Molecular Phylogenetics and Evolution 51 (2009) 373–381

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Molecular Phylogenetics and Evolution

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Nuclear and mitochondrial diversification in two native California minnows:insights into taxonomic identity and regional phylogeography

Andres Aguilar a,*, W. Joe Jones b

a School of Natural Sciences & Sierra Nevada Research Institute, University of California, Merced, P.O. Box 2039, Merced, CA 95344, USAb Department of Environmental Health Sciences, Public Health Research Center, University of South Carolina, Room 413, 921 Assembly Street, Columbia, SC 29208, USA

a r t i c l e i n f o

Article history:Received 10 September 2008Revised 25 November 2008Accepted 28 November 2008Available online 1 February 2009

Keywords:AMOVALaviniaMicrosatelliteMitochondrial DNAPhylogeography

1055-7903/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ympev.2008.11.028

* Corresponding author. Fax: +1 209 228 4060.E-mail address: aaguilar2@ucmerced.edu (A. Aguil

a b s t r a c t

Diversification of the California icthyofauna has been greatly influenced by a complex geomorphologicalhistory and past fluctuations in climate regimes. This complex history has resulted in areas of high ende-mism for a number of taxa. Here we present data on the two species in the genus Lavinia, the Californiaroach (Lavinia symmetricus) and hitch (Lavinia exilicauda), that are widespread throughout the region.Individuals were sequenced at two mitochondrial DNA fragments and genotyped at eight microsatelliteloci. Mitochondrial DNA indicated the presence to two highly divergent clades representing roach fromthe Gualala and Pit Rivers, which diverged from all other Lavinia �3–6 MYA. Support was also foundfor roach from the Navarro River, Tomales Bay region, the Red Hills region, and the Russian River-ClearLake basin. We found no evidence for any geographical groupings of mtDNA haplotypes for roach andhitch from the Monterey Bay region and the Sacramento–San Joaquin River drainages. Additionally roachand hitch from these two areas could not be readily distinguished by mtDNA data. Analysis of microsat-ellite DNA recovered all groupings found in the mtDNA analysis and was able to separate out roach, hitch,and all currently recognized subspecies of each species. These results indicate that hybridization mayobscure the phylogenetic/phylogeographic informativeness of mtDNA in this group. Additionally, theseresults suggest that differentiation in this group occurs at the river basin level and that the describedgenetic entities constitute distinct units and should be conserved as such.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Past climatic events and geomorphological changes havegreatly influenced the species-wide genetic structure for a numberof North American organisms (Avise et al., 1998; Bernatchez andWilson, 1998). The dramatic climate fluctuations that occurredthroughout the Pliocene and Pleistocene modified historical geo-graphic connections and composition of terrestrial and aquaticenvironments in western North America (Brunsfeld et al., 2001).Extreme periods of wetness and aridity led to the formation andloss of large inland water bodies that influenced the large-scalehistorical distribution of many aquatic species (Minckley et al.,1986). Additionally geomorphologic changes, in the form of tec-tonic activity and uplift, have contributed greatly to the distribu-tion of genetic structure for species with low dispersalcapabilities including species that are restricted to aquatic or mesichabitats (Bernatchez and Wilson, 1998; Brunsfeld et al., 2001). Thecombined effects of dramatic shifts in climate regimes coupledwith changes in geomorphology may also be important in smaller

ll rights reserved.

ar).

scale phenomena (e.g. headwater stream capture) that could haveprofound implications for the structure of aquatic species.

Past work on the phylogeography of the California floristic prov-ince has been focused on a variety of terrestrial vertebrate andinvertebrate species. Comparative analyses (Calsbeek et al., 2003;Lapointe and Rissler, 2005) and studies of individual species(Maldonado et al., 2001; Martinez-Solano et al., 2007; Starrettand Hedin, 2007) have found that diversification of a number ofCalifornia taxa coincides with mountain range uplift and periodsof extreme aridity in California. While great insight has beengained on the historical factors influencing the geographic distri-bution of genetic variation for some species, there remains a pau-city of information on the historical phylogeography of California’sfreshwater icthyofauna. Even within freshwater teleosts, a major-ity of the work has been focused on salmonids (Bagley and Gall,1998; Aguilar and Garza, 2006) with very few studies investigatingphylogeographic structure of stream-dwelling fishes, especiallythose that are restricted to lower order streams (Whiteheadet al., 2003; Baerwald et al., 2007; Chen et al., 2007).

The icthyofauna of the California region contains a high degreeof endemism. This can be attributed primarily to complex geolog-ical history and historical fluctuations in aridity of the area (Moyle,2002). These two factors may have served to increase the

374 A. Aguilar, W.J. Jones / Molecular Phylogenetics and Evolution 51 (2009) 373–381

occurrence of isolation among populations of freshwater fishes,leading to the contemporary species diversity and endemism ofthis region. However, there is not enough information to determinethe importance of specific historical effects on the speciation pro-cess in California freshwater fishes. Additionally, the native Califor-nia icthyofauna is experiencing dramatic declines in the abundanceand occurrence of many species. This is due to dramatic habitatalterations (primarily from anthropogenic influences) and theintroduction of non-native predators and competitors (Moyle andWilliams, 1990). Increased knowledge of historical and contempo-rary connectivity/isolation among stream-dwelling fishes will notonly provide greater insight into the evolutionary history of these

Table 1Sampling locations, site codes, and samples sizes for mitochondrial DNA (Nmt) and micrhaplotype diversity (h)) and microsatellites (expected—HE and observed heterozygosity—H

Species Subspeciesa Location Code

Roach Sacramento–San Joaquin (L. s. symmetricus) Little Chico Creek LCCCosumnes River COSAsh Creek ASHDye Creek DYECoyote Creek COYLos Gatos Creek LOSWoods Creek WOCCurtis Creek CUROrestimba Creek OREDeer Creek DECPeoria Creek PEO

Clear Lake–Russian River (L. s. spp) Kelsey Creek KELHendrick’s Creek HENSeigler Creek SEIRussian River RUSFeliz Creek FERMark West Creek MWC

Red Hills (L. s. spp) Horton Creek HORRoach Creek ROA

Monterey (L. s. subditus) San Lorenzo River SLRLlagas Creek LLAUvas Creek UVAPescadero Creek PES

Navarro (L. s. navarroensis) Indian Creek INCNavarro River-1 NAV1Navarro River-2 NAV2

Tomales (L. s. spp) Walker Creek WALLagunitas Creek-1 LAG1Salmon Creek SALLagunitas Creek-2 LAG2

Gualala (L. s. parvipinnis) Gualala River GUANorth Fork Gualala River NFGSouth Fork Gualala River SFG

Pit (L. s. mitrulus) Ana River ANADry Creek DRYPit River PIT

Total

Hitch Monterey (L. e. harengus) Pajaro River PAJSalinas River SAL

Central Valley (L. e. exilicauda) Rock Creek ROCWillow Creek WILLittle Butte Creek LBCCosumnes River COSSacramento River SACPiney Creek PIN

Clear Lake (L. e. chi) Adobe Creek ADOMiddle Creek MID

Total

a Sub-specific designations based on Hopkirk (1973), Miller (1945) and Moyle (2002)b California Academy of Science sample accession numbers.

organisms, but will also aid in the conservation and managementof these systems.

The genus Lavinia is ideal to understand both the evolutionaryhistory of interconnections among northern California river drain-ages (i.e., stream capture) and the endemism of California’s ict-hyofauna. The genus is widely distributed throughout Californiafrom coastal watersheds in northern California to inland streamsin the southern Central Valley (Table 1 and Fig. 1). Currently thegenus is in need of more extensive morphological and genetic eval-uation of the proposed taxonomic units (Moyle, 2002). Addition-ally all roach subspecies possess ‘species of special concern’status by the California Department of Fish and Game (Moyle

osatellite analysis (Nl). Summary statistics for mtDNA (Nucleotide diversity (p) andO). Standard errors are given in parentheses for p and h.

Nmt p h Nl HE HO CASb

10 0.003(0.002) 0.778(0.137) 19 0.6876 0.6746 208,00810 0.000 0.000 10 0.4951 0.6000 213,814 and 213,811

12 0.6870 0.690829 0.6861 0.6563

5 0.005(0.003) 0.800(0.164) 207,99716 0.6695 0.7250

5 0.003(0.003) 0.900(0.161) 208,0025 0.005(0.004) 0.800(0.164)

10 0.003(0.002) 0.778(0.091) 208,00310 0.001(0.001) 0.467(0.132) 209,043

19 0.6275 0.6667

5 0.005(0.004) 0.800(0.164) 13 0.7366 0.7045 207,9985 0.004(0.003) 0.800(0.164) 209,044

8 0.6874 0.700010 0.004(0.003) 0.933(0.077) 209,050 and 209,053

15 0.7194 0.710912 0.6645 0.7386

5 0.002(0.002) 0.400(0.237) 22 0.6588 0.61515 0.004(0.003) 0.800(0.164)

10 0.003(0.002) 0.200(0.154) 17 0.6326 0.6917 213,817 and 213,8235 0.004(0.003) 0.600(0.175) 32 0.7121 0.7444 213,8185 0.003(0.002) 0.700(0.218) 213,8081 0.000 - 213,819

5 0.002(0.002) 0.700(0.218) 10 0.6612 0.7153 213,8215 0.005(0.003) 1.000(0.127) 8 0.6759 0.6896 213,810

10 0.6353 0.7143

5 0.001(0.001) 0.600(0.175) 209,07324 0.7460 0.8051 209,067

5 0.000 0.000 213,8065 0.000 0.000 8 0.6832 0.7250

10 0.003(0.002) 0.844(0.103) 10 0.6428 0.6389 209,06310 0.6264 0.636110 0.6511 0.6786

10 0.011(0.007) 0.756(0.129) 11 0.7484 0.711810 0.004(0.002) 0.844(0.079) 10 0.6775 0.6595

3 0.000 0.000 10 0.6006 0.5806

164 345

10 0.002(0.002) 0.644(0.152) 28 0.6842 0.7458 213,82012 0.001(0.001) 0.318(0.164) 11 0.6993 0.7705 213,822

5 0.004(0.003) 1.000(0.126) 8 0.7379 0.7344 209,0455 0.003(0.003) 1.000(0.126) 10 0.7733 0.6750 209,0495 0.004(0.003) 1.000(0.126) 10 0.7868 0.7222 209,0614 0.002(0.002) 0.833(0.222) 11 0.7175 0.8111 214,1694 0.000 0.000 23 0.6953 0.6970 214,1717 0.003(0.002) 0.714(0.181) 217,170

14 0.002(0.002) 0.593(0.144) 21 0.7543 0.743423 0.7548 0.7826

66 145

and their respective locations.

DRY

PIT

ANA

ASHDYE

LCCROC

WICLBC

SAC CPR

COS

PEOHOR

ROA

CURWOCPIN

DEC

ORE

SAL

PESSLR

LOS

COY

LLA,UVAPAJ

WAL,LAG1SAL,LAG2

GUA,NFG,SFG

INC,NAV1NAV2

RUS,FERMWC

KEL,HENSEI

ADO,MID

Monterey BayDrainage

Pit RiverDrainage

San JoaquinDrainage

Sacramento RiverDrainageNavaro

River

GualalaRiver

Tomales BayDrainage

RussianRiver

Clear LakeBasin

A.

B.

Fig. 1. (A) Map of regions where the two species are found. (B) Map of sampling locations. Site codes are listed in Table 1. Only water bodies from which samples were takenare shown.

A. Aguilar, W.J. Jones / Molecular Phylogenetics and Evolution 51 (2009) 373–381 375

et al., 1995). Several roach subspecies have more intense conserva-tion needs due to limited distributions and the influence of habitatalteration (e.g. Red Hills roach). One subspecies of hitch (Lavinia

exilicauda chi) is restricted to Clear Lake and associated tributaries.Recently, this subspecies has also been classed as a ‘species of spe-cial concern’ (Moyle et al., 1995). Unlike the Sacramento hitch

376 A. Aguilar, W.J. Jones / Molecular Phylogenetics and Evolution 51 (2009) 373–381

(Lavinia exilicauda exilicauda) and the Monterey hitch (Laviniaexilicauda. harengus), the Clear Lake subspecies is the only knownpopulation of hitch that migrates into streams to spawn (Nicola,1974; Geary, 1978). Clear Lake hitch spawning runs havedecreased markedly in the past 20 years due to dam construction,pollution (including DDT), and habitat loss.

Previous genetic work on Lavinia has concentrated on under-standing the hybridization dynamics between the two species. Thiswork was focused on a single drainage in the Monterey Bay region(Avise et al., 1975). Avise et al. (1975) found that although morpho-logically distinct, hybridization between roach and hitch could bequite extensive. They also found that diagnostic allozyme loci wererare for the two species, indicating extensive hybridization orrecent speciation between the two species. The inclusion of DNAsequence data and polymorphic microsatellite DNA makers tothe roach/hitch system is needed to provide greater insight intothe genetic distinctiveness at the species and subspecies levels.

We studied the range-wide genetic structure in the two cur-rently recognized species of Lavinia. This will provide an indepen-dent, but not exclusive, representation of the distinct geneticentities that exist within the two recognized species and detectgroups that are reproductively isolated and representative of a spe-cies’ evolutionary history (Avise and Wollenburg 1997, Grady andQuattro 1999, Avise 2000). Additionally this information can offerunique insight into the diversification of the California icthyofauna.We also employed a rigorous analysis of nuclear (microsatellite)and mitochondrial DNA data to gain insight into the historical phy-logeography and taxonomic identity of Lavinia.

2. Methods

2.1. Sampling

Roach and hitch samples were collected by seine, backpackelectroshocker, and baited minnow traps. We sampled eitherwhole individuals or in most instances fin clips. Samples of allputative subspecies of roach and hitch were collected from thetype localities whenever possible (Fig. 1 and Table 1). Samples ofthe Pit roach (Ana River, OR and Dry Creek, OR) were obtained fromthe Oregon State University Museum. Voucher specimens of wholeindividuals were deposited in the Ichthyology Collection of theCalifornia Academy of Sciences.

2.2. DNA extraction

DNA was extracted by one of two methods. For the firstapproach utilized approximately 100 mg of white muscle or fin clipwas digested overnight in extraction buffer (10 mM Tris–HCl, pH8.2; 10 mM EDTA; 200 mM NaCl; 0.5% SDS; 200 mg/ml ProteinaseK). The digest was extracted once with an equal volume of phe-nol:chloroform:isoamyl alcohol (25:24:1) and once with an equalvolume of chloroform:isoamyl alcohol (24:1). DNA and proteinwere precipitated with 200 mM NaCl and the genomic DNA precip-itated with 2 volumes of 100% ethanol and centrifuged at13,000 rpm for 30 min at room temperature. The pellet waswashed with 70% ethanol (�20 �C), dried in a vacuum centrifuge,and re-suspended in 100 ll of sterile water. The second approachwas done solely on fin clip and utilized the Qiagen DNAeasy96kit on a QIABOT. 1:5 dilutions of the extracted DNA were used insubsequent microsatellite amplifications.

2.3. Mitochondrial DNA

Polymerase chain reactions (PCRs) were performed as 50-llreactions in a Perkin-Elmer Thermal Cycler (480 or 9700) with

negative controls. Mitochondrial genes (NADH-2 subunit (ND2)and control region (CR)) were amplified using the following prim-ers (NADH-2: ND2F—GGRGCRTAYTGGAGRAYAAYRAYYATTCA andND2R—CARGCCACCGCRKCRGCYAT; CR: primers ‘A’ and ‘E’ from(Lee et al., 1995)). PCR thermocycling profiles were the same forthe two primer sets: initial denaturation at 94 �C for 5 min fol-lowed by 30 cycles of 94 �C for 1 min, 55 �C for 1 min, and 72 �Cfor 2 min and a final extension of 72 �C for 7 min. The quality ofthe PCR product was checked by electrophoresing 5 ll of the PCRproduct on a 2% 1� TBE agarose gel and the bands visualized withethidium bromide. Products were cleaned with the Qiagen PCRcleanup kit and sequenced on either an Applied Biosystems Inc.(ABI) 373 or 377 automated sequencer. ND2 sequences werealigned by eye and control region sequences were aligned withCLUSTAL-W. Subsequent phylogenetic analysis treated gaps asmissing data.

We used the Aikake Information Criteria in ModelTest (version3.7) (Posada and Crandall, 1998; Posada and Buckley, 2004) to eval-uate proper models of sequence evolution for the control region andND2 alignments independently. Bayesian phylogenetic reconstruc-tion was performed with MRBAYES (version 3.1.2) (Ronquist andHuelsenbeck, 2003) partitioning on the control region and ND2 frag-ments. The analysis was run for 10,000,000 generations samplingtrees every 100 generations (100,000 total trees sampled). We dis-carded the first 25,000 sampled trees as burn in. Analyses were pre-formed on the entire data set and a ‘trimmed’ data set that did notcontain all Gualala and Pit roach sequences (see Results).

2.4. Microsatellites

We used 10 microsatellite loci developed from other NorthAmerican cyprinids: CypG8, CypG12, CypG23, CypG29, CypG30,CypG32, and CypG49 (Baerwald and May, 2004); Gbi-34 and Gbi-294 (Meredith and May, 2002); Rhca-20 (Girard and Angers,2006). Reaction conditions were the same for each locus: 4 ll ofdiluted DNA, 1� Amplitaq PCR buffer (ABI), 2.5 mM MgCl2,0.1 mM of each dNTPs, 0.3 pmol of each primer, and 0.4 U of Amp-litaq (ABI). One of the primers was end-labeled with one of fourcommercially available fluorescent dyes (ABI). Thermal cycler con-ditions for each locus were identical and consisted of an initialdenaturation period at 95 �C for 3 min followed by 35 cycles of a‘touch-down’ PCR protocol. The first cycle of the touch down pro-tocol consisted of a 30-s denaturation at 95 �C, a 30-s annealingstep at 60 �C and a 45-s extension at 72 �C. For the next 20 cyclesthe annealing temperature was reduced by 0.5 �C each cycle. Fol-lowing the 20 touch-down cycles, 15 standard PCR cycles (with a50 �C annealing temperature) were performed. PCR products werepooled post-amplification (pool 1: CypG49, CypG23, CypG32; pool2: CypG8, CypG12, CypG30, CypG29; pool 3: Gbi-34, Gbi-294,Rhca-20). Pooled products were run out on an ABI 3130xl auto-mated sequencer, with LIZ600 internal size standard, for sizing.Allele size calls were made in GENEMAPPER (ABI).

Microsatellite data was imported into GENETIX (version 4.5.2)(Belkhir et al., 2004) for the estimation of allele frequencies,observed and expected heterozygosity. The Markov chain-basedmethods of Gou and Thompson (1992) and Raymond and Rousset(1995) were used to assess Hardy–Weinberg equilibrium (HWE)linkage equilibrium for each locus within each population in GENE-POP’007 (Rousset, 2008). Ten thousand dememorizations followedby 20 batches of 1000 iterations per batch were run in all analyses.Pairwise chord distances (Cavalli-Sforza and Edwards, 1967)between all populations and neighbor joining (NJ) trees were esti-mated with PHYLIP (version 3.67) (http://evolution.genetics.washington.edu/phylip.html). Node support was evaluated with500 bootstrap replicates. Trees were exported into TREEVIEW(version 1.6.6) for viewing and manipulation.

A. Aguilar, W.J. Jones / Molecular Phylogenetics and Evolution 51 (2009) 373–381 377

We used STRUCTURE (version 2.2) (Pritchard et al., 2000) todetermine the number of genetic units in the Lavinia species com-plex without a priori designation of individuals to populations. Foreach run 100,000 burn-in steps was followed by 500,000 steps ofthe MCMC. We used the admixture model with correlated allelefrequencies and ran STRUCTURE with K = 2 through K = 15. Foreach K, 15 independent runs were performed to assure the conver-gence of the Markov chain and used to estimate mean and stan-dard deviations for each K. Due to the ‘plateauing’ nature of themean logPr(X|K) values for each K (see Section 3) we explored avariety Ks with similar mean logPr(X|K) values (K = 7–9). We em-ployed the approach of Jackobsen and Rosenburg (2007) to deter-mine the optimal alignment of clusters across individual runs foreach K. We utilized the program CLUMPP (version 1.1.1) (Jackob-sen and Rosenberg, 2007) with the Greedy algorithm and 10,000random input orders of the 15 independent STRUCTURE runs.Results from CLUMPP were imported into DISTRUCT (version 1.1)(Rosenberg, 2004) for viewing.

2.5. AMOVA

ARLEQUIN 3.11 (Excoffier et al., 2005) was used to perform ananalysis of molecular variance (AMOVA) for mtDNA haplotypesand the microsatellite loci. For mtDNA pairwise distances wereused and for microsatellites FST was used. For both marker sets10,000 replicates were used to assess significance of the AMOVA.Populations were grouped in three possible ways: (1) by species,(2) by subspecies, and (3) by drainage. Analyses were preformed

N61P14N60P11

0.4

Pit Roach

Gualala Roach

0.02

Fig. 2. Bayesian phylograms of the L. exilicauda and L. symmetricus mtDNA haplotypes. Btree: trimmed dataset (see Sections 2 and 3) with roach subspecies labeled and Pit roaccorrespond to recognized sub-species are labeled by common sub-specific name. For alphylogram of all haplotypes; Gualala and Pit haplotypes are indicated. Branches leading

on hitch and roach separately (case 2) and only roach was groupedby drainage (case 3). Grouping our sampled hitch populations bydrainage was equivalent to grouping then by sub-species.

3. Results

3.1. Mitochondrial DNA

We sequenced 66 hitch and 164 roach individuals for a 727-bpfragment of the mitochondrial ND2 and control region genes. A to-tal of 79 haplotypes were observed (Appendix 1—Supplementarymaterial; Genbank accession numbers: AF392455–AF392487 andAF392491–AF392551). The HKY + I (0.835) and TrNef + G (0.245)models were the most appropriate models of sequence evolutionfor the control region and ND2 alignments based on the AIC. Bayes-ian phylogenetic reconstruction of all haplotypes revealed twohighly divergent clades with high posterior probabilities (Fig. 2, in-set). These clades correspond to haplotypes from Gualala and Pitroach. Due to the highly divergent nature of haplotypes from thesetwo subspecies we re-analyzed a ‘trimmed’ dataset which weremoved the Gualala and Pit haplotypes and used two Pit haplo-types (N60P11 and N61P14) as outgroups. The AIC indicated thatthe TrN + I (0.838) and the GTR + G (0.239) were the best modelsof sequence evolution for the trimmed control region and ND2fragments, respectively. Four highly supported clades that corre-spond to roach subspecies were recovered in the analysis of thetrimmed dataset: Tomales, Red Hills, Navarro, and Russian River-Clear Lake haplotypes (Fig. 2). Elevated support was also found

N56P1-LBC

N5P1-LCC

N33P20

N33P1

N36P23

N8P1-PAJ

N24P35

N33P25

N32P17

N1P1-LCC,WOC,CUR,OSC,CPR,BAY,PIN

N40P34

N14P1

N12P1-CLH

N31P1-OSC

N21P29-DEC

N39P32

N10P27-LLA,UVA

N20P8

N37P24

N30P1-COY,SLR

N57P1-SAL

N48P1-ROC

N18P1

N52P1-WIC

N17P8

N9P1-UVA,CPR,BAY,PIN

N4P1-CLH

N9P26-UVA

N50P1-WIC

N49P1-ROC

N21P28-DEC

N16P7

N33P6

N22P35

N27P36N24P37

N3P1-LCC

N19P1

N54P1-LBC

N2P1-LCC,OSC,PAJ,SAL,WIC, PIN,CLH

N30P4-COY

N33P22

N13P1-CPR,CLH

N58P18

N26P36

N35P6

N58P19

N38P32

N53P1-LBC

N11P1-ROC,CPR,CLH

N15P1

N55P1-LBC

N28P1-COS

N25P36

N7P1-PAJ,SAL,WIC,LBC

N51P1-WIC

N46P1-ROC

N23P36

N6P1-LCC

N34P6

N29P1-PES,SLR

N47P1-ROC

N33P21

Red Hills Roach

Navarro Roach

Russian River/Clear LakeRoach

Tomales Roach

WOC,CUR

ranches with posterior probabilities greater than 0.8 are shown as thick lines. Largeh haplotypes used as outgroups. Haplotypes that form well supported groups andl other haplotypes, site(s) code is indicated after haplotype code. (Inset) Unrootedto these two groups have posterior probabilities of 1.0.

Table 2Mean LogPr(X|K) of 15 independent STRUCTURE runs, with K varying from 2 to 15.Values level off at K = 7–9 (bold).

K Mean lnPr(X|K) SD lnPr(X|K)

2 �16309.5 86.483 �15659.3 13.724 �15344.2 4.205 �15238.5 40.396 �15081.7 29.407 �14970.8 10.568 �14952.7 11.309 �14944.6 37.29

10 �15265.3 911.9811 �15149.6 644.2512 �15565.8 259.0613 �16778.7 31.7414 �16828.9 344.0815 �16608.4 674.10

378 A. Aguilar, W.J. Jones / Molecular Phylogenetics and Evolution 51 (2009) 373–381

for two separate groups of haplotypes, one found in the upper Tuo-lumne River drainage (Curtis and Woods Creeks—CUR/WOC) theother from Deer Creek in the southern San Joaquin Valley (Fig. 2).No significant groupings were recovered for any hitch haplotypes.

3.2. Microsatellites

A total of 145 hitch and 365 roach were typed and scored at 10microsatellite loci. Sampled sites exhibited moderate microsatel-lite heterozygosity and allelic variation (Table 1). The CosumnesRiver sample exhibited the lowest heterozygosity and lowest meannumber of alleles (Table 1). Two of the loci (CypG12 and CypG49)were significantly out of HWE in 18 and 14 population samples,respectively, after correcting for multiple tests (p < 0.17) (Benja-mini and Yekutieli, 2001; Narum, 2006). These loci were removedfrom all subsequent analyses. Two of the pairwise comparisons forlinkage disequilibrium were statistically significant after correc-tion for multiple comparisons (p < 0.017).

The neighbor joining tree indicated strong to moderate supportfor all of the currently recognized hitch and roach sub-species(Fig. 3). All of the L. exilicauda samples formed a group with 65%bootstrap support. Elevated bootstrap support (77%) was alsofound for the Clear Lake hitch samples (sites ADO and MID). Withinthe hitch cluster there was also evidence of genetic associations forsites from the Monterey Bay area with bootstrap support greaterthan 63% (sites PAJ and SAL). High bootstrap support (93% and91%, respectively) was found for both the Pit roach (sites ANC,DRY, and PIT) and Gualala roach (sites GUA, NFG and SFG)(Fig. 3). Elevated support (>50%) was also found for groupings ofthe Navarro roach (sites NAV, INC and PDC), Tomales roach (sitesLGO and LAG), and separate groupings of roach from the Clear Lake(sites KEL and SEI) area and the Russian River (sites FER and MWC).

Mean LogPr(X|K) values rose from K = 2 to 7 and plateaued, sub-sequently falling off again after K = 9 (Table 2). The most biologicallymeaningful result comes from K = 8. Clearly defined groups based onthis K include the Gualala, Navarro, Pit, and Red Hills roach sub-spe-

0.01

ADOMID

LBCROCWIC

BAYCPR

PAJSAL

COSLCC

DYCASC

PEOSLR

GATULC

LGOLAG

FERMWC

NAVPDC

INCGUA

NGUSFG

ANCDRYPIT

HORKEL

SEI

7494

93

91

81

53

65

63

77

55

5852

Pit Roach

Gualala Roach

Navarro Roach

Tomales Roach

Sacramento/San Joaquin Roach

Monterey Roach

‘Russian River’ Roach

‘Clear Lake’ Roach

Red Hills Roach

Clear Lake Hitch

Hitch

Fig. 3. Neighbor-joining tree based on chord distances of eight microsatellite locifor all samples hitch and roach populations. Bootstrap values above 50% are shown.Site codes are given in Fig. 1 and Table 1.

cies, as well as roach from Peoria Creek and the Cosumnes River(Fig. 4). Monterey and Tomales roach individuals belonged toanother cluster (Fig. 4). Ancestry of hitch individuals was predomi-nately from a separate cluster with evidence of ancestry from theRed Hills and Cosumnes roach populations (Fig. 4). Results at K = 7were similar except that Navarro and Cosumnes River populationsgrouped together (Fig. 4). While the results of K = 9 gave rise to anadditional cluster found in hitch that was not informative (Fig. 4).

3.3. AMOVA

The AMOVAs indicated significant partitioning of variation, forboth mtDNA and microsatellites, for most analyses (Table 3).Grouping populations by sub-species increased the amount ofamong group variation for both roach and hitch. Grouping roachpopulations by sub-species or drainage did not significantly changethe amount of among group variation (Table 3). The only non-sig-nificant partitions were the among species comparisons for allLavinia and the among sub-species comparison for hitch.

4. Discussion

The genetic entities that we find below the species level, with afew exceptions, mirror the sub-species described by Murphy(1948) and Moyle (2002) that are based primarily on morphology.Likewise data from the microsatellite loci readily separate roachand hitch across the range of the two species, a result that is notsupported by mitochondrial DNA or past work on allozyme loci(Avise et al., 1975). These results suggest a complicated patternof historical isolation that has implications for better understand-ing the genetic structure of the endemic freshwater fauna of theCalifornia region and establishing conservation units for the twospecies.

A long history of isolation is evident for roach from the Pit andGualala River drainages. Both the Pit and Gualala roach have recip-rocally monophyletic mitochondrial DNA haplotypes and showstrong differentiation based on nuclear microsatellites. Weobserved 12.1% corrected sequence divergence between Pit roachand all other Lavinia (minus Gualala roach) for the mitochondrialsequence data. Assuming 1.5% sequence divergence per millionyears for teleost mitochondrial genes (Bermingham et al., 1997)the Pit roach have been isolated for approximately 8 million years.Historically, the Pit drainage was isolated from the SacramentoRiver drainage and connected with the Klamath drainage (Dupreet al., 1991). During the Pliocene the connection between the Pitand Sacramento drainages was established (Minckley et al.,1986; Dupre et al., 1991). The large amount of divergence observedfor Pit roach may be a result of an early colonization of the Pit

Hitc

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Sac

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y Ro

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Cosu

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s Ri

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oach

Red

Hills

Roa

ch

Peor

ia C

reek

Roa

chM

onte

rey

Roac

hCl

ear L

ake

Roac

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Russ

ian

Rive

r Roa

chTo

mal

es R

oach

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rro R

oach

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K=9

K=8

K=7

Fig. 4. Results of the STRUCTURE/CLUMPP analysis (K = 7–9) on 145 L exilicauda and 345 L. symmetricus individuals (see Section 2 for details).

Table 3Analysis of molecular variation (AMOVA) of mtDNA haplotypes and eight microsatellite loci. Variation is partitioned among species, among sub-species for L. exilicauda and L.symmetricus, and among drainages for L. symmetricus.

mtDNA Microsatellites

df Percent of variation p df Percent of variation p

LaviniaAmong species 1 4.19 0.1095 1 1.14 0.0088Within species 32 87.22 <0.0001 31 6.38 <0.0001Within populations 196 8.59 <0.0001 881 92.48 <0.0001

L. exilicaudaAmong sub-species 2 15.83 0.1237 2 1.08 0.0195Within sub-species 6 16.55 0.001 6 0.44 0.0166Within populations 57 67.11 <0.0001 277 98.48 <0.0001

L. symmetricusAmong sub-species 7 84.02 <0.0001 7 8.03 <0.0001Within sub-species 17 9.49 <0.0001 16 1.82 <0.0001Within populations 139 6.48 <0.0001 604 90.15 <0.0001

L. symmetricusAmong drainage 10 84.16 <0.0001 9 5.1 0.001Within drainage 14 8.98 <0.0001 14 4.2 <0.0001Within populations 139 6.85 <0.0001 604 90.7 <0.0001

A. Aguilar, W.J. Jones / Molecular Phylogenetics and Evolution 51 (2009) 373–381 379

drainage when the connection to the Sacramento was first estab-lished followed by a long period of isolation. Clearly more intenseevaluation of this species and others within the region (e.g. ende-mic Pit River sculpins), with nuclear sequence data, will elucidateputative invasion histories of the Sacramento basin ichthyofaunainto the Pit River basin (and vice versa). The high degree of isola-tion that has occurred for the roach that inhabit the Pit River drain-age may be indicative of isolation for other species that occur inthis drainage, such as the endemic species Pit sculpin (Cottus piten-sis), Rough sculpin (Cottus asperrimus), Pit-Klamath Brook Lamprey(Lampetra lithophaga), and Modoc sucker (Catostomus microps)(Moyle 2002).

We also observe an interesting an unexpected pattern for theGualala roach—high genetic divergence, much like the Pit roach,with both nuclear and mitochondrial markers. This observation issurprising given the geographic proximity to other roach bearingstreams (Fig. 1). Fish from the nearby Navarro, Tomales, andRussian River drainages also show some degree of distinctiveness,though they do not display the degree of divergence that the Gual-ala roach do (for all analyses). The observed mean sequence diver-gence between Gualala haplotypes and all other Laviniamitochondrial haplotypes (except Pit roach) is 7.3% which, assum-ing 1.5% sequence divergence per million years (Bermingham et al.,1997), translates into 4.9 million years of divergence. Unfortu-

nately there are no available geological explanations for isolationof the Gualala River basin or comparative phylogeographic datafor other aquatic taxa with similar distributions as roach in coastalnorthern California. Other sympatric freshwater fish species alsopossess the ability for limited marine dispersal (Cottus asper, Cottusaleuticus, Gasteroceus aculeatus, and Oncorhynchus mykiss) and willundoubtedly not show the same degree of divergence that Gualalaroach displays.

The proposed roach sub-species of Moyle (2002) are reflected inthe analysis of nuclear microsatellite loci. Mitochondrial DNA didindicate some degree of reciprocal monophyly for the Navarro,Red Hills, and Tomales roach, though many of these grouping didnot have high statistical support. The neighbor-joining tree (basedon microsatellite distances) support Moyle’s sub-specific designa-tions, with a separation of Russian River roach (sites FER andMWC) and Clear Lake roach (sites KEL and SEI). Additionally theSTRUCTURE analysis clearly distinguished the Red Hills, Pit andGualala roach. Likewise the AMOVA indicated a significant amountof genetic variation, for both mtDNA and microsatellite, was parti-tioned among roach sub-species. This indicates that morphologicaland genetic differentiation for roach occur on drainage or basin-wide scales. Even in areas where hybridization between roachand hitch is known to occur at high frequencies (Pajaro River,Sacramento and San Joaquin River drainages) and mitochondrial

380 A. Aguilar, W.J. Jones / Molecular Phylogenetics and Evolution 51 (2009) 373–381

DNA is not diagnostic between the two species or among the roachsub-species, we were still able to recover the various roach groups.Roach found in the Clear Lake and Russian River areas have tradi-tionally been grouped together based on morphological similaritiesand this is supported by a historical connection between these twobasins (Minckley et al., 1986; Dupre et al., 1991). Our results sug-gest that there may be fine-scale differentiation between roachpopulations that inhabit these two areas. This observation, coupledwith the genetic distinctiveness of the Clear Lake hitch (and roach),has implications for the isolation and evolution of the Clear Lakeichthyofauna, which has historically possessed a number of ‘ende-mic’ species or sub-species.

The historical connection between the San Francisco Bay drain-age and the Monterey Bay drainage (Hopkirk, 1973) is also evidentin the genetic relationships of roach populations from theseregions. Roach from Los Gatos Creek (GAT), which drains into theSan Francisco Bay, shows a close affinity to the Llagas Creek(LLA) population that flow into the Monterey Bay. Additionallyroach from Coyote Creek (COY), which also feeds into the San Fran-cisco Bay, share an mtDNA haplotype (ND9P1) with roach from Lla-gas Creek and Uvas Creek (Monterey Bay drainage). It is knownthat up until 1.5 million years ago rivers that are currently partof the Pajaro Basin flowed north into the San Francisco Bay (Dupre,2000). This historical connection could give rise to the genetic sim-ilarities observed between contemporary Monterey Bay drainageand San Francisco Bay drainage roach populations.

We did not find a conclusive association of roach from the Sac-ramento and San Joaquin basins. Four of the six populations fromthese basins grouped together in the microsatellite tree (sitesASC, PEO, DYC, and LCC) and these populations tended to clustertogether in the STRUCTURE analysis. Conversely Horton Creek,Peoria Creek, and the Cosumnes River populations do not grouptogether, despite their close geographic proximity. This is true inthe microsatellite tree and in the STRUCTURE analysis (K = 8)where each assign to separate clusters. We also observed well sup-ported clades of haplotypes from the upper Tuolumne River (CURand WOC) and the southern San Joaquin Valley (DEC). Though allof these samples together do constitute two separate sub-species(Red Hills roach and Sacramento–San Joaquin roach) these resultsindicate that a higher amount of within basin differentiation mayoccur in the San Joaquin Valley and that a number of San JoaquinValley populations may constitute distinct evolutionary units.Roach do exist further south in the San Joaquin basin than we havesampled (Tulare, Kings, and Kaweah Rivers), and subsequent anal-ysis of these populations may shed further light on the historicalassociations and distinctiveness of populations from the southernSan Joaquin Valley.

Hitch show elevated bootstrap support for being a distinct ge-netic entity and also show genetic support for two recognizedsub-species (Clear Lake hitch—L. e. chi and Monterey hitch—L. e.harangus) with the microsatellite but not mtDNA data. This is evi-dent in both the phylogenetic analysis and AMOVA of mtDNA hap-lotypes. Past genetic studies of roach and hitch were not able touncover diagnostic molecular makers for these species (Aviseet al., 1975). Additionally there is a lack of reciprocal monophylyof mtDNA sequences and high degree of shared mtDNA sequencesbetween roach and hitch (Jones, 2001). This lack of any clear genet-ic distinctiveness, despite being morphologically unique, could bedue to high amounts of introgression or incomplete lineage sortingfor mtDNA loci. It is clear the hybridization rates can be high insome areas, particularly the Monterey and Sacramento–San Joa-quin regions (Avise et al., 1975; Jones, 2001), and that this is mostlikely the cause of the observed mtDNA patterns between roachand hitch. Though we cannot currently test this with the availabledataset, it appears that nuclear genes may not move across roach-hitch hybrid zones as readily as mtDNA does. The propensity for

mtDNA haplotypes to move across hybrid zones, when comparedto nuclear genes, has been observed in other North American cyp-rinids (Dowling and Hoeh, 1991) and limits our ability to assessabsolute divergences between roach and hitch solely based onmtDNA.

We did find evidence for a geographic component to the group-ings within hitch based on the analysis of microsatellites. The ClearLake hitch (L. e. chi) was distinct based on the neighbor joining treeof microsatellite genetic distances. This is not surprising given thegeographic isolation of this population from other hitch popula-tions, the extensive morphological differentiation between ClearLake hitch and all other hitch (Hopkirk, 1973; Moyle, 2002), andthe lack of observed hybridization between roach and hitch inthe Clear lake basin (Jones, 2001). Additionally the grouping ofhitch from the Monterey area (sites PAJ and SAL) in the microsat-ellite tree and a significant AMOVA when hitch populations aregrouped by sub-species tentatively indicates that ‘pure’ (based onmorphology) hitch from the Monterey Bay area may be a geneti-cally distinct entity supporting the notion that individuals fromthis region are in fact a recognizable sub-species. Notably sitesfrom the Sacramento River basin do not group together (sitesCPR, LBC, ROC, and WIC). This could be due to a lack of informationin the microsatellite loci to resolve these relationships at this levelor due to the introgression of divergent roach alleles. The STUC-TURE analysis does not indicate a roach component in many ofour hitch samples, indicating that additional informative locishould resolve this. Analysis of more samples from the Sacramentoand San Joaquin basins would allow for more definitive designa-tion of unique groups in this area.

4.1. Phylogeography of the California icthyofauna

While no other studies exist for a rigorous comparative analysis,some salient findings do come out of this study that has implicationsfor better understanding the evolution of the California icthyofauna.The high divergence observed for the Pit roach may be indicative ofisolation for other taxa in this region. Oakey et al. (2004) did find agrouping of speckled dace from the Pit and Sacramento drainages;however their sampling could not assess absolute levels of diver-gence between Pit and Sacramento populations. Our finding ofgenetic similarity between streams/rivers that empty into the SanFrancisco Bay and the Monterey Bay regions is supported by anotherfreshwater fish (Oakey et al., 2004) and strong geological evidence(Dupre, 2000). The reported historical connection between the SanJoaquin and Monterey Bay drainages was not evident from ourresults. However, there is biogeographical evidence to support thisconnection (Snyder, 1913; Moyle, 2002) and future work shouldconcentrate on associations between populations from the westernportion of the San Joaquin drainage and Monterey Bay drainage pop-ulations. Lastly, the high divergence observed between the Gualalaroach populations and all other roach populations is at this timeenigmatic. Comparative analyses of other taxa, especially freshwaterrestricted species, will provide valuable information on patterns ofhistorical isolation for this region.

4.2. Conservation implications

This study provides evidence that the sub-species previouslydescribed for hitch and roach are genetically distinct units andshould be managed as such. Currently the Red Hills roach (L. s.ssp) is listed as endangered and the Clear Lake hitch (L. e. chi)and Pit roach (L. s. mitrulus) are listed as species of special concernby the state of California. Additional conservation attention shouldalso be given to the Gualala roach (L. s. parvipinnis) as it displayshigh degree of genetic distinctiveness and may constitute a smallisolated population. We would also argue that future studies

A. Aguilar, W.J. Jones / Molecular Phylogenetics and Evolution 51 (2009) 373–381 381

should investigate fine-scale genetic structure for each of the sub-species in order to gain a better handle on standing genetic varia-tion, anthropogenic influences on movement patterns and the ex-tent of hybridization among species and sub-species (Avise et al.,1975; Jones, 2001). This will be especially useful in delineatingboundaries for sub-species that have limited geographic rangeslike the Red Hills roach (Jones et al., 2002).

Acknowledgments

We thank S. Reid for collecting the Pit River L. symmetricus sam-ples. D. Cooper assisted with the laboratory work. K. Kamer andtwo anonymous reviewers made valuable comments that improvedthe quality of this manuscript. All specimens collected by W.J. Joneswere collected under CDFG Permit Nos. 803053-02, 803036-03, ad803026-05 and a Memorandum of Understanding following the cri-teria of the Chancellor’s Animal Research Committee (CARC).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2008.11.028.

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