Restriction Fragment Length Polymorphism Analysis Reveals High Levels of Genetic Divergence Among the Light Organ Symbionts of Flashlight Fish

9 Pages·1991·3.6 MB·English

by  C J Wolfe

Reference: Bwl Bull 181: 135-143. (August, 1991)
Restriction Fragment Length Polymorphism Analysis
Reveals High Levels of Genetic Divergence Among
the Light Organ Symbionts of Flashlight Fish
CONNIE J. WOLFE AND MARGO G. HAYGOOD
Scripps Institution ofOceanography, University ofCalifornia, San Diego. California 92093
Abstract. Restriction fragment length polymorphisms anomalopid fishescontain fivespecies:Anomalopskatop-
within the lu.\and 16S ribosomal RNAgeneregionswere tron, Photoblepharon palpebratus, Photoblepharon stein-
used to compare unculturable bacterial light organ sym- il:i, Kryptophanaron alfredi, and Phthanophaneron har-
bionts ofseveral anomalopid fish species. The method of vevi. Evidence from comparative morphology and bio-
Nei and Li (1979) was used to calculate phylogenetic dis- geography suggests the following familial phylogeny
tance from the patterns ofrestriction fragment lengths of (Johnson and Rosenblatt, 1988). Theanomalopidsappear
the luxA. and 16S rRNA regions. Phylogenetic trees con- to have branched off of the Trachithyid lineage during
structed from each distance matrix (luxA. and 16S rDNA the Cretaceous period. The genus Anomalops probably
data) have similar branching orders. The levels ofdiver- diverged later in the Cretaceous and is radically different
gence among the symbionts, relative to other culturable from the other genera. Phthanophaneron diverged in the
luminous bacteria, suggests that the symbionts differ at Miocene with Kryptophanaron. and Photoblepharon di-
the level ofspecies among host fish genera. Symbiont re- verged most recently, in the Pliocene.
latedness and host geographic location do not seem to be Luminousbacterial symbiontsin several otherfamilies
correlated, and the symbionts do notappearto be strains offishes have been cultured. In these instances, a single
ofcommon, free-living, luminous bacteria. In addition, species ofsymbiont is associated with an entire family of
the small number ofhybridizing fragments within the 16S host fish. These bacteria belong to species that have also
rRNA region ofthesymbionts, compared with that ofthe been isolated directly from seawater, and thejuvenile host
free-living species, suggests adecrease in copy number of may acquire itssymbionts from the surroundingwaterat
rRNA operons relative to free-living species. At this level each generation (Ruby and Nealson, 1976; Fitzgerald,
ofinvestigation, thesymbiontphylogenyisconsistent with 1977; Reichelt el al. 1977; Leis and Bullock, 1986; Wei
theproposed phylogenyofthehostfish familyandsuggests and Young, 1989). Other light organ symbioses, in cer-
thateach symbiont strain coevolvedwith itshost fish spe- atioid fishes and pyrosomes, contain unculturable sym-
cies. bionts, and the identity ofthe symbionts is unknown in
thosecases.Theanomalopidsymbiontshave,asyet,defied
Introduction all ofour attempts to culture them in laboratory media.
Our inability to culture the symbionts, combined with
Members ofthe teleost fish family Anomalopidae har- the extensive morphological adaptations ofthe host fish,
bor luminous bacterial symbionts in specialized light or- suggest an obligate symbiosis in which the bacteria may
gans, located under each eye. Light is produced contin- have coevolved with the fish host.
uously by the bacteria through the action ofbacterial lu- We have compared the symbionts in four species of
ciferase; but depending on the species, the light may be anomalopid fish by analyzing restriction fragment length
occluded, either by rotation ofthe entire light organ, or polymorphisms (RFLPs) of fragments containing either
by a shutter mechanism (Kessel, 1977; Leisman el a!., the luciferase structural genes (/j/.vAB), or the 16S ribo-
1980;Johnson and Rosenblatt, 1988). The fourgenera of somal RNA genes. RFLPs ofother structural genes have
frequently been usedto compare speciesand populations
Received 20November 1990:accepted 18 March 1991. ofbacteria (Stanley el al.. 1985; Franche and Cohen-Ba-
135
136 C. J. WOLFE AND M. G. HAYGOOD
zire, 1987; Denny el a!., 1988; Lindblad el at., 1989). palpebratus #1 is dark, has the white area on the opercle
Ribosomal RNA genes are very conserved, and compar- characteristic ofthe species, and the white reflectingscales
isons of rRNA gene restriction patterns had previously and fin edgings shared with P. steinitii. P. palpebratus#2
been used todifferentiate between species, aswell as sub- isgrey, and lackscontrasted white areas on the body and
species, ofbacteria (Gottlieband Rudner, 1985;Grimont fins (R. H. Rosenblatt, pers. comm.). The P. steinitzi
and Grimont, 1986; Saunders et a/., 1988; Stull el a/., specimen wasobtainedat Eilat, Israel, andisthusvirtually
1988; Buyser cl al, 1989). Our goal was to analyze both certainly P. steinitii, although the specimen is no longer
gene regions to determine: (1) whether the symbionts are available for positive identification.
the same species in all ofthe anomalopids or are specific
to fish species, genus, or geographic location; (2) the in- DNA preparation
terrelationshipsofthe symbiontscompared with the phy- DNA
lhyopgoetnhyeosfist;heahnodst(3f)isht,hetobsruopapdoerrtoprhyrleofguteenetthieccoafefviolliuattiioonns ligShtamoprglaenssoafs in HawyegroeopdreapnadreCdohfrno(m19s8in6g)l.e,ToenttailreD,NfiAsh
ofthe symbionts within the luminous bacteria. yDiNelAd per light organ ranged from 5 to 63 ^g of DNA.
from the other luminous bacterial strains was iso-
lated by the method ofDitta et al. (1980). DNA concen-
Materials and Methods
tration was determined spectrophotometrically by absor-
Bacterialstrains bance at 260 nm.
Bacterialstrainsusedinthisstudyaredescribed inTable DNA and
digestion, electrophoresis, hybridization
I. The free-living luminescent strains weregrown in com-
DNA
plex seawater broth (SWC: Ruby and Nealson, 1978) at samples were digested with the following en-
25C. zymes: Hpal Seal Bglll, Kpnl, Pst\, C/al, Hindi and
Sphl for lu\ RFLP analysis; and EcoRl, Bglll, Hindlll
Fish collection and Neil forthe 16S rRNA genecomparison. Eachdigest
consisted ofabout 200 ngofDNA. The digests were sep-
Fish were hand-collected by SCUBA divers in the lo- arated by0.8% agarose gel electrophoresis in 40 mA/Tris
cations shown in Table I. Samples offour species offish acetate/1 mA/EDTA pH 8.0. Theagarosegelsweredried
were obtained, including two species (.-1. katoptron and at 60C, denatured, neutralized (Tsao et al., 1983), and
P. palpebratus) from two genera collected at the same prehybridized at 60C in 6 X SSC (sodium chloride/so-
location in Papua New Guinea. Both specimens ofPho- dium citrate; Maniatis et al.. 1982), 2.5 mA/EDTA. 200
toblepharon from Papua NewGuinea would be identified yug/mldenaturedsalmonsperm DNAand0.5 mg/mldried
as P. palpebratus on the criteria given by McCosker and milk. Inpreviouswork,theA', alfredisymbiont luxregion
Rosenblatt(1986). Thetwospecimensaresimilarin mer- was cloned into pDR720 (Haygood and Cohn, 1986). A
isticsand overall morphology, but differ in coloration. P. 1.73 kb Hpa\/Kpn\ fragment containing ln.\A and halfof
Table I
OriginsolD.\.l .si/m/>/c.v
Bacterial species
FLASHLIGHT FISH SYMBIONT RFLPS 137
Table II
Pairwisecomparisonsofthepercentageconservedfragmentsandthegeneticdistances(seeMaterialsandMethods) within theluxgeneregions
<>/Vibrioharveyi andtheanomalopidsymbionts
Strain
138 C. J. WOLFE AND M. G. HAYGOOD
Kpn\ Hpa\ Bgl\\ izingfragments inthe luxcomparisonsareconsistentwith
the presence ofasingle copy ofthe luxgenes, in both the
12345 12345 12345 symbionts and the culturable strain. In the 16S compar-
kb isons, the low number of symbiont fragments indicates
that a mixed symbiont culture is unlikely. As shown by
the fractions ofconserved hybridizing fragments (Table
III. lower left), the 16S probe generally yielded a higher
23.13 number ofconserved fragments than the lux probe. Dis-
tancescalculated from hybridization patternsamongcul-
9.42 turable species range from 0.062 to 0.216, with a mean
of0.1 13 0.040 (the standard deviation was calculated
from values in Table III). As in Table II, the d values
shown in Table III have been multiplied by 100 to ease
visual comparison. The symbiontsdiffer from thecultur-
6.68
able specieswith distances ranging from 0.099 to >0.180.
Distances among symbionts from fish ofdifferent genera
4.36- range from >0.1 17 to >0.151, contrasting strongly with
the level ofdifference seen between strains ofculturable
species(0.029 0.021). Thus,thesymbiontsinthisfamily
do not appear to be members ofa single species.
In two instances, a strain-level relationship was seen
between symbionts. The two specimens ofA. katoptron
were indistinguishable, and distance values for symbiont
labKeilgeudre1.7I.3 kbAu/tH.oVrpardoiboegrtaomDoNbAtaienxetdracftreodmfhryobmridI'iizhartiioohnarovfeytiheB31!9P2- cDlNosAe,swiftrhoma cP.alcstueliantietdzidiasntdanPc.epoafl0p.e0b2r3a.tuTshu#s1, abreecaaulsseo
(al1f)racdnid#t1he(3l)i.ghPthoorlgoahnlscopfhaArnoonmapfalappeshrkaattuosp#t2nm(4#)1, a(2n),dKPrhyopltMopehpahnaarroonn these values are within the range that we have seen when
xlcinil:i(5). DNA wasdigested with Kpn\. Hpa\ and B,?/II. comparing strains of the same species of Vibrio (Table
III), these two symbiont strains should be considered as
are most similar, with a distance of0.109. The A. katop-
tron specimen has no conserved hybridizing fragments ^Vibrio harveyi B392
with any of the other anomalopid symbionts; thus the
calculated distances are underestimates ofan unknown
distance. The divergence (d) among symbionts and be-
tween symbionts and I". han>eyi ranges from 0.109 to
>().178 whencalculatedwith Nei and Li'smethod(Table
II, upper right). Values for(/have been multiplied by 100
in Table II, to facilitateeasy visual comparison. The con-
sensus tree is shown in Figure 2.
Comparison ofRFLPs detectedby the 16S rRNA
geneprobe
In DNA digestsofevery bacterial strain tested, the 568
bp 16S rRNA gene probe hybridized with between 1 and
12 restriction fragments generated by enzymes with six-
base-pair recognition sequences (Fig. 3). Digestion ofthe
1'. liarveyi strains generated the highest number of hy-
bridizing fragments (~9/digest), while digestion of the Figure 2. Phylogenetic tree for anomalopid symbiont strains and
symbiont DNAs, particularly fromAnomalopsandKryp- I'ibrioharveyiconstructed from theluxregiongeneticdistancedata in
loplumaron, resulted in fewer hybridizing fragments (~2/ Table II. The tree is a majority rule consensus tree derived from the
digest). In boththeluxand 16Scomparisons, the number CleOngNtShsEwNeSreEopbrtaoignreadmforfomPHthYeLdIaPta(isneeTaMbalteerIiIaulssiangndthMeeFthIoTdCsH).prBorgarnacmh
ofhybridizing fragments indicates that a single symbiont ofPHYLIP. The arrow indicates that the followingbranch lengthsare
inhabitseach lightorganorthatanyadditional symbionts minimum estimates: there were few or no conserved hybridizing frag-
arepresentat undetectable levels. The numberofhybrid- mentsbetween thisgroupofstrainsand theotherscompared.
FLASHLIGHT FISH SYMBIONT RFLPS 139
EcoR! H/ndlll
Sg/ll
12345678910111213 12345678910111213 12345678910111213
kb
-21.23
21 .23-
Figure3. Autoradiogramobtainedfrom hybridizationofthe32P-labeled 16SrRNAgeneprobetoDNA
extractedfromthelightorgansofKryptiiphanaron allrcdi#16(1).Anomalopskaloplron#2(2).Anomalops
katoptron #1 (3), Photoblepharon palpebratus #2 (4), Photoblepharon palpebratus #\ (5). Photoblepharon
steinit:i(6),andfromlaboratoryculturesofI'ibriotischcriB61 (7), VibriofischeriMJ1 (8), I'ibriooricnhili\
#2(9). I'ibrioorienialis#\ (10). I'ibrioImn-eyi BB7 (11),and I'ibriohan-eyiB392 (12). DNA wasdigested
with f<)RI. %/II and Hind\\\. Molecularweight markersarethesameforboth EcoR] and fi.tf/II.
belonging to the same species. All ofthe Photoblephanm than to the other P. palpebratus sample (P. p. #2). The
symbionts(P. p. #1, P. p. #2,and P. s. #1)aremoresimilar degree ofdifference between the symbionts ofthe P. pal-
toeachotherthantoA", alfrediandA. katoptron. However, pebratus specimens is even more anomalous when com-
two samples from different individuals ofP. palpebratus pared to the total conservation of restriction fragments
(P. p. #1 and P. p. #2) collected in the same geographic from the DNA oftwoAnomalops specimens from differ-
location showed considerable differences in their restric- ent geographic locations. The possibility of inadvertent
tion patterns. Moreover, oneP. palpebratussample (P. p. exchange ofsamples was investigated and it does not ap-
#1) is more closely related to the sample of P. steinitzi pearto haveoccurred; theexperimentswere repeatedwith
140 C. J. WOLFE AND M. G. HAVGOOD
identical results. The consensus tree generated is shown
in Figure 4.
^ Discussion
<",-5- V%O OONO opd \pC * C draOmuarticdaaltlayiwnidtihciantethtehaftamtihleybAanctoemrailalopsiydmabei.onBtasseddiffoenr
'< il RFLP analysis within the /.vgene region, the symbionts
from different genera are almost as different from one
another as the Kryptophanaron symbiont is from I', har-
veyi. Comparison ofthesymbiontrestriction patternswith
s,i those of known species of luminous bacteria within the
.- a 16S rRNA gene region strongly supports the conclusion,
vOv-N> OivNn pO Eofo^ dgrreaawtenrtfhraonmsttrhaeinlulexvedlatbae,twteheant gtehneersaymwbiithoinntsthdeifffaemrilayt
Anomalopidae.
2
In other light organ symbioses, in which the symbionts
havebeen cultured, all membersofthe fish familycontain
ON o od od r-j o o the same bacterial species. RFLP analysis supports this
V V V assertion for Photobacterium phosphoreum, a bacterial
i! species that inhabits light organs of several families of
s J fishes. The genetic distances between three strains ofP.
rv~>! *~"""": O"! O~T o~-' rn. '-dJ r"-^' o*-? O"!""O' phosphort'iim from two species of opisthoproctid fishes
V V V i 3 (Opisthoproctus gnnuildii and Opisthoproctus soleatus)
and P. phosphoreum NZ1 1D, a symbiont from a macro-
urid fish, were determined using the same 16S RFLP
analysis method, and ranged from 0.002 to 0.006 (data
not shown). The small genetic distances observed forthe
opisthoproctid symbionts contrast greatly with the large
differences observed among the anomalopid symbionts.
Clearly, theanomalopidsdo notappeartohostasingle
F i species. Symbionts from differentanomalopidgeneradif-
1030 = ferat least asmuch asspeciesofculturable luminousbac-
e teria. In the 16S rRNA analysis, the observed distances
N so" r-' r--' od < ^s -rc between the anomalopid symbionts are considerably
greaterthan thedistancebetween I'. harveyiand ('. orien-
talis, approachingthedistance between I'. harveyiand I".
": ""; P "* P "O P P ^ fischeri, two highlydivergent membersofthegenus I'ibrio
(Baumann el a/.. 1980). In contrast, there is a high level
of conservation among strains of characterized Vibrio
species, even among the free-living and monocentrid
_ symbiont strains of I'. fischeri from different locations
, r*-( . 1 (strains B61 and MJ1, Table I and Fig. 4).
SS! Within theanomalopid family, equivalent strain levels
ofsimilarity are found only within genera, in two ofthe
three samples from the genus Photoblepharon (P. p. #1
and P. s. #1) and the samples from different specimens
ofA. katoptmn. In addition, we recently collected several
specimens ofKryptophanaron alfredifrom Roatan, Hon-
duras, and compared five light organ samples, using the
16S probe and the same restriction enzymes previously
\f: Si
S used in thisstudy. The specimens were indistinguishable,
'-< t/i
-o with all fragments conserved. Thus, although we do not
have additional samples of each fish species within the
anomalopid family, it seems likely that symbionts within
FLASHLIGHT FISH SYMBIONT RFLPS 141
least 60 million years before the divergence of Krypto-
plmnawnand Photoblepharon(<5 million yearsago). The
Kryptophanaron symbiont also differs from the Photo-
blepharon symbionts, but does share some conserved hy-
bridizing fragments with Photoblepharon symbionts in
both the luxand rRNA analyses. These data suggest that
the symbionts may have coevolved with the host fish.
However, the divergence between the two P. palpebratus
P f^lfrbrolulfl2 samples raises the possibility that the apparent corre-
spondence between symbiont and host trees could dis-
appear when more samples are examined; i.e., that ex-
treme divergence may occur in symbionts within host
species as well as between host genera. However, this hy-
pothesisisnot supported bythe identity observed between
the twoAnomalops samples from different locations, the
similarity ofP. palpebratus #1 and P. steinitzi, and our
Figure 4. Phylogenetic tree foranomalopid symbionts and several comparison offive K. alfredi samples. Equally likely al-
free-livingluminousbacteriaconstructedfromthe 16SrRNAgeneregion ternatives are that (1) the genus Photoblepharon is more
geneticdistancedatain Table III. Thetree isa majority ruleconsensus complex thancurrentsystematicswould suggest,andthat
treederivedfromtheCONSENSEprogramofPHYLIP. Branchlengths more than one type ofPhotoblepharon may occurat our
awPreHerYeLmIoibPnt.iaimAnurermdowfesrstooimnmatttheweso;doatfthtaehreienbwrTeaarnbeclheefsIeIwIinuodsriicnangtoettchheoantFsteIhrTevCebHdrahpnyrcbohrgilrdeainzmgitnhogsf rcoelalrercatinognemseitnet(isneethMeatPe.rpiaallpseabrnadtuMset#h2osdysm)bioron(t2)realagteinvee
fragmentsbetween thesestrainsand theotherscompared. to the other anomalopid symbionts could give an over-
estimate ofgeneticdistance. Unfortunately, theadditional
specimens needed to resolve this anomaly are not cur-
each hostspeciescompriseasinglespecies. Theanomalous rently available and will be difficult to obtain. Further
result with P. palpebratus #2 is discussed below. study ofboth the systematics ofPhotoblepharon and the
famTihley Aonbosmeravleodpiddiaveersiistnyotamasosnogciastyemdbwiiotnhtsgeowgirtahpihnictahle genAentoicthreerlatiendtneersesstoinfgthaespseycmtbioofnttsheis1r6eSquirrRedN.A RFLP
location, and this might be expected ifthe symbiontsen- analysis is the variation in number ofhybridizing restric-
gageingeneticexchangewith bacterial populationsinthe tion fragments between symbiont and free-livingbacterial
environment. DNA from specimensofP. palpebratusand DNA. In all cases, the symbionts show fewer hybridizing
A. katoptron collected from the same location in Papua fragments to the 16S probe than the free-living strains.
NewGuineaare no more similartoeach otherthan each Thelownumberofhybridizing fragmentsrevealed inthis
is to the K. alfredi symbiont from the Caribbean. In fact, study suggests the presence ofa bacterial monoculture in
they areconsiderably less similarthan thesymbionts from the light organ. In addition, these data could indicate a
Photoblepharon specimens from the Red Sea and Papua difference in copy number of the rRNA genes; i.e., the
NewGuinea(P. p. #1 andP. s. #1). Inaddition, symbionts symbionts havealowernumberofoperonsthan the free-
from twosamples ofA. katoptron, collected from different living luminescent strains. Similar studies on pea-aphid
locations in Papua New Guinea (~800 miles apart) ap- endosymbiontsrevealedthattheendosymbiontshaveonly
pear identical within the 16S rRNA gene region. Thus, a singlecopyoftherRNA operon, while related free-living
thegeneticsimilarityofsymbiontsseemstocorrelate more organisms haveseveral (Unterman and Baumann, 1990).
DNA
with host phylogeny than geographic location. Restriction fragment analyses of mycobacteria 1
Precise comparisons ofthe symbiont branching order have shown that the number ofrRNA copies correlates
to the proposed evolutionary history ofthe host fish are with growth rate, i.e.. slow growing species have fewer
difficult because the branch lengths are very rough esti- copiesofthe rRNA operon than thosegrowing morerap-
mates. However, the branching order, is similar in both idly (Bercovier et a!., 1986; Suzuki etal, 1987). Previous
analysesand isconsistent with that ofthe fish host (John- studies ofanomalopids suggested that symbionts in the
son and Rosenblatt, 1988). These highly conservative es- light organ have slow growth rates [doubling times of8-
timatesofdistance indicatethattheAnomalopssymbiont 23 hours: (Haygood et al., 1984)]. A reduced rRNA op-
differs from the other symbionts at the specific, and pos- eron copy number in the anomalopid symbionts may
sibly the generic, level. Anomalops is very different from represent a permanent adaptation to light organ condi-
the other genera in the family Anomalopidae and is tions, such that the growth rate is inherently low in the
thought to have diverged from the ancestral lineage at anomalopid symbionts.
142 C. J. WOLFE AND M. G. HAYGOOD
The taxonomic identity ofthe anomalopid symbionts The number of symbiont samples examined in this
remainsunknown. Noneofthe luminousspeciesincluded study is sub-optimal, due to the difficulty and expense
in the 16S rRNA region analysis appear to be the same involved in obtaining specimens, but we have attempted
species as the symbionts, which fall somewhere between to compensate forthe small sample size. The large inter-
I'. orientalisand F iisi-lieri. The 16SrRNA regionanalysis generic differences among the anomalopid symbionts is
suggeststhat the symbionts are more closely related to V. supported by 17 independent comparisons, i.e., each
orientalis than to I harveyi. Ln.\A nucleotide sequence symbiont sample compared to each sample from a dif-
data for I'. harveyi, I'.fischeri, and the Kryptophanaron ferent fishgenus. Even iff. palpebratus#2 wereexcluded
aljredi symbiont reveal a closer relationship between V. due to a possible gene rearrangement, there are still 15
harveyi and the A", aljredi symbiont (75% identity) than independent comparisons in support ofour conclusion.
between the symbiont and I', fischeri [63% (Haygood, Despite the limitations ofthe analysis, several conclu-
1990)]. This relationship is not observed in the 16S data, sions can be drawn. First, the symbionts are obviously
which indicate that the symbionts are equally related to not the same species in all members ofthe family Anom-
both r. harveyi and ('. fischeri. This discrepancy could alopidae. The differences are so extreme that even the
indicatethatthesimilarity ofthe symbiontwith I' harveyi largestpossibleestimatesofvariancecannotobscurethem.
is found onlywithin the luxregion and isnot distinguish- Thisresultstandsindistinctcontrasttoallthosesymbioses
able at the rRNA level. in other fish light organs in which symbiont identity has
The RFLPapproach hassome limitations, due toerrors been studied. In addition, nogeographicalconnection be-
in the estimation of distance. These errors result from tween hostlocationandsymbiontspeciesisapparent, im-
small, undetected fragments, or fragments ofslightly dif- plyingthat nosignificantgeneticexchange occursbetween
ferent lengths, thatare indistinguishable atthelevel ofgel symbionts from different hosts via free-living populations.
electrophoresis. The major virtue ofthe RFLP approach Furthermore, the symbiont branching order isconsistent
is that many samples can be analyzed directly and si- with the proposed sequenceofevolution within the family
multaneously. The calculations of genetic distance are Anomalopidae. Thus, at this level ofanalysis, the sym-
based on assumptionsabout thesubstitution rateand fre- bionts appear to have coevolved with the host fish. The
quency of the four nucleotides within a sequence (Nei, large difference between P. pa/pehratus #1 and #2 could
1987). Theerrorsin theseassumptionsare negligiblewhen be due to a rearrangement or unrecognized difference in
(tNheei,est1i9m87a)t]e,dbnuutclheoatviedeansuibnsctrietaustiinognly(d)lairsgesmeaflflect[>a0s.1d; tthhee h1o6sSt frisRhNesA. Foipnaelrloyn, tihnetahpepasryemnbtiolnotwscompayynurempbreesrenotf
increases, resultinginan underestimation ofthedistances geneticadaptation tothelightorgan environment. Inany
and a corresponding decrease in accuracy. The distances case, thesymbiontdivergence observed in theassociation
between these luminescent bacteria and the anomalopid
between the symbionts in this study are large, approach-
ing, and in some cases exceeding, the upper limit ofac- fishes appears to be novel among previously characterized
curacy for this method. light organ symbioses.
Differences in copy number are also a problem in the
16S RFLP analysis, because these differences represent Acknowledgments
differences in genome organization ratherthan actual se- WethankA.C. Arnesonandthe DepartmentofMarine
quencedivergence. Includingall fragments in an analysis Sciences ofthe University of Puerto Rico for assistance
between two samples with different copy numbers will in collecting A', aljredi, A. C. Arneson and P. Colin for
overestimate the calculated divergence by inflating the the A. katoptron and P. palpebratus specimens, and the
denominator in the fraction of conserved hybridizing staffofthe H. Steinitz Marine Biological Laboratory, Eilat,
fragments. There is no valid way to normalize the data, Israel, for the P. sleinitzi sample. We are grateful to
buttotesttherobustnessofourconclusions, weattempted Dr.G. Wilson forhelpin obtainingand runningthecom-
a conservative compensation for copy number discrep- puter programs, and Dr. R. Rosson and B. Tebo fortheir
ancies by recalculating divergence with the denominator helpful suggestions. We also thank Dr. R. Rosenblatt for
normalized to the smallest number offragments in each his identification of the Photoblepharon specimens and
sample pair. The values obtained by these calculations critical reading of the manuscript. Thanks are also due
should be the lowest possible estimates of divergence to M. P. Quaranta for help in writing the distance esti-
within each pair. Using this method of estimation, the mation program. This research was funded by the Office
intergeneric divergences among the symbionts are not ofNaval Research, grant no. N00014-89-J-1742.
significantlychanged [rangingfrom0.1 12 to>0.122(cor-
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FLASHLIGHT FISH SVMBIONT RFLPS 143
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