculpin among caves would result in replicate popu-lations and alter the conservation priority to a broaderscale. The purpose of this study was to investigate thephylogenetic placement and population genetic struc-ture among grotto sculpin populations within the BoisBrule drainage cave systems using mitochondrial se-quence data. Here we provide pertinent information fordevelopment of a management plan that seeks to con-serve the maximum amount of genetic variation presentin these populations while also contributing to the lim-ited data available regarding the evolution of cave-adapted species.
Methods
Study species
The genus Cottus currently contains approximately 33freshwater species, although many are poorly under-stood and the true number of species is probably muchhigher (Adams et al. 2013; Eschmeyer 1998). Grottosculpin are limited to caves and their correspondingresurgence streams (Cinque Hommes Creek and BlueSpring) within the Bois Brule watershed in PerryCounty, Missouri. Perry County contains 656 knowncaves, one of the highest concentrations of caves in theUnited States, with terrain typified by sinkholes, caves,disappearing streams, and springs representative of asinkhole plain (Schooler 1996). In this unique land-scape, subterranean streams outnumber surface streams,
driving water drainage underground and increasing thepotential for both aquatic organisms and organic matterto enter cave environments.
DNA isolation and sequencing
All known localities for grotto sculpins within the BoisBrule drainage were sampled and specific sample loca-tions within each system were selected based on ease ofentry, relative location to other sites, and current land-owner permissions. Sampling cave streams can betreacherous with subterranean conditions varying con-siderably. Though every effort was made to samplethoroughly, our sample sizes were limited by safetyconcerns and occurrence of fish. Banded sculpin(Cottus carolinae) collections were made in streams inclose geographic proximity to grotto sculpin streams,and included samples from the Black River andMidlands races. Twenty-one localities were sampled(Table 1; Fig. 1). Fin clips or whole fish were collectedfrom each sampling site. Specimens were caught using acombination of seining and dip netting, depending onconditions and ceiling height. Whole specimens weresacrificed by MS-222 overdose, then preserved in 95 %ethanol in the field, or frozen whole at –30 °C in thelaboratory. Fin clips were preserved in the field using95 % non-denatured ethanol.
DNAwas extracted from 1.0 g caudal peduncle or finclip using a standard phenol/chloroform protocol or celllysis method (Hillis et al. 1996). DNA quality wasconfirmed by gel electrophoresis using 1.0 % agarosegels stained with ethidium bromide and visualized viaUV light. Approximately 725 bp of mitochondrial con-trol region (mtCR) were amplified using CottusPro: 5′-TTCCACCTCTAACTACCCAAAGCTAG-3′andCaarh: 5′-AAGCACATTTTTCGCCCC-3′. Polymerasechain reaction (PCR) amplifications were optimized(Cobb and Clarkson 1994) and performed using MJMinicyclers. Twenty μl reactions consisted of 1–3 μlDNA template, 1 μl 10 mM each primer, 7 μl REDTaqDNA Polymerase (Thermo Scientific), and sterile waterto a final volume of 20 μl. PCR conditions consisted ofan initial 95ºC denaturation for 2 min, followed by 35cycles of 95ºC for 1 min, 58ºC for 1 min, and 72ºC for1 min, and a final 6-min extension at 72ºC. Prior toimplementing this standard program, thermal gradientreactions were performed to alleviate misinterpretationsdue to gene duplication or thermally preferential alleles.
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A negative control was included with eachthermalcycler program run, and PCR products wereconfirmed and sized by gel electrophoresis. Reactionsyielding bright, sharp bands were purified for sequencingusing Exonuclease I and Shrimp Alkaline Phosphataseand cycle sequenced onABI 3130Automated Sequencerusing both primers. Cycle sequencing and direct se-quencing reactions using both primers were performedby the University of California at Davis (UCD) COREfacility. New, unique haplotypes were verified by se-quencing several individual PCR products in forwardand reverse directions. Sequences have been submittedto GenBank (KF746038-55).
Phylogenetic analysis
Sequences were aligned and edited using Sequencherversion 4.8. Cottus bairdii (mottled sculpin) was
s e l e c t e d a s t h e ou t g r o up f r om GenBank(GQ290456.1). Phylogenetic analyses were performedusing maximum likelihood (ML), maximum parsimony(MP), and Bayesian optimality criterion. ModelTestversion 3.7 (Posada and Crandall 1998) was used toselect the appropriate model of sequence evolution.ML searches were executed heuristically with 1,000replications of a random stepwise addition of taxa andTBR branch swapping using HKY + G model parame-ters (ti:tv = 1.7687; α = 3.7892; Hasegawa et al. 1985),as specified by ModelTest. To evaluate node support,bootstrap analysis was performed heuristically with1,000 pseudoreplicates of a heuristic search with 100replicates of random stepwise addition and TBR branchswapping.
Unweighted MP searches were conducted using aheuristic search with TBR branch swapping on initialtrees, which were obtained by random stepwise additionof taxa for 1,000 replicates. Bootstrapping was per-formed heuristically with TBR branch swapping, ran-dom stepwise addi t ion of taxa , and 1,000pseudoreplicates. Bayesian analyses were conductedusing MRBAYES version 3.1.2 (Huelsenbeck andRonquist 2001). Four chains were run for 10 milliongenerations each, and trees were sampled every 1,000generations. The first 1,000 trees were discarded asburn-in, and results analyzed in Tracer to (http://evolve.zoo.ox.ac.uk/software/tracer/) construct amajority-rule consensus tree. In all analyses we consid-ered bootstrap support 70 % or higher and Bayesianposterior probability 0.95 or higher to indicate signifi-cant support for the node in question. Each analysis wasrun on at least three separate occasions to ensure con-sistency and validity of these results. Finally, Network(http://www.fluxus-engineering.com/sharenet.htm) wasused to construct a median joining diagram of grottosculpin haplotypes.
Population structure
Analysis of grotto sculpin genetic diversity, includinghaplotype diversity (h), and nucleotide diversity (π),ΦST and FST was calculated in ARLEQUIN version3.5.1.2 (Schneider et al. 2000; Excoffier et al. 2005)and DnaSP 4.0 (Rozas et al. 2003). Genetic divergenceamong haplotypes was assessed by calculating percentsequence divergence, or uncorrected p-distances, amongpairs of samples in PAUP (Swofford 2002) and MEGA4.0 (Kumar et al. 2004). Analysis of molecular variance
Table 1 Cave, resurgence, and surface streams sampled includedwith the number of Cottus specus individuals sequenced for mi-tochondrial control region (mtCR). Regions are listed only for siteswithin the Bois Brule drainage and correspond to geographiclocations in relation to Cinque Hommes Creek
(AMOVA, Excoffier et al. 1992) was used to test forpopulation structure and subdivision. AMOVAs werefirst performed among all sampled locations with noassigned groupings. Next, caves and resurgence streamsin the Bois Brule drainage, (BS, MC, RB, RR, CC, TM,MR TH, ME, HC, and CH), which encompasses theknown range of the grotto sculpin, was compared to alllocations outside this drainage. Hydrologically relatedlocations within the Bois Brule drainage were thengrouped as southern (MC, RR, RB and TH), northern(BS, CC,ME and TM) and resurgences (MR, CH and itstributaries). Finally, individual location were attempted.
Results
A total of 767 bp of mtCR were analyzed after removalof primer sequences. A total of 224 individual fish wereanalyzed from 21 sample locations and sample sizes perlocation ranged from two to nineteen individuals.Heuristic analyses found 98 variable characters, ofwhich 71 were parsimony-informative in the dataset,85 variable sites (68 parsimony-informative) excludingthe outgroup, and 31 variable sites (23 parsimony-informative) among Bois Brule haplotypes. Of the sam-ple locations included, 18 total haplotypes were ob-served and nine locations contained unique haplotypes.
A total of seven haplotypes were recovered withinthe Bois Brule drainage. Five haplotypes were restrictedto the Bois Brule drainage, two of which were onlyfound in caves. Mystery Cave and Whitewater Riverwere fixed with a single haplotype while all remainingsites had a minimum of two haplotypes (Table 2).Haplotype diversity in the Bois Brule drainage rangedfrom 0.000 to 0.778, after removing the two locationsthat were fixed (Table 2; MC, WW). Most individualssampled (91 %) in the drainage were represented by oneof the three primary haplotypes GR1, GR2 and GR3(Fig. 3). Within these haplotypes, GR1 and GR2 weresimilar with a pairwise distance of 0.1 % (Table 3).Haplotype GR3 was found at three of twelve locationswith haplotypes GR1 and GR2 and had a pairwisedistance of 2.3 % (16 substitutions) to 2.4 % (17 substi-tutions), respectively.
Phylogenetic analysis
Each of the three phylogenetic methods employed re-covered the same set of major nodes (Fig. 2). Tree
topologies were also comparable, however Bayesiananalysis was found to resolve within lineage variationmore consistently than ML or MP. Across analyses wefound consistent groupings of three geographically dis-tinct sets of haplotypes. The most basal lineage includedtwo haplotypes from Greasy Creek (Fig. 2), whichcorrespond with the Midlands race of C. carolinae.The Current River system consisted of three haplotypes(BR1, BR2, and BR3), which correspondwith the BlackRiver race of C. carolinae. Divergence of both basalC. carolinae lineages from the remaining C. specus taxawas fully supported by all analyses.
Considerable haplotype diversity was evident in thethird C. specus lineage, which almost exclusively rep-resents Perry County haplotypes and includes the BoisBrule drainage (Table 2; Fig. 3). The divergence of twodistinct groups within this lineage was well supported.The first of these groupings represents caves and resur-gences in northern Perry County, including Blue Spring,Tom Moore Cave, Crevice Cave, and Cinque HommesCreek (Fig. 2; GR1, GR2, GR, GR9, GR10, GR11,GR12 and GR13). The second grouping encompasseshaplotypes found primarily in caves and resurgences insouthern Perry County at Running Bull Cave,Thunderhole Resurgence, Mystery Cave, andRimstone River Cave (Fig. 2; GR3, GR4, GR5, GR6,GR7, and GR8). Several sites were found to containhaplotypes from both lineages, including Hot Caverns,Dry Fork Creek, Mystery Resurgence, Gerler Spring,and Apple Creek. And, while some haplotypes from thenorthern lineage were found at locations outside PerryCounty, all six haplotypes comprising the southern lin-eage were restricted solely to Perry County (Table 2).
Population structure
Limited sample sizes precluded the use of populationgenetic methods to determine fine-scale relationshipsbetween individual sample locations. However, we wereable to reveal and support structure at the lineage andgeographic boundary levels and results are reportedaccordingly. A significant degree of population structurewas observed in the overall analysis with all locationsincluded and no groupings assigned (ΦST=0.387,P<0.001; FST=0.431, P<0.001). The Bois Brule drain-age was significantly different when compared to alllocations outside the drainage (ΦST=0.405, P<0.001).Within the Bois Brule drainage there was significantstructuring based on hydrological connectivity with
Environ Biol Fish
Tab
le2
Geographicdistributio
nof
CottusmtCRhaplotypes
andcorrespondinghaplotypediversity
measuresfoundam
ongcave,resurgence,andsurfacestream
samplesites
Location
GR1
GR2
GR3
GR4
GR5
GR6
GR7
GR8
GR9
GR10
GR11
GR12
GR13
MD1
MD2
BR1
BR2
BR3
Nh
π
Tom
Moore
Cave
[TM]
58
00
00
00
00
00
00
00
00
130.513±0.0822
0.0335
±0.0177
BlueSp
ring
[BS]
115
00
00
00
00
00
00
00
00
160.458±0.0954
0.0299
±0.0156
Crevice
Cave[CC]
52
00
00
00
00
00
00
00
00
70.476±0.171
0.0311
±0.0179
Crevice-M
ertzCave
[ME]
85
00
00
00
00
00
00
00
00
130.513±0.0822
0.0335
±0.0177
CinqueHom
mes
Creek
[CH]
55
00
00
00
00
00
00
00
00
100.556±0.0745
0.0363
±0.0197
Mystery
Cave[M
C]
00
170
00
00
00
00
00
00
00
170.000
0.000
Mystery
Resurgence
[MR]
25
20
00
00
00
00
00
00
00
90.667±0.132
0.0336
±0.0186
Dry
Fork
Creek
[DF]
11
50
00
00
00
00
00
00
00
70.524±0.209
0.0269
±0.0156
Running
Bull
Cave[RB]
00
118
00
00
00
00
00
00
00
190.515±0.0517
0.00431±0.00262
Thunderhole[TH]
00
10
01
00
00
00
00
00
00
21.000±0.0500
0.0321
±0.0328
Rim
stoneRiver
Cave[RR]
00
120
20
00
00
00
00
00
00
140.264±0.136
0.0223
±0.0119
Hot
Caverns
[HC]
42
30
00
10
00
00
00
00
00
100.778±0.0907
0.0430
±0.0233
GreasyCreek
[GC]
00
00
00
00
00
00
02
50
00
70.476±0.171
0.0166
±0.00980
Current
River
[CR]
00
00
00
00
00
00
00
02
71
100.511±0.164
0.00678±0.00408
Salin
eCreek
[SC]
40
00
00
00
00
03
30
00
00
100.733±0.0764
0.0361
±0.0196
GerlerSp
ring
[GS]
70
03
00
00
00
00
00
00
00
100.467±0.132
0.0337
±0.0183
AppleCreek
[AC]
50
60
00
04
00
00
00
00
00
150.705±0.0535
0.0421
±0.0219
Brazeau
Creek
[BC]
40
00
00
00
00
05
30
00
00
120.712±0.0691
0.0330
±0.0176
WolfCreek
[WC]
00
00
00
00
04
80
00
00
00
120.485±0.106
0.0343
±0.0183
Whitewater
River
[WW]
00
00
00
00
20
00
00
00
00
20.000
0.000
SouthforkSalin
eRiver
[SF]
00
00
00
00
05
40
00
00
00
90.556±0.0902
0.0393
±0.0213
Total
6133
5711
21
14
29
128
62
52
71
224
Environ Biol Fish
caves to the south of Cinque Hommes genetically dis-tinct from those to the north of Cinque Hommes (ΦST=0.466, P<0.001; FST=0.550, P<0.001; Table 3) andCinque Hommes was genetically intermediate to the
caves, which we suspect to be due to the presence ofhaplotypes from both southern and northern lineages.Surface locations within Bois Brule exhibited signifi-cant structuring (ΦST=0.216, P=0.004) and when all
Table 3 Significant results of ungrouped and geographic lineage groupings of C. specus AMOVA calculations, respectively
Fig. 2 Phylogram of the relationships among the 18 uniqueCottus mtCR haplotypes and one outgroup (Bayesian topologyshown). Numbers on each branch correspond to bootstrap values ≥70 based on 1,000 pseudoreplicates for ML and MP analyses,followed by posterior probabilities. Insignificant support
placeholders are indicated by dashes. Lineages representing theBlack River and Midlands races of Cottus carolinae are represent-ed, as is the Cottus specus lineage. Shaded boxes are indicative ofhabitats where haplotypes are found, and clade icons match thoseused in Adams et al. 2013 and Kinziger et al. 2007
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sample locations combined within Cinque Hommeswere compared to northern caves (ΦST=0.179, P=0.003) and southern caves (ΦST=0.395, P<0.001) theywere significantly different. However, resurgencesfound in close proximity to northern caves were notsignificantly different from northern caves (ΦST=−0.005, P=0.317). Within the northern caves therewas no significant genetic structuring observed (ΦST=0.074, P=0.157). Pairwise comparisons within thesouthern caves revealed significant genetic structuringamong all sites, with each of the three caves (RimstoneRiver, Running Bull and Mystery Cave) significantlydifferent (P<0.001).
Pairwise distance comparisons among allCottus hap-lotypes ranged from 0.10 to 8.0 % (1 to 52 parsimony-informative), and reflected the variation betweenC. specus and C. carolinae samples outside the BoisBrule drainage. Pairwise distance comparisons amonggrotto sculpin haplotypes ranged from 0.10 to 2.7 % (1to 17 characters) (Fig. 3). Base pair frequencies usingHKY + G (%A=31.14, %C=21.07, %G=17.15, %T=30.64) did not differ significantly, both within the BoisBrule drainage and among all haplotypes.
Discussion
This study represents the first comprehensive assess-ment of genetic diversity in the newly described andfederally endangered (USFWS 2013) grotto sculpin andits geographically related sister taxa. Given the consid-erable obstacles present when sampling subterraneanpopulations with restricted ranges in highly variablehabitats, we are cautious to draw fine-scale conclusionsabout the evolutionary history of grotto sculpin. Cavespecies are often recognized as in need of protectionlong after calls for study have been made (Niemilleret al. 2013) and we are excited to share these findingsin a timely manner to aid in the conservation of thisunique, new species.
We were able to resolve relationships between twolineages within the Cottus carolinae species complex, inaddition to describing substantial diversity within theC. specus lineage (Fig. 2). Two distinct grotto sculpinlineages were found, one primarily to the north ofCinque Hommes Creek and the other to the south.Although seven haplotypes were recovered in the north-ern group, all cave and resurgence locations sampled
Fig. 3 Haplotype network diagram of 13 Cottus specus mtCRhaplotypes constructed using median joining methods. Haplotypefrequencies are proportionate to node sizes, and geographic distri-butions are represented by three categories that correspond with
lineage presences: northern, southern, andmixed sample locations.Parsimony-informative differences are displayed along node con-nections, and linkages nodes numbered mv1 through 4
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contained the same two haplotypes (GR1 and GR2).The remaining five haplotypes were found exclusivelyin surface streams in close proximity to northern caves.Based on its frequency and wide geographic distributionwe hypothesize that GR1 was once the primary haplo-type in this region. In contrast, GR2 was restricted tocave and resurgence habitats, and may represent a de-rived cave haplotype. The apparent nesting of easternPerry County haplotypes within the northern grottosculpin clade may indicate that closer scrutiny of thegeographic boundaries of Ozark Highlands sculpins iswarranted.
The southern grotto sculpin lineage was comprised ofsix related haplotypes. Haplotype diversificationthrough isolation may have been more widespread insouthern caves and is supported by the presence of threehaplotypes (GR5, GR6, and GR7) that were recoveredfrom a single cave or resurgence locations. Significantgenetic structuring was observed among the southerncaves that was not observed in northern caves. Wesuggest that the southern lineage may signify a morerecent cave invasion that occurred with the formation ofnew caves during the Pleistocene, when fish residing inCinque Hommes Creek would have been forced toexpand their niche habitats to include subterraneanstreams in order to persist.
Sculpins from the Bois Brule drainage were highlydivergent and showed no evidence of recent maternalgene flow between either Black River or Midlands racesculpins, supporting the presence of discrete geneticunits. Considerable population structure was detectedwithin the Bois Brule drainage itself, with indicationsof low maternal gene flow among sample locationswithin the drainage marked by the presence of bothlineages at four locations (MR, DF, HC, and AC) thatmay represent intermediate populations or areas of lin-eage mixing.
The physical conditions or behaviors that producedisolation between northern and southern lineages remainunknown. Haplotypes belonging to each respectivegroup exhibited a marked degree of divergence fromboth banded sculpin races (8.0 %) that was approxi-mately equal to the degree of divergence from eachother (7.7 %). Similarly, population genetic analysesrevealed significant structuring among sites harboringthese lineages. This implies that in addition toexperiencing a deep divergence from other membersof the C. carolinae species complex, grotto sculpin alsopossess a substantial amount of genetic variability
within and among their populations. We suggest furtheranalyses investigate the possibility that multiple speciesare present within the range of C. specus.
Grotto sculpin in close proximity to Cinque HommesCreek remain perplexing given the presence of haplo-types from both northern and southern cave systems. Itis also possible sculpin colonized the region after extir-pation of the surface population or due to niche avail-ability in Cinque Hommes prior to colonization bygrotto sculpin. Divergence between haplotypes inPerry County may be reflective of secondary admixturebetween haplotypes that evolved in allopatric popula-tions, supporting multiple invasions of sculpin intocaves. An alternative explanation postulates that con-temporary populations are small, isolated remnants of alarge, widespread population that contained higher ge-netic diversity and subsequently lost the majority of itsdiversity as a result of population fragmentation duringPleistocene cave formation.
The dynamic evolutionary nature of grotto sculpinrevealed by genetic analyses substantiates ecologicaldata and reinforces a more unstable natural history thatis likely influenced to varying degrees by hydrology,geology, and biology. Recent population ecology stud-ies have demonstrated a unique demography, in whichfish utilize both cave and resurgence streams for com-pletion of their life cycle inconsistently across theirrange, possibly as a function of stochastic hydrographicconditions (Day, unpubl. data) that often characterizeshallow karst systems. Such variable patterns of ecologysuggest that grotto sculpins are obligate cave dwellers,but are proficient at utilizing multiple habitat types. It istherefore possible genetic sampling may be skewed byseasonal variability in reproductive output and levels ofsubterranean water movement.
Both major grotto sculpin lineages provide evi-dence of multiple invasions or colonizations ofcaves. Areas of mixing between the two dominantlineages, correspond with either intermediate cavesystems or areas where several cave resurgencesoccur within close proximity to each other, and arethe only localities at which lineage mixing occurs.For conservation and taxonomic purposes, this is acritical observation. Grouping of the northern andsouthern lineages into a single designation mayonly preserve a portion of the diversity we identi-fied, particularly if management efforts are geo-graphically centered or isolated, and supports fur-ther inquiry.
Environ Biol Fish
Conservation implications
Similar to bullhead (Cottus gobio) populations (Knapenet al. 2003), the phylogenetic relationships amongOzark Highlands sculpin populations clearly depictsthe extent to which they are geographically contiguousyet highly evolutionarily divergent. Our results illustratethat populations situated within close proximity candiffer substantially in their genetic make-up, and in thecase of grotto sculpin, clearly correspond to differentialevolutionary paths. Such patterns are not widespread buthave certainly been found across a variety of taxa(Møller-Hansen 1993; Grandjean et al. 1997; Estoup1998; Manceau et al. 1999; Negro and Torres 1999;Knapen et al. 2003). Due to low sample sizes, however,caution should be used in the interpretation of fine-scalestructuring.
Throughout the range of grotto sculpin, sinkholesremain one of the greatest potential threats to theirpopulations. It has been estimated that over half of thenumerous sinkholes in Perry County are “trash-cansinkholes” (Burr et al. 2001). Due to the unstable natureof the cave environment and prevalence of these sink-holes, grotto sculpins are highly susceptible to pollutionfrom suspected agricultural and waste runoff. Pesticides,herbicides, metals, and organic waste that leak intosubterranean streams via sinkholes are all likely to havedetrimental effects on grotto sculpin habitat and popu-lations. Although the causes remain unknown, two sep-arate fish kills were observed in the grotto sculpin caveswithin the last decade. These kills prompted the U.S.Fish and Wildlife Service to list the grotto sculpin forhigh priority listing under the Endangered Species Act.Furthermore, the presence of two dominant haplotypesin the Bois Brule drainage found on opposing planes ofCinque Hommes Creek suggests there is need to assessthreats to these populations independently. Both north-ern and southern caves experience intensive agriculturalrunoff; however caves north of Cinque Hommes Creekmay be subject to higher levels of urban runoff (Vandike1985; Fox et al. 2010).
To date, studies of both cave-adapted fish and Cottidmolecular ecology and in-depth phylogenetic relation-ships remain largely undocumented. Our results suggestsubterranean fishes, particularly recent cave invaders,may have more complicated evolutionary histories thanthe simplicity of the cave environment may suggest. Westrongly recommend that their evolutionary ecology begiven closer examination with respect to conservation
initiatives and unresolved taxonomic positions prior todrawing broad-reaching conclusions.
Acknowledgments Support for this project was provided bygrants from the Missouri Department of Conservation and U.S.Fish and Wildlife Service. The diligence and dedication of BradPobst made this project possible and successful. We are grateful tothe Perry County MDC office for use of their facilities, the LittleEgypt Grotto for their support, numerous landowners who allowedaccess to sites, and PhilipMoss of Ozark Underground Laboratoryfor his unending guidance. We also thank UCA students KiraBennett and Sarah Vestal for lab assistance, Jean Krejca andBrooks Burr for tissue samples, and Ed Heist for critical feedback.All work was conducted according to UCA IACUC animal carestandards.
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