Stability of Deep-Sea Hydrothermal Vent Polychaetes (Alvinellidae): a Possible Selection by Habitat

Home , Alvinella pompejana, Alvinellidae

MARINE ECOLOGY PROGRESS SERIES Vol. 123: 125-136, 1995 1 Published July 20 Mar Ecol Prog Ser

Evidence for differences in the allozyme thermostability of deep-sea hydrothermal vent polychaetes (Alvinellidae): a possible selection by habitat

Didier ~ollivet',Daniel ~esbruyeres~,Christine ad rat^,Lucien ~aubier~

'Plymouth Marine Laboratory, Molecular Biology Department, Citadel Hill. Plymouth PL1 2PB, United Kingdom 21FREMER Centre de Brest, DROIEP, F-29280 Plouzane, France 31nstitut Oceanographique, 195, rue Saint-Jacques, F-75005 Paris, France

ABSTRACT Alv~nelhdpolychaetes are, to date, restncted to deep-sea hydrothermal vents of the eastern and the western ndges of the Pacific Ocean These organisms live In vanous sulfide-rich habitats, Including the hottest part of the hydrothermal envlronment (l e chimneys) They expenence transient anoxia, h~ghlevels of heavy metals and H2S, natural radioactivity and temperatures ranging from 5 to 80°C which vary greatly wlth tlme The Alvinellidae, as many vent organisms, have developed spec~fic adaptations to cope wlth this harsh and unstable envlronment Enzyme systems are good markers of the adaptation of ectotherms to temperature, which acts on both enzyme kinet~csand proteln denaturation We est~matedgenet~c distances between 11 alvlnellid species using a data set of allozymes and studied in vltro allozyme thermostabillties of aspartate-amino transferase (AAT), glucose-6-phosphate isomerase (GPI) and phosphoglucomutase (PGM) which may play a role In onentatlng aerobic versus anaerobic metabolism pathways, for 8 species using the most common homozygous genotypes Results show great genetlc d~vergencesbetween species llving in distinct mlcrohabitats as well as stiong thermostability differences with~nand between specles which also rely on ddferent enzymatic strategies (phenotyp~cplasticity versus genetlc variab~lity)Allelic f~tnessto temperature in a hlghly fluctuating environment may explain the high level of polymorphism found In alvinellids and may have also provided sufficient genetic divergence between ind~v~dualslivlng in dist~nctthermal regimes to produce speciation

KEYWORDS: Deep-sea hydrothermal vents . Allozymes . Aspartate-am~notransferase . Glucose-6- phosphate isomerase . Phosphoglucomutase . Temperature . Selection

INTRODUCTION The environment is also characterized by great spatial and temporal instabilities due to both tectonic and Fauna inhabiting deep-sea hydrothermal vents may magmatic events or the dynamic properties of the experience high temperatures (Chevaldonne et al. hydrothermal convection through the basaltic crust of 1991, 1992), natural radioactivity (Cherry et al. 1992), the fast spreading centers (Hessler et al. 1988, hypoxia and high concentrations of sulfide (Johnson Watremez & Kervevan 1990, Haymon et al. 1993, Jol- et al. 1988), ammonia and heavy metals which greatly livet 1993).The hydrothermal activity shifts along the exceed those recorded for other marine or terrestrial spreading axes and generates numerous short-lived environments (Edmond et al. 1982, Michard et al. vents (1 to 100 yr: Lalou 1991), thus causing cata- 1984). These environmental conditions seem to be strophic and chaotic extlnctions in populations which highly selective and able to induce peculiar adapta- induce the continuous need to colonize new active tions (Felbeck 1981, Powell & Somero 1986, Arp et al. vents (Hessler et al. 1988, Tunnicliffe & Juniper 1990, 1987, Dahlhoff & Somero 1991, Dixon et al. 1992). Jollivet 19931.

0 Inter-Research 1995 Resale of full article not permitted 126 Mar Ecol Prog Ser 123: 125-136. 1995

The Alvinellidae is a family of polychaete worms of alvinelllds. Indeed, these polychaetes exhibit a restricted to the deep-sea hot hydrothermal vents peculiar reproductive behaviour and a development located along the oceanic ridges of the eastern and which may result in a short or absent planktonic phase western Pacific. A total of 12 species have so far been (viz, larval dispersal) (Desbruyeres & Laubier 1986, collected and described from vents of the East Pacific 1991, McHugh 1989, Zal et al. 1994, 1995). The het- Rise including the Guaymas basin (5 species: Des- erozygote deficiencies might be better explained as an bruyeres & Laubier 1980, 1982, 1986, 1991), the ridges adaptive response of the polychaetes to the hlgh tem- of the northeastern Pacific (4 species: Desbruyeres & poral fluctuations of the vent conditions (Johnson et al. Laubier 1986, Detinova 1988, Tunnicliffe et al. 1993) 1988, Little et al. 1988, Chevaldonne et al. 1991). and the ridges of the western Pacific back-arc basins (3 On the other hand, the genetic differentiation of species: Desbruyeres & Laubier 1989, 1993, Miura & alvinellid populations separated by distances varying Ohta 1991). from several metres to more than 1000 km is of the Alvinellid polychaetes are sedentary worms and live same magnitude for all the alvinellid species collected inside a tube or a mucus sheath. Most of them live on or at vents of the East Pacific Rise and the ridges of the at the base of the tubes of vestimentiferan worms in northern Pacific (Tunnicliffe et al. 1993, Jollivet et al. areas of diffuse low-temperature (5 to 25°C) venting, 1995). These results strongly support the hypothesis but a few others (Alvinella pompejana, Alvinella cau- that genetic drift enforced by high rates of extinction/ dd ld, ?dral vin ells sulfincola dnd Paraivin ella hessleri) recolonisation is balanced by a uniform selective pres- live on the walls of hydrothermal chimneys where hot, sure stemming from the chemical and thermal homo- anoxic, sulfide-rich fluid is mixing with cold (2°C) geneity of vents on a large spatial scale (viz. East oxygenated seawater. These latter species inhabit the Pacific Rise; Edmond et al. 1982, Michard et al. 1984). hottest zones of the hydrothermal-vent environment The aim of this study was to investigate the possible (Desbruyeres et al. 1982, Chevaldonne & Jollivet 1993). role of temperature both on the maintenance of a Recently, Tunnicliffe et al. (1993) and Jollivet et al. genetic structure and on the promotion of the genetic (1995) reported that these organisms display a genehc isolation of the alvinellid populations sampled from variability higher than that of most eucaryotes (Hart1 various microhabitats. To achieve this aim, the genetic 1988): they recorded polymorphism varying from 30 to relationships of 11 alvinellid species were analysed 75% under the 5% criterion and observed heterozy- using a set of enzyme loci, and a comparative study gosltles from 0.1 to 0.3. They also reported a strong was performed on the allozyme thermostability of the tendency towards a heterozygote deficiency systemat- most common homozygous genotypes using 2 poly- ically occurring at the same loci in nearly all the popu- morphic enzyme loci, Aat (aspartate-amino transfer- lation~. Such a genetic structure does not fit with ase) and Pgm (phosphoglucomutase), and the mono- repeated founder effects which could be expected both morphic Gpi locus (glucose-6-phosphate isomerase) from the vent patchiness and the low dispersal ability which is diagnostic in alvinellids.

Fig. 1 iMap of the deep- sea hydrothermal-vent areas where alvlnell~dspecies have been sampled. (A.c.)Alvinella caudata; (A.p.)A. pornpejana; (P.d.) Paralvinella dela; (P.I) P. fijiensis; (P.g.1 P. grasslei; (P.h.) P. hesslen; (P.p.1 P palmiformrs: (P.p.j.1P pandorae idandel, (P.pp.) P p. pandorae; (P.s.) P sulfincola; (P.u.) P. unidentata Jollivet et al.: Allozyme thermostab~hty of vent polychaetes 127

MATERIALS AND METHODS supernatant separated into 2 aliquots, one for the electrophoresis identification of allozymes and the other for Sample collection. Alvinellid samples were col- biochemical experiments. EDTA was used in excess to lected at deep-sea hydrothermal vents of the East chelate heavy metals found at high levels in the Pacific Rise ('Hydronaut' 1987 and 'MMVT' 1990 tvorms' tissues. Electrophoresis was conducted for 4 to cruises), the Juan de Fuca and Gorda ridge complex of 6 h at 80 mA using 11% starch gels refrigerated the northern Pacific ('Axial Seamount Hydrothermal between 2 ice-water cooling plates using 2 continuous Expeditions, ASHES' 1986, 1987, and 'Gorda Ridge buffer systems: tris-citrate pH 8.0 and tris-HC1 pH 8.5, Expedition' 1988), and the western Pacific ridges of the and 2 discontinuous buffer systems: tris-citrate pH Mariana and North Fiji back-arc basins ('Mariana 6.7/6.3 and tris-citrate-borate pH 8.7/8.2. The Alvinella Trench Vent Expedition, MTVE' 1987 and 'Starmer' pompejana specimens collected at the site Pogonord in 1989 cruise) (Fig. 1). The number of individuals used 1987 (see Table 1) were arbitrarily chosen as our refer- per species as well as location, depth and vent habitat ence population, and the following 14 enzyme loci are listed in Table 1. At least 3 main habitats have been were examined. Acid phosphatase (EC 3.1.3.2, locus distinguished: (1) the walls of active hydrothermal- Acp), aconitase (EC 4.2.1.3, loci Acoh-1, Acoh-Z), vent chimneys, (2) the top of the tubes of vesti- aspartate-amino transferase (EC 2.6.1.1, locus Aat), mentiferan worms, and (3) basaltic cracks at diffuse- adenylate kinase (EC 2.7.4.3, locus Ak), glyceralde- venting areas or at the base of active chimneys. hyde-3-phosphate dehydrogenase (EC 1.2.1.12, locus Specimens were sampled using the hydraulic arms of Capdh), glucose-6-phosphate isomerase (EC 5.3.1.9, the deep-sea manned submersibles Nautile and Alvin, locus Gpi), isocitrate dehydrogenase (EC 1.1.1.42, brought to the surface in an insulated box, and frozen locus Idh-l), cytosolic leucine aminopeptidase (EC in liquid nitrogen. 3.4.11.1, loci Cap-l, Cap-Z), malate dehydrogenase Electrophoresis. Each piece of tissue was homoge- (EC 1.1.1.37, locus Mdh- l ), mannose-6-phosphate iso- nized in an equal volume of a 0.01 M Tris/HCl pH 6.8 merase (EC 5.3.1.8, locus Mpi], phosphoglucomutase extraction buffer containing 2 mM EDTA, 0.5 mM (EC 5.4.2.2, locus Pgm) and superoxide dismutase (EC NADP and 0.05 % P-mercaptoethanol, Homogenates 1.15.1.1, locus Sod- l ) were scored for each individual. were centrifuged for 30 min at 25000 X g and the Alleles were numbered according to their anodal

Table 1. Location and habitat characteristics of 11 alvinellid species collected along the oceanic ridges of the Pacific. N: number of individuals sampled; 'generic and specific name of a vestirnentiferan tube-worm which provides a suitable habitat for an alvinellid species; EPR: East Pacific Rise

Species Site Location Depth Habitat Alvinella pompejana Pogonord 12°48.61'N (13"N/EPR) 103O56.62' W 2635 m Top of the chimney Alvinella caudata Pogonord 12048.613N (13ON/EPR) 103"56.62'W Top of the chimney Paralvinella grasslei Genesis 12'48.58'N ' (13"NEPR) 103"56.47'W Tubes of Riftia pachyptila Paralv~nella pandorae irlandei Genesis 1Z048.58'N Cracks at the base of the (13"N/EPR) 103O56.47'W tubes of Tevnia jerichonana' Paralvinella palrmformis Nesca 41°00.40'N Tubes of Rldgeia piscesae' (Gorda Ridge) 127O29.3O'W Paralvinella sulfincola Lamphere 45"59.00'N Top of the chimney (Juan de Fuca Ridge) 13O0O3.80'W Paralvinella pandorae pandorae Max 45"55.50'N Cracks at the base of the tubes (Juan de Fuca kdge) 13O0O2.50'W of Ridgeia piscesae ' Paralvinella hessleri Illium 18"12.80'N Base of the chimney (Mariana Ridge) 144'42.40'E Paralvinella dela Bouquet 45"55.50'N Cracks at the base of the tubes (Juan de Fuca Ridge) 13O0O2.50'W of Ridgeia piscesae' Paralvinella fijiensis White Lady 16O59.5O'S Top of the chimney (North Fiji Basin) 173"55.47'W Paralvinella unidentata White Lady Base of the chimney (North Fiji Basin) 16"59.50'S 173O55.47'W 128 Mar Ecol Prog Ser 123: 125-136, 1995

mobility relative to the most frequent allele. The stain- species using starch-gel zymograms. For the small ing recipes were those provided in Harns & Hopkinson alvinellid species (i.e. Paralvinella hessleri), individu- (1976) and Pasteur et al. (1987). als were totally homogenized, and, in order to have a Thermostability theoretical background. Enzyme reasonable amount of crude enzyme extract per geno- systems are good markers of the adaptation of ecto- type, the aliquots containing the same homozygous therms to temperature, which acts both on enzymatic genotype were grouped into 3 pools of 3 individuals. In rates and protein denaturation. Thermosensitivity of addition, the protein concentration of each enzyme enzymes can be assessed by searching for the temper- extract was measured according to the method of ature for which enzyme activity is optimal, by follow- Lowry et al. (1951) to specifically express the enzyme ing the evolution of the K,,, (an indicator of the enzyme activity (optical density: OD unit mg-' protein). affinity for its substrate) with temperature (Hochachka Allozyme incubation and enzyme assays. Enzymatic & Somero 1984), or by following the protein stability mixtures were diluted in Tris-HC1 0.1 M pH 8.0 buffer with increasing incubation temperatures through (15) and sub-divided into 0.3 m1 aliquots which were changes in the residual enzyme activity (Fullbrook simultaneously incubated for 1 h at 4, 20, 35,45, 50, 55, 1983). Enzymatic thermostability can be measured at 60 and 65°C. Experiments were also conducted with constant pH and temperature by following the alter- fresh enzyme extracts at 4, 60, 62, 68 and 72°C when ation in time of the enzyme. For our purposes, the time allozymes showed a strong resistance to temperature. of incubation was set to 1 h (time for which all the alio- The NAD appearance or the NADK disappeararlce zymes displayed 20% of their original activity at 45°C) rates were simultaneously measured at 340 nm using and incubations were performed at different tempera- a 35°C-thermostated, multi-cuve spectrophotometer tures. (UVIKON 930, Kontron) for each set of thermal treat- Enzyme preparation. Thermostability was assessed ments including 2 controls, one without substrate and in 8 alvinellid species for the most common homo- one without enzyme extract. Assays were repeated zygous genotypes encoded for aspartate-amino trans- using increasing amounts of extract (45, 90, 120 p1) to ferase (AAT: EC 2.6.1.1; 7 genotypes), glucose-6-phos- minimize errors associated with the calculation of the phate isomerase (GPI: EC 5.3.1.9; 6 genotypes) and enzyme activity. The final reaction mixtures (2.5 ml) phosphoglucomutase (PGM: EC 5.4.2.2; 9 genotypes). consisted of AAT [aspartic acid (215 mM), ketoglutaric These enzymes play a key role in glycolysis and acid (l5mM), pyndoxal-5-phosphate (0.6 mM), NADH anaerobic fermentative metabolism pathways, such as (0.55 mM) and malate dehydrogenase (10 units ml-')l, proteolysis and neoglucogenesis, and usually display GP1 [fructose-6-phosphate (6.5 mM), MgC12 (20 mM), allozymes known from shallow-water organisms to NAD (1 mM) and glucose-6-phosphate dehydrogenase have a differential tolerance to temperature and pollu- (5 units ml-l)], and PGM [glucose-l-phosphate (40 mM), tants (Hummel & Patarnello 1994). Aspartate-amino MgC12 (20 mM), NAD (1 mM) and glucose-6-phos- transferase (AAT) (or glutamate oxaloacetate transam- phate dehydrogenase (5 units ml-l)], all dissolved in inase, GOT) is a transaminase catalysing the reversible Tris-HC1 0.1 M pH 8.0 buffer. Concentrations of NAD amino group transfer from L-aspartic acid and ketoglu- or NADH, the other substrates and co-enzymes (G-6- taric acid to L-glutamate and oxaloacetate, L-gluta- PDH or MDH) were previously adjusted to be optimal. mate often being an ammonlac carrier. Phospho- We calculated the percentage of the incubated-extract glucomutase (PGM) catalyses the interconversion of residual activity (RA%) for each allozyme using the glucose-l-phosphate and glucose-6-phosphate in gly- formula RA% = 100 (Ao - AT)/Ao in which AT refers to colysis and neoglucogenesis. Glucose-6-phosphate the enzyme activity (OD unit mg-' protein) measured isomerase (GPI) catalyses the reversible isomerization spectrophotometrically at 35°C after a previous 1 h of fructose-6-phosphate and glucose-6-phosphate in incubation at a selected temperature and A. to the ini- glycolysis and may provide a suitable substrate for tial enzyme activity. neoglucogenesis. Kinetic differences between allozymes of these enzyme systems both in vivo and in vitro may provide adaptation to transient hot and RESULTS hypoxic conditions through individual fitness differences. For Alvinella spp. which have epibiotic bacteria Genetic distances on their dorsal part (Desbruyeres & Laubier 1986), and for species which have a large maximum size, enzyme The allelic frequencies of each alvinellid sample are assays were prepared only from the gills and anterior given in Jollivet (1993) and have been published in part of the body. At least 3 replicates corresponding to Tunnicliffe et al. (1993) and Jollivet et al. (1995) for individuals having the same homozygous genotypes Alvinella pompejana, A. ca udata, Paralvinella grasslei, for at least 1 of the enzymes were selected for each P. palmiformis and P. sulfincola. The unbiased Nei's Jollivet et al.: Allozyme thermostability of vent polychaetes 129

Table 2. Alvinella and Paralvinellaspp. Unbiased Nei's genetic identities (above the diagonal) and distances (below the diagonal) estimated from alvinellid species pairwise comparisons using a set of 14 enzyme loci

Species 9

P' 9- 9. Q. 9 9,

A. pompejana A. caudata P. sulfincola P. fijiensis P. grasslei P. palmiformis P. p. pandorae P, p. irlandej P. unidentata P. hesslen P. dela

genetic identities and distances (Nei 1978), calculated pandorae pandorae and P. p. irlandei and the sibling using the program BIOSYS-1 (Swofford & Selander species P. palmiformis and P. grasslei occurred 35 mil- 1989), are given in Table 2 and provided the data base lion years ago when the Farallon plate was subducted for an UPGMA phenogram (Fig. 2).Genetic distances below the American plate, resulting in the separation obtained between species varied from 0.61 (subspe- of the northern and the southern East Pacific ridges cific level) to 4.87 (generic level). The 2 genera are (Tunnicliffe 1988). The correlation between genetic monophyletic and the Paralvinella genus is cleaved distance and divergence time indicates that the cleav- into 3 sister groups. Each group contains a set of age of the 3 sister groups of Paralvinella occurred allopatric species which are morphologically alike and before the opening of the Atlantic ocean, during the live in similar microhabitats. We estimated the diver- Mesozoic, 150 million years ago. gence time between the 3 sister groups of Paralvinella using Nei's formula IXy= 10Xy.e-2u'where I,, and Ioxv correspond to current and initial genetic identities of Thermostability of allozymes the populations X and Y, a is the mutation rate and t the divergence time. The calculation was done assum- Figs. 3, 4 & 5 display the thermostability curves ob- ing that both separation between the subspecies P. tained using allozymes of AAT, GP1 and PGM for 8

A.pompejona A. ca~tdaro n F(Prager & Wilson 1976) = 27.1 % SD (Filch & Margoliash 1967) = 50.7 K = 0.786 P. grasslei m

Fig. 2. Alvinella and Paralvinellaspp. UPGMA phenogram of 11 alvinellid species using Nei's (1978) modified genetic distances (F and %SD correspond to the maximum likelihood cri- P. unidenrora 0 teria defined by Prager & Wilson P.hessleri (1976) and Fitch & Margoliash (1967), U respectively; R corresponds to the P. dela m cophenetic- correlation coefficient). Habitat characteristics of each species: (m)top of actlve hydrothermal chimneys; (m)tubes of vestirnentiferan I I I I I I I I I I I worms; 13)diffuse vent,ng areas at 3-40 3.06 2.72 2.38 2.04 170 1.36 1.02 0.68 0.34 000 the base of hydrothermal chimneys or of vest~mentiferantube-worms Nei'sgenetic distance (D) 130 Mar Ecol Prog Ser 123: 125-136, 1995

A. pompejana bation. TaO,usually sets the limit at which the enzyme begins to denature and Tzo, 1olz;-n;:n-9;l\, sets the limit at which the enzyme is 60 totally denatured. Results are summarized 3 6 40 in Table and Fig. in which TBOXis plot- ted against T20%to distinguish species in 20 terms of 'hot' versus 'cold' adapted geno- 0 0 20 40 60 80 types. P. hessleri Intraspecific variation. Monomorphic species at either Aat, Gpi or Pgm loci always exhibit an allozyme which displays a Tso%similar to that of the highest thermoresistant allozymes of their polymor- phc counterparts. This is especially the case for most GP1 allozymes and the AAT allozyrnes of the 2 Alvinella species studied here. In the other cases, species are P.palmiformis P.grasslei usually polymorphic but alternative alleles are often rare and found only as heterozygotes. As a consequence, it was not possible to assess the thermostability of the homozygous genotypes. However, in a few cases, alternative alleles were more frequent within samples and allowed us to 0 20 40 60 SO 0 20 40 60 80 find enough homozygous genotypes to P.pandorae pandorae P.pandoroe irlandei make comparisons (see Fig. 3: at'^"^, and at'^^'"^ in Paralvinella palmiformis and P. sulfincola; and Fig. 5: pgm66/66, pgm78/78, pgm90/90 and pgm 100/100 in P. sulfincola and A. pompejana). Results show the occurrence of at least 2 allozymes displaying T8,, differences rang-

0 20 40 60 SO 0 20 40 60 80 ing from 10 to 20°C, which indicates that allozyme thermostability varies greatly Temperature ('C) within species from individual to individual according to genotype. Fig. 3. Alvinella and Paralvinellaspp. Thermostability curves obtained for variation' allozymes corresponding to hornozygous genotypes at the locus Aat in 8 Interspecific Enzyme alvinellid sDecles (bars corresaond to the standard errors associated with ties of the 2 Alvinella species studied the percentage of residual activity) here still persist after 1 h incubation at temperatures exceeding 50°C (GPI) and 60°C (AAT). Paralvinella hessleri and P. alvinellid species. The shape of the curve vanes sulfincola also exhibit a Tso%greater than 45°C (GPI) slightly according to the enzyme system. Allozymes of and 55°C (AAT), whereas other congeneric species AAT and GP1 are characterized by a plateau followed have much lower values (Table 3). This is particularly by a rapid decrease of the enzyme activity with in- obvious in Fig. 6 in which all the species living on the creasing incubation temperatures whereas the curves walls of actlve hydrothermal chimneys (A. pompe- obtained for the allozymes of PGM are sigmoidal. Fur- jana, A. caudata, P. sulfincola and P. hesslen] display thermore, the half-life time (tlI2)of AAT allozymes thermoresistant allozymes at least for the AAT and incubated at 45°C was respectively twice and 4 times GP1 enzyme systems, whereas species living in greater than those of GP1 and PGM when we 'cooler' diffuse venting conditions (P grasslei, P. attempted to fix the incubation time. To compare the palmiformis, P. pandorae pandorae and P. p. irlandei) thermostabllity of allozymes encoded by distinct geno- are characterized by somewhat more sensitive alleles. types with~nand between alvinellid species, we used The PGM allozymes appear to be less informative and T80,',,and Tzor36,temperatures at which the enzyme con- do not provide reliable markers to distinguish eco- serves 80% and 20 % of its initial activity after 1 h incu- types because most of the species are polymorphic Jollivet et al.. Allozyme thermostability of vent polychaetes 131

A.caudara organisms to changing environments can be achieved by a phenotypic regulation of the genetic expression or by reduction of the genetic diversity of populations through elimination of individuals lacking the advantageous alleles (Hochachka & Somero 1984). As a consequence, the adaptive nature of enzyme genotypes to

P. hessleri temperature has been largely supposed to explain deviations from the Hardy- ljnx;-n;fi,\@= , Weinberg equilibrium in shallow-water populations subjected to ecological or 40 anthropological stresses (Nevo et al. 1977, 20 Patarnello et al. 1989, 1991). 0 At deep-sea vents, temperature is a 0 20 40 60 80 semiconservative factor of dilution be-

P.palmifarmis tween the acidic, hot, hydrothermal fluid and the cold, oxygenated seawater. At a local scale, it is well correlated with H2S concentrations and negatively correlated with oxygen concentrations (Johnson et al. 1988) and thus reflects changes for most environmental parameters affecting the 0 20 40 60 80 physiology of the vent organisms. Our

P. pandoraepandorae P,pandorae rrlandei results demonstrate that some alleles may encode for differences in the allozyme sensitivity to temperature within alvinellid species exhibiting polymorphism. Such results have already been reported by Place & Powers (1979),who found that the cytosolic allozymes LDHBaBaand LDH~~~~ of the teleost fish Fundulus heteroclitus 0 20 40 60 80 0 20 40 60 80 are sensitive to temperature, and by Temperature('C) Wilkins et al. (1978), who found thermoresistant and thermosensitive allozvmes in Fig. 4. Alvinella and ParaIvineLla spp. Thermostability curves obtained for shallow-water limpets and littorinid gas- allozymes corresponding to homozygous genotypes at the locus Gpi in 8 alvinellid species [bars correspond to the standard errors associated with the tropods (which might their genetic percentage of residual activity) diversity). Rapid changes in temperature, exposure to waves and salinity with time are therefore known to be responsible for and exhibit 2 or 3 equifrequent alleles coding for increasing genetic variability in marine invertebrates strong differences in the protein thermostability. living in the intertidal zone or in estuaries (Hummel & Patarnello 1994). Similarly, such an allelic fitness may explain the high levels of genetic variability recorded DISCUSSION in alvinellids (Tunnicliffe et al. 1993, Jollivet et al. 1995). Genetic variability of alvinellids and short- and The alvinellid environment is not only characterized long-term fluctuations in venting in terms of 'hot' and 'warm' habitats but also in changes with time. Time series of temperature Protein polymorphism may indicate a genetic recorded at the surface of alvinellid colonies indicate 2 response of ectotherm populations to selection in vari- kinds of temporal fluctuations wherein a high-fre- ous environments. Ectotherms, especially marine quency signal is superimposed on one with a much invertebrates, need to regulate their body temperature longer period clearly associated with tidal cycles in order to improve their metabolic functions, espe- (Chevaldonnh et al. 1991). Temperature increases dra- cially enzyme kinetics. Physiological adaptations of matically 5 to 10 cm inside a colony. Desbruyeres et al. 132 Mar Ecol Prog Ser 123 125-136, 1995

tage to the other one and vice versa. As a consequence, heterozygotes will get a loo: 975- selective advantage because of their less 80 - {\ energy-demanding maintenance, as pre- 60 : lmlm dicted by Koehn & Bayne (1989), when 40 - environmental conditions vary greatly. 7n - In addition, environmental conditions also evolve rapidly during the lifetime of a vent: in situ observations have shown that a vent may evolve from birth to death within a decade (Jollivet 1993). During that period, hydrothermal activity pro- gressively decreases and the venting fluid becomes cooler and cooler until venting stops completely. As a consequence, the first colonizers may experience higher temperatures than other successive cohorts, and thus modifications in the fre- quency of thermosensitive alleles could arise with time. Such variations have been reported by Monti et al. (1986), who observed rapid changes in the nlalate dehydrogenase (Mdh)genotypic frequencies within a population of the shallow- water bivalve Ruditapes decussatus in

P. pandorae pandorae P.patdorue rriandei response to increasing temperatures and hypoxia during the summer period. Simi- larly, the differential sensitivity to temperature of allozymes encoded by AatJo5and at'" seems to explain genotypic modifications observed by Tunnicliffe et al. (1993) in a population of Paralvinella palmiformis sampled before and after (1 yr) the vent site Mushroom unexpectedly Temperature("C) warmed up. In that particular case, the fre- Fig. 5. Alvinella and Paralvinella spp. Thermostabhty curves obtained for quency of the allele at"^ increased from allozyrnes corresponding to homozygous genotypes at the locus Pgm in 8 0.042 to 0.433 within the same population alvinellid species (bars correspond to the standard errors associated with of P. palmiformis between 1986 and 1981. the percentage of residual activity)

(1982) reported a temperature range from 20°C (at the Microhabitats and alvinellid speciation tube openings) to 100°C (10 cm inside). More recently, Chevaldonne et al. (1992) provided in situ discrete Genetic distances and their derived phenogram indi- temperature measurements simultaneously recorded cate important evolutionary divergences, which are on videotapes, which varied from 20 to 45OC when the consistent with relationships obtained using morpho- probe was placed close to the gills of the worms and logical characters (Desbruyeres & Laubier 1991, 1993), from 40 to 80°C when the probe was inserted 2 to 3 cm among alvinellid species sampled so far. Even if allo- inside a tube. They also reported a temperature mea- zymes are not the most appropriate indicators of the surement at 105°C while a dislodged, live Alvinella phylogenetic relationships between genera or highest pompejana was coiled around the probe for a few min- taxa, such high genetic distances may stem from very utes without showing any change in its behaviour. old cleavages spurred by the oceanic plate movement. Thus, it is likely that short-term fluctuations in the oxy- Indeed, some vent fauna is known to have direct gen concentration and temperature could subse- ancestors from the early Mesozoic (Newman 1985), quently affect one of the 2 homozygous genotypes at a and fossil burials attributed to alvinellid polychaetes non-neutral diallelic locus, giving a selective advan- have been reported from sulfide ore structures dated Jollivet et al.. Allozyme thermostability of vent polychaetes 133

Table 3. Alvinellaand Paralvinellaspp TBO.,.,,and T20..:, (temperatures at which the enzyme conserves 80% and 20% of its initial activity) obtained for allozymes of the AAT, GP1 and PGM for 8 alvinellid species. Temperatures were graphically estimated on the thermostability curves

Species Aspartate-amino transferase Glucose-6-phosphate isomerase Phosphoglucomutase Genotype TRo T?" Genotype T T2" Genotype TB,) T2(,

A. pompejana

A. caudata P. hessleri P. sulfincola

P. pandoraejrlandei P. pandoraepandorae P. grasslei P. palnnformis

Mean SE from the Cretaceous and the Devonian periods (Hay- 80 - Aspartate-amino transferase mon et al. 1984, Oudin & Constantinou 1984, Kuz- , 0 , netsov et al. 1991). U' ,, However, cinepatry (i.e. species isolation due to the D' ,972 oceanic plate movement) does not appear to be the 60 - ,. ,2+ only evolutionary process acting on hydrothermal-vent species isolation. Our calculations indicate that species which compose the genus Paralvinella evolved in 3 sis- 40 t ter groups approximately when the 2 alvinellid genera 20 40 60 80 diverged some 150 million years ago; such a divergence may have been favoured by different ecological T80% ('c) constraints. Indeed, morphologically closely related - / species such as P. palmiformis and P. sulfincola, which g0d have very different ecological preferenda, display a 90 high level of genetic divergence (TunnicMfe et al. 1993). In an opposite fashion, closely related species such as P. dela and P. hessleri or P. sulfincola and P. fijiensis, which live in similar habitats but have been geographically isolated for a long period of time Glucose-6-phosphate isomerase (eastern Pacific versus western Pacific), display great genetic similarities. Desbruyeres & Laubier (1993) have already proposed that the occurrence of morphologically closely related species of ParalvineLla living in similar habitats over the 3 main hydrothermi- - 0 0 0 cally active areas of the Pacific (eastern Pacific, north- 0 eastern Pacific and western Pacific) may be due to the 0 / %l'r _0=6 Q ,sq*'. ,, 29.62 Fig. 6. Thermostability of AAT, GP1 and PGM allozymes along a gradient of incubation temperatures obtained from a linear Phosphoglucornutase regression (r2. correlation coefficient) against the temperatures T20n;,at which an allozyme conserves 20% of its initial activity, and T80..0,at which an allozyme conserves 80% of its initial activity. Symbols as in Fig. 2 134 Mar Ecol Prog Ser 123: 125-136, 1995

parallel evolution of 3 ancestral types living in 3 dis- which encodes for the most thermostable PGM allo- tinct hydrothermal-vent habitats. As a consequence, zyme ever recorded for alvinellid polychaetes. we may infer that ecological constraints could have Such results are in agreement with ecological and provided evolutionary forces which drove alvinellid physiological data previously recorded on vent organ- species toward isolation. isms, especially on alvinellid polychaetes. Dahlhoff & Adaptive responses of allozymes to temperature Somero (1991) have already predicted that deep-sea have been previously proposed as explanations of spe- hydrothermal vent organisms living on chimneys ciation in 4 species of eastern Pacific barracudas Living emitting high temperature fluids have sustained body in different thermal environments (Graves & Somero temperatures which are 20 to 25OC higher than those 1982). To determine if functionally significant molecu- of vent species living in cooler diffuse vent habitats. lar evolution has occurred in response to different ther- Similarly, Dixon et al. (1992) found differences in mal microregimes, allozyme thermostabilities of the the thermal stability of nbosomal DNA sequences diagnostic locus (Gpi) and polymorphic loci displaying of hydrothermal-vent organisms including several shared alleles between species (Aat and Pgm) were alvinellid species which fit our data very well: melting measured. rDNA temperatures varying from 88°C (both species of The level of thermal stability obtained from our set of Alvinella) to 82OC (Paralvinella grasslei and P. pando- enzymes appeared to be well correlated to the maxi- rae). Finally, Taghon (1988) reported great differences mum temperature each aivinellid species might expen- in tne levels of polyunsaturated fatty acids between P. ence within its habitat. T,,, values, which ranged from palmiformis and P. sulfincola, which was explained as 22°C (Pgm78'78)to 62°C at'^^"^^), are also in good a specific adaptive response of the latter species to agreement with several in vitro comparative physiolog- increasing habitat temperature. As a consequence, ical studies conducted on the body-temperature range although temperature strongly varies with time on that vent organisms could sustain. Haemoglobin O2 short- and long-term scales, comparisons between affinity (Toulmond et al. 1990) and collagen melting- congeneric paralvinellid species using enzyme ther- temperature (Gaill et al. 1991) studies conducted on the mostability indicate that differences in habitat are suf- 2 Alvinella species gave an optimal temperature range ficient to favour molecular adaptations in functional of 20 to 50°C whereas results obtained from enzyme genes which may result in species isolation. kinetics varied from 25 to 40°C (Dahlhoff & Somero In conclusion, we can infer from these results that 1991, Dahlhoff et al. 1991). However, caution must be temperature fluctuations and the aerobidanaerobic taken when an attempt is made to interpret our ther- alternation of hydrothermal vent conditions seem to mostability values because (1) Tsocx neither reflects the play a determinant role in the evolution of the genetic maximal nor the optimal temperature of enzymes and structure of vent organisms. Vent instability may be (2) we used crude instead of purified enzyme extracts responsible for maintaining a high genetic diversity of which did not allow us to discard the influence of the the alvinellids, whereas the spatial heterogeneity of cell micro-environment (Le, the more or less important the thermal regime within a vent site seems to have efficiency of proteases during incubation and/or the provided an evolutionary driving force to produce measurement of activity, presence or absence of genetic divergences between species living in distinct specific inhibitors such as cytosolic heavy metals). Nev- microhabitats. ertheless, enzyme activities are reproducible across replicates and different enough between genotypes to Acknowledgements. We are indebted to the captains and the discount the influence of the cell environment. crews of the NO 'Nadir' and RV 'Atlantis II', and the Nautile This study is therefore the first attempt to compare and Alvin pilots and crews, without whose expertise and congeneric hydrothermal vent species living in 'hot' assistance this work would not have been possible. Cruises were organized by IFREMER (France), Rutgers Univers~ty, and 'warm' habitats. Results obtained for the AAT, GP1 New Jersey, University of California, Santa Barbara, and the and PGM enzyme systems indicate that species living Woods Hole Oceanograph~cInstitution (USA). We are very on the walls of hydrothermal vent chimneys display grateful to A.-M.Alayse, R. C Vrijenhoek and R.A. Lutz, co- thermoresistant allozymes whereas species living in chief scientists of the 'Hydronaut' 1987 and 'MMVT' 1990 'cooler' conditions (diffuse venting areas) display allo- cruises who kindly provided the biological material collected along the East Pacific Rise. We are also indebted to V Tunni- zymes more sensitive to temperature. Moreover, there cliffe, who participated to the Axial Seamount Hydrothermal also exist electromorphic and thermostability conver- Expeditions (ASHES) in 1986 and 1987, and to R. R. Hessler gences at the Aat and Pgm loci between alvinellid and P. Boudrias, who participated inthe Gorda Ridge Expedi- species which are not closely related but which live in tion and the Mariana Trench Vent Expedition [MTVE),for sampling and storing the biological material for us. CVe also similar microhabitats. Indeed, Alvinella pompejana thank P. Chevaldonne,A. Rogers and P. Lemaire for their crit- and Paralvinella sulfincola, which both live on the ical review of the manuscript and their useful comments.This walls of hydrothermal chimneys, share the allele PgrngO work was supported by IFREMER Jollivet et al.. Allozyme thermo stablllty of vent polychaetes 135

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This article was submitted to the editor Manuscript first received: February 16, 1995 Revised version accepted: March 2, 1995