Reference: Biol. Bull. 204: 205-209. (April 2003) 2003 Marine Biological Laboratory From Genes to Genomes: Beyond Biodiversity in Spain's Rio Tinto LINDA A. AMARAL ZETTLER1 MARK A. MESSERLI1~, ABBY D. LAATSCH1 , , PETER J. S. SMITH2. AND MITCHELL L. SOGIN1 * 1 The Josephine Ba\ Paul Centerfor Comparative Molecular Biology and Evolution, Marine Biological Laboratory. 7 MBL Street, Woods Hole. Massachusetts: and 2 The BioCurrents Center, Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts Spain's Rio Tinto, or Red River, an example ofan ex- microorganisms. We have been exploring the genetic and tremely acidic (pH 1.7-2.5) environment with a high metal physiological diversity oforganisms living at pH extremes, content, teems with prokaryotic and eukaryotic microbial both acidic (pH < 3) and alkaline (pH > 10). Our research life. Our recent studies basedon small-subunit rRNA genes ranges from environments like the warm (45 C), acidic (pH reveal an unexpectedly high eukaryoticphylogenetic diver- 2.7) Nymph Creek in Yellowstone National Park to tem- sity in the river when compared to the relatively low pro- perate alkaline lakes in the Sandhills region of western karyotic diversity. Protists can therefore thrive in and domi- Nebraska. The focus of this report is the acidic, heavy- nate extremely acidic, heavy-metal-laden environments. metal-rich Rio Tinto in southwestern Spain. Further, because we have discoveredprotistan acidophiles The Rio Tinto flows 100 km through the world's largest closely related to neutrophiles, we can hypothesize that the pyritic (FeS->) belt. The rivergets its red colorfrom the high transitionfrom neutral to acidic environments occurs rapidly levels of iron dissolved in its acidic waters (pH 2.0). oavdearptgeedoltoogsiuccahl etnivmierosncmaleenst.s?HoWewahraevceurtrheenstelyoerxgpalnoirsimnsg HFeSrrOic4 h(ydprKoaxi2d.0e) a<cFte3a+s/bFuef(fOerHs)3t,o pmaKianta2i.5n)thaendpHSoOf42th~e/ the alterations in physiological mechanisms that might al- river at about 2. The concentration ofiron can be as high as lowforgrowth ofeukaryotic microbes at acid extremes. To 20 g/1, and the river also contains other heavy metals at this end, we are isolating phylogenetically diverse protists concentrations orders of magnitude higher than those in in order to characterize and compare ion-transporting typical freshwater environments. AphTiPlaicsecsoufnrtoemrpacrutlst.ureWdeacpirdeodpihcitletshawtitshpetchioaslepfrroopmernteiuetsroo-f onMtuhechprookfarthyeotpeasstthratespelaarychanonimtphoertRainot rToilnetoinhsahsapfioncgustehde these ion transporters allow protists to sun'ive in the Rio acidic environment of the Rio Tinto through their metabo- Tinto. Earth harbors many extreme environments. Previous in- lism of iron-rich pyrite and chalcopyrite. Recent paleonto- vestigationsofthe microbial diversity in these environments logical research shows that iron-oxidizing bacteria existed have been constrained by preconceived notions about the in the Rio Tinto river basin 300,000 years ago, long before range of habitability for both eukaryotic and prokaryotic its 5000-year mining history (Leblanc et al., 2000). Other chemolithotrophs such as sulfur-oxidizing bacteria and ar- chaea also contribute to the river's probably ancient eco- *To whom correspondence should be addressed. E-mail: sogin@ system structure (Gonzalez-Toril et al., 2001). evol5.mbl.edu The paper was originally presented at a workshop titled Outcomes of Some of these prokaryotes, along with fungi, contribute Genome-Genome Interactions. The workshop, which was held at the J. to the formation of biofilms on the surface of rocks. These Erik Jonsson Center ofthe National Academy ofSciences, Woods Hole. biofilms, in turn, are the site of metal and mineral precipi- Massachusetts, from 1-3 May 2002, was sponsored by the Center for tation that ultimately forms stromatolites. Biofilms provide Advanced Studies in the Space Life Sciences at the Marine Biological Laboratory, and funded by the National Aeronautics and Space Adminis- a substrate for communities to develop within the river. tration underCooperative Agreement NCC 2-1266 However, in many parts of this river basin, eukaryotic 205 206 AMARAL ZETTLER ET AL nl.. 1993, 1994, 1995; Lopez-Archilla and Amils. 1999). This makes the Rio Tinto system unique among acidic environments described to date. Figure 2 shows a phylogenetic tree depicting the breadth of eukaryotic diversity in the river. In our rRNA gene diversity survey ofbiofilm samples, we obtained sequences representing most of the major eukaryotic lineages, includ- ing the fungi, animals, green algae, land plants, strameno- piles, and alveolates (Amaral Zettler, 2002). Many of the sequences from the Rio Tinto represent photosynthetic lin- eages that were previously identified employing light mi- croscopy (Lopez-Archilla el ul., 2001). These included members of the Euglenozoa (euglenids), Chlorophyta (chlamydomonad and chlorella-like relatives). Viridiplantae (Zygneraz-relatives), and Bacillariophyta (diatoms). How- ever, our moleculardata also unveiled a significant nonpho- tosynthetic component to the Rio Tinto. Phagotrophic lin- eages such as the ciliates, cercomonads. and vahlkampriid amoebae, as well as heterotrophic fungi and possibly myx- otrophic lineages as known to occur in members of the stramenopiles such as Ochnmumas (Porter el til.. 1985) also populate the Rio Tinto. We discovered a diversity offungi that escaped detection using traditional identification methods. None ofour fungal clones showed high similarity to those species already de- scribed from the Rio Tinto. Fungi undoubtedly play an important role in community structure in the Tinto since most of them are metal resistant and can sequester specific metals (Duran et ul.. 1999). Such sequestration of poten- tially toxic metals could allow less tolerant species to exist where they might not have otherwise, possibly enhancing biodiversity in these areas. Similar studies on metal seques- tration have not been conducted on protists living in the Tinto. We hope to apply new tools in genome science to Figure 1. Some representative eukaryotes found in the RioTinto. (A) address these questions surrounding the rich microbial di- = iAnmgartaoilutihZeectfg.tleemnruuksainhAdicltiDisan;voipsdhcraPylaset;tbesarcrsaolne.b(1aB0r) A/=Milhl1.e0l0Pioh/zojot.amo.nm,iPcmhrooostgtormaliipckhreolybgyrbaepLlhionnbdgy-a verOsiutry sotfudtiheesRailosoTirnetvoe.aled a diversity that we were not Linda Amoral Zettler and Erik Zettler. able to readily characterize using molecular techniques. Clones such as RT5iinl6 and RT5iinl4 are most likely examples ofnovel eukaryotic lineages that at best branch at microbes (Fig. 1) are the major contributors of biomass the base of the animal-fungal-nucleariid radiation. Other (Lopez-Archilla et nl.. 2001 ). Eukaryotes not only form the clones (RT5iin21 and RT5iin44) branched with the recently foundations ofsome ofthese biofilm communities, but they sequenced filose amoeba Filtiinoehti noltint/i, whose own are also conspicuous inhabitants ofthem (Lopez-Archilla et taxonomic placement is equivocal. Because our study was Figure 2. A minimum evolution phylogeny for small xuhumt rRNA genes using a likelihood model. Bold letters indicate environmental clones. "RT" indicates the sequence is from the Rio Tinto. and "cul" indicates cultured species. Underlined ta\a represent genera that have been identified in the river based on microscopic observation. Sampling sites were as follows: RT1, La Raima; RT3. Berrocal Upper; RT5i. the Origin, black filamentousbiolilm; RTSii,theOrigin, green filamentousbiofilm; RT7i. Anabel'sGardengreen biofilm; RTVii. Anabel's Garden yellow biofilm. Bootstrap support values are shown, and the scale bar represents the number ofsubstitutions per site. GenBank accession numbers AY082%9-AYO!OOOI. BEYOND BIODIVERSITY IN RIO TINTO 207 ^Botrvosphaertaribis it Scyialidiumhyalinun 581 Hortaea werneckn - RT3n2 I"", RT3nS liy~L RT5in6 i EuaNsecuormoyscpeotreascirassa Ascomycota . RTSiin23 RTSinl Peniallium chrysogenum Candida alhi Pneurnocvstiscarinii Fungi Tremella gluhisppra CrypItn(>ttii.\alhitlus Basidiomycota Rhudolorula Spi~elloni\cesacitmtnuJits Chytridiomycota -Morlierelltipolycephala Zygomycota Gigasporamargdrila RTSiin3 Scvphaciliala Animals Mnemiopsisleid\i Diaphanoecagranais' Ichthyophonushoferi RTSiinW RT5iinl6 Nucleariasp. Gracilariasp. Red Algae RTlnl4cul Chondruscrispus RTlnl Chlamyilnmonasnoclieama Unknown Chlamvdomonasreinhardtn Volvoxcarleri RT5iin2 Green Algae RT5iin49 Chlorellaminulissima Nanochlorumeitcarvotum RTSiinlO RTSiinS RT5in45 Z\gnernacircumcannata Cylindrocystisbrebissonii KlebZs-eoarmmaivdsiumflacciditm Land Plants "Orvzasaliva Acantnamoebacaslellann HeteTrhoamiulmaatsqlmoboonsaassp. RTSiinl9 RTSiinlO -EuePlavunlhianer/olatucnhdraomatophora Testate Pilose Amoebae mdReTn3tinfli9edeukaryote(UEU130856) CercomonasATCC50316 Cercomonads Massisreriamarina RTSiin4 RT5iin35 RT5in4 RT5in36 -Polerioochromonasmalhainensis -Ochromonasdanica -Hibberdiamagna -RTln9 .. Stramenopiles RT7in48 Eolimnaminima Pseuao-nitzschiapungens _ Unknown Proteromonaslacertae Cafeteria roenbergensis RT5iin25 Lab\rinthuloidesminulaG,-,l,aucoma ch,altom m<rColpidiumcampylum Vorticellamicrostoma Opislhonectahennegu\i Alveolates O.\\trichagraniilifera RT7iinl Toxoplasmagondii Sarcocystismuris Alexandrium lamarense Prorocenlrummicans Dictyosteliumdiscoideum Physarumpolycephalum Gephyramoebasp. RTSiin21 RT5iin44 Filamoebanolandi Masligamoeba(Phreatamoeba)balamuthi Entamoebahistolytica .Paravahlkamnfiausliana RTS5iin38 Naegleriagruberi Euglenamutabilis RT8n7cul Euglenagracilis . 0.05 substitutions/site 21IS AMARAL ZETTLER ET AL. not exhaustive, we surmise that there are still more undis- nini. which has the ability to switch between living in a covered novel lineages in the river. neutral environment, pH 7.5. as a promastigote (flagellated Despite ourgrowing knowledge ofthe Tinto's eukaryotic stage) and in an acidic environment, pH 5.0, as an amasti- diversity, we know little about the role eukaryotes play in gote (nonrlagellated stage) (Meade etal., 1989). The plasma shaping the varied ecosystems that occur along the river. membrane of this organism contains a P-type ATPase that For example, we do not know ifthese biofilm communities has two isoforms with slightly different sequences. Isoform have microenvironments that enhance survival of their la is expressed in both promastigotes and amastigotes, members. Could fungal metal sequestration protect nontol- whereas isoform Ib is expressed more abundantly in the erant species'? Furthermore, we know little about how these amastigotes (Meade et al.. 1989). This difference suggests organisms have evolved adaptations to extreme concentra- the use of a sequence change to accommodate the acidic tions of acid and metals. condition. Modifications to ion regulatory machinery might To explore these questions, we have been isolating or- be reflected by convergent amino acid substitution patterns ganisms from the river for e.\ situ physiological experi- or by accelerated rates of change in acidophilic protist ments. We have established monocultures of Chlamydomo- lineages, as revealed in phylogenetic analyses. Forexample, nas sp., Euglena cf. mutabilis, Chlorella sp., and Vannella portions of membrane-bound V- and P-type ATPases that sp. isolated from enrichments of river water and are cur- are exposed to the acidic external environment may display rently exploring the physiology of these protists from ex- different amino acid substitution patterns than do domains treme environments. that face the cytoplasm. We have initiated our physiological studies on an acido- We are currently using degenerate primers designed philic species of a chlamydomonad alga isolated from the against two conserved regions, the phosphorylation site and river Chlamydomonas sp. Our first question about the the ATP-binding site, to amplify members of the P-type physiology of the Tinto acidophiles was the nature of the superfamily of ion transporters. Thus far, all of our clones cytosolic pH (pH,). There are published reports of acido- fall into the heavy-metal P-type class but may represent philes from all domains of life with internal pH values that different metal transporters. We have found more diverse deviate from neutral these include the archaebacterium sequences in the acidophilic Chlamydomonas than in the Picniphilus oshimac, pH, = 4.6 (van de Vossenberg et ai. neutrophilic C. reinhardtii. We are screening additional 1998); theeubacterium Bacillusacidocaldarius, pH; = 5.6- clones for H+-transporting ATPases. 5.8 (Thomas et al., 1976); and the eukaryotic alga Euglena Once we obtain ion-transporter sequence information wntabilis. pH, = 5.0-6.4 (Lane and Burris. 1981). Using from these acidophiles, we will focus on correlating the the fluorescent H+ indicator BCECF, we determined that expression of these transporters in space and time to bio- our acidophilic chlamydomonad isolate maintains an aver- geochemical characteristics in the river. This will bring us age internal pH of 6.6 at an external pH of 1 (M. A. beyond the study ofbiodiversity in the river to questions at Messerli, L. A. Amaral Zettler. S.-K. Jung. P. J. S. Smith, the heart of potential genomic interactions between mem- and M. L. Sogin. unpubl.). Our other isolates await similar bers ofthe microbial consortia. With this kind ofapproach, measurements. we may also be able to determine whether symbiotic inter- Given that there is a 40,000-fold difference in hydrogen actions are occurring in this environment. ion activity between the inside and the outside ofthesecells, we propose the existence of active transport mechanisms Acknowledgments We thhyaptothheelspiztehetsheatorngoavenlismdisverresgiutlyatien tHhe+i-rAiTnPtaesrneasl mpaH.y ex- datTihoins'swLoerxkewnasPrsougprpaomrtDedEBb-y00th8e54N8at6i.ontahle SNcAieSnAceAFsoturno-- plloawinpHth,ehaibgihli-tmyetoafldiRfifoerTeinnttporoetnivstirsopnemceinets.toThtehrreivearienttwhoe biology Program NCC2-1054, and an NIH:NCRR 01395. majorfamilies ofH+-ATPases: the V/F/A-ATPases and the The authors wish to acknowledge the support of the Amils P-type-ATPases. The V-type ATPases can occur in the lab at the Autonomous University of Madrid and the tech- plasma membrane of eukaryotes (but are more commonly nical assistance of Brendan Keenan and Erik Zettler. associated with vacuolar membranes) and consist ofat least 1 1 subunits and a molecular mass approaching 106 Da. In Literature Cited contrast, eukaryotic P-type ATPases consist of either mo- AmaralZettler.I,.A.,F.Gomez,E.R.Zettler,B.G.Keenan,R.Amils, nosubunits (aswith H+-ATPases) orahetero-subunit (alpha andM.L.Soj-in.21)02. EukaryoticdiversityinSpain'sRiverofFire. and beta, as found in the Na'/K ' -ATPases and H+/K+- Nuiiiri- 417: 137. ATPases); have a molecular weight of about 100 kDa; and Duraacni,doCp"h.,ilIi.cMfaurnigin.,Papn.d5R2.1-A5m3i0lsi.n1B9i9o9h.ydrSopmeectiafliclumregtyalansdeqtuheestEenrviin-g form a phosphorylated intermediate during the course of i-HiiiiH'iit T\mrilx the Mining ofthe 21st Century. Proceedings ofthe ATP hydrolysis. 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