Molecular Ecology (2008) 17, 3136–3146 doi: 10.1111/j.1365-294X.2008.03806.x

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd

Blackwell Publishing LtdMolecular evidence for broad-scale distributions in bdelloid rotifers: everything is not everywhere but most things are very widespread

DIEGO FONTANETO,*† TIMOTHY G. BARRACLOUGH,*‡ KIMBERLY CHEN,* CLAUDIA RICCI† and ELISABETH A. HERNIOU**Division of Biology, Silwood Park Campus, Imperial College London, Ascot, Berkshire SL5 7PY, UK, †Dipartimento di Biologia, Università degli Studi di Milano, Via Celoria 26, I-20133 Milano, Italy, ‡Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK

Abstract

The Baas-Becking’s hypothesis, also known by the term ‘everything is everywhere’ (EisE),states that microscopic organisms such as bacteria and protists are globally distributed anddo not show biogeographical patterns, due to their high dispersal potential. We tested theprediction of the EisE hypothesis on bdelloid rotifers, microscopic animals similar toprotists in size and ecology that present one of the best cases among animals for the plausibilityof global dispersal. Geographical range sizes and patterns of isolation by distance wereestimated for global collections of the genera Adineta and Rotaria, using different taxonomicunits: (i) traditional species based on morphology, (ii) the most inclusive monophyleticlineages from a cytochrome oxidase I phylogeny comprising just a single traditional species,and (iii) genetic clusters indicative of independently evolving lineages. Although there arecases of truly cosmopolitan distribution, even at the most finely resolved taxonomic level,most genetic clusters are distributed at continental or lower scales. Nevertheless, although‘everything is not everywhere’, bdelloid rotifers do display broad distributions typical ofthose of other microscopic organisms. Broad dispersal and large population sizes might befactors lessening the evolutionary cost of long-term abstinence from sexual reproduction inthis famous group of obligate parthenogens.

Keywords: cosmopolitism, ‘everything is everywhere’ hypothesis, global distribution, microscopicanimals, Rotifera Bdelloidea, spatial pattern

Received 28 November 2007; revision received 6 April 2008; accepted 17 April 2008

Introduction

Zoologists always hope to find weird and interesting neworganisms in exotic places. Over the last few centuries,scientific expeditions in remote places have indeed discov-ered new species and even higher taxa of limited distribution(e.g. Klass et al. 2002; Ribera et al. 2002; Jenkins et al. 2004).In contrast, scientists working on microscopic animals andprotists have mostly found organisms that could be ascribedto taxa already known in their home country (Fenchel &Finlay 2003). The recently described microscopic animalphyla, Loricifera from marine sediments off the coast of

France (Kristensen 1983), Cycliophora on the mouthpartsof lobsters in Denmark (Funch & Kristensen 1995) andMicrognathozoa in cold springs in Antarctica (Kristensen& Funch 2000), have all been suddenly found in regionsvery distant from the original type locality, but in similarpreviously unexplored habitats (Todaro & Kristensen 1998;De Smet 2002; Nedved 2004). So why do we observe sucha discrepancy between the global distribution of microscopicand macroscopic organisms?

The Baas-Becking (1934)’s hypothesis, also known as the‘everything is everywhere’ (EisE) hypothesis, encapsulatesthe classical view that microscopic organisms are globallydistributed due to their high dispersal potential (Kellogg &Griffin 2006). Small size and an ability to enter dormancy,and thus to produce dormant propagules (Cáceres 1997;

Correspondence: Diego Fontaneto, Fax: +44(0)20 7594 2339; E-mail: d.fontaneto@imperial.ac.uk

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Bohonak & Jenkins 2003; Fenchel & Finlay 2004) mightexplain why prokaryotes and some microscopic eukaryotes,such as protists and small invertebrates, have acquiredglobal distributions. The assumption that organisms smallerthan 2 mm have cosmopolitan distribution often holdstrue when species are defined using traditional taxonomy.However, the EisE hypothesis has been challenged recentlyas molecular evidence of distance–decay relationshiprevealed a high degree of cryptic diversity and restricteddispersal in a variety of microscopic organisms, includingprokaryotes (Cho & Tiedje 2000; Whitaker et al. 2003),protists (Darling et al. 2004; Foissner 2006; Telford et al.2006), and fungi (Taylor et al. 2006). Other studies havefound cases of restricted distributions solely by re-evaluatingmorphological evidence within species previously assumedto have cosmopolitan distributions (Smith & Wilkinson2007).

The current debate on the EisE hypothesis divides scien-tists in two major groups (Whitfield 2005). On one side,scientists following the EisE hypothesis in its original formassume that species differences in samples from differentareas occur because of environmental differences, and notbecause of restricted dispersal. Thus, ‘everything is every-where, but the environment selects’ is considered the rulefor microorganisms (e.g. de Wit & Bouvier 2006; Fenchel &Finlay 2006). On the other hand, other scientists propose thatclassical morphological taxonomy of microscopic organismsis not able to resolve their actual diversity, and thereforethat cosmopolitan ranges result from misidentification andlumping of spatially isolated lineages (e.g. Coleman 2002;Foissner 2006; Taylor et al. 2006). Thus, cosmopolitanism isconsidered as an exception in microorganisms, as it is inmacroorganisms.

The EisE hypothesis incorporating environmental selectionis difficult to falsify because there could be unmeasuredaspects of the environment that differ consistently amongregions (Foissner 2006). However, if we assume a densesample of equivalent habitats across sampling regions,the hypothesis makes clear predictions about genotypedistributions. If EisE is the rule, the degree of geneticrelatedness between two individuals should be independentof the geographical distance between them, except thatindividuals within a habitat patch might be more closelyrelated to one another than those in different habitatpatches. Conversely, if EisE does not hold true, spatiallyexplicit models should work in the same way as they dofor macroorganisms, and genetic diversity should berelated to geographical distances by a classical distance–decay relationship.

We test the EisE hypothesis in an interesting test-case,the bdelloid rotifers. Bdelloids are microscopic animals (thevast majority smaller than 1 mm) that have been regardedtraditionally to have cosmopolitan species: more than 20%of all described bdelloid species have been found in a small

Italian valley (Fontaneto et al. 2006); no distance–decayrelationship in species composition is observed amongcommunities (Fontaneto & Ricci 2006); and most tradi-tional morphological species seem to have cosmopolitandistribution (Fontaneto et al. 2007a). However, thesestudies were based on morphological species identificationonly. Several other small invertebrate species traditionallyseen as having cosmopolitan ranges have been recognizedas assemblages of cryptic species with regionally morerestricted entities, both in the marine and continentalhabitats (De Meester et al. 2002; Gómez et al. 2002; Suatoniet al. 2006). Yet, among all microscopic animals, bdelloidspresent the most plausible case for truly cosmopolitandistributions: adults can survive desiccation in a state ofanhydrobiosis without the need to produce dormant eggstages (Cáceres 1997; Ricci et al. 2007) and adult bdelloidsare found in aerial plankton collected from windsocks(Jenkins & Underwood 1998). Moreover, the populationstructure of bdelloids is of considerable interest because oftheir status as ancient asexuals (Mark Welch & Meselson2000; Birky 2004). Recent work has shown that bdelloidshave indeed diversified into discrete genetic and morpho-logical clusters indicative of independently evolving species(Birky et al. 2005; Fontaneto et al. 2007b, 2008). Whetherthose species have global or locally restricted distributionsis an important parameter for understanding any costsand benefits associated with their strict asexual lifestyle(Barraclough et al. 2007).

We used cytochrome oxidase I (COX1) sequence datafrom intensive samples of two genera of bdelloids, Adinetaand Rotaria, to test whether bdelloid species display trulycosmopolitan distributions. In order to compare results fromtraditional species identification and molecular evidence,we analysed geographical ranges and distance–decayrelationships at three taxonomically nested levels: traditionalspecies identified from morphology; monophyletic cladeson the DNA tree belonging to a single traditional species;and finally, genetic clusters indicative of independentlyevolving populations. We find evidence for the true globaldistribution of some bdelloids at all levels of resolution,not just for morphologically defined species. Nevertheless,although the results support very widespread dispersalin bdelloids, most clades identified by molecular datado display significant evidence of isolation by distance.Evolutionary processes mostly operate within populationsdistributed at continental scales or below.

Materials and methods

Sampling

We sampled living animals of the bdelloid genera Adineta(family Adinetidae) and Rotaria (family Philodinidae) fromwater in rivers, ponds and water bodies, and dormant

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animals in dry mosses and lichens. Our sample effort wasapproximately nested, with greatest sampling effort inItaly and the UK, then lesser sampling effort acrossseven other countries in Europe, finally with 25 samplesfrom Africa, Antarctica, Australia, New Zealand, NorthAmerica and Southeast Asia (Fig. 1, Table S1, Supplementarymaterial).

Species identification

We followed the most recent taxonomic revision for thegroup (Donner 1965) to identify animals, where possible, totaxonomic species. Identification relies on morphologicaldifferences between the shape and size of many features,such as the head, corona, rostrum, eyes, trunk sculpture,foot, toes, and spurs. Fourteen species of Adineta and 24species of Rotaria have been described based on morphologyalone (Segers 2007), although many of them are only knownfrom their original description or rarely found.

Monophyletic taxonomic lineages

We isolated DNA using a Chelex preparation and poly-merase chain reaction (PCR) amplified a 657-bp fragmentof cytochrome oxidase I (COX1) as described in Fontanetoet al. (2007b) using the primers LCOI (5′-GGTCAACAAAT-CATAAAGATATTGG-3′) and HCOI (5′-TAAACTTCAGGGT-GACCAAAAAATCA-3′) (Folmer et al. 1994). The sequenceswere edited and assembled in sequencher 4.1 and alignedin clustal w and macclade 4.07. Sequences were depositedin GenBank (Table S1).

Bayesian analyses were run in mrbayes 3.1.1 for 5million generations with two parallel searches, using aGTR + inv + gamma model, separately for the two datasets (Adineta and Rotaria) (see Barraclough et al. 2007 fordetails on model selection and phylogenetic reconstruction).Two individuals from the related genus Bradyscela wereincluded as outgroup for Adineta, and eight individualsfrom the genus Dissotrocha were included as outgroup forRotaria (Table S1). These taxa are supported as close out-groups in combined analysis of COX1 data for a larger sampleof bdelloids (D. Fontaneto, E. A. Herniou, T. G. Barraclough,unpublished). Monophyletic taxonomic lineages wereidentified as the most inclusive taxonomically monospecificclades with Bayesian support values higher than 0.80.

Cluster delimitation

We used the recently developed approach by Pons et al.(2006), already applied to bdelloids (Barraclough et al.2007; Fontaneto et al. 2007b), to identify genetic clusters,indicative of independently evolving sets of individuals.The model optimizes a threshold age, T, such that nodesbefore the threshold are considered to be diversification

events whereas branches crossing the threshold define kgenetic clusters each obeying a separate coalescent process.Models were fitted using an R script available from T.G.B.to an ultrametric tree (a rooted additive tree with terminalnodes equally distant from the root) obtained by ratesmoothing the DNA trees using penalized likelihood in r8sand cross-validation to choose the optimal smoothingparameter for each tree (Sanderson 2002). We assumed auniform branching rate for coalescence in all of the clusters.The identified clusters represent putative populationunits identified solely from the DNA data; hence, there isno circularity in terms of assuming that species must begeographically coherent.

Geographical analysis

For all three levels of resolution, that is, (i) traditional species,(ii) taxonomic monophyletic lineages, and (iii) independentgenetic clusters, we estimated range size as the maximumdistance between the samples. One potential bias forcomparing the different levels of resolution is that geogra-phical range size might decline at more resolved levelssimply because fewer individuals have been sampled. Tocheck this, we permuted locality data among individualsand compared the observed range size distributions tothose obtained by randomization.

For all groups with more than three individuals from atleast two different sampling locations, we tested whetherpairwise genetic distances within each group were relatedto geographical distances. Analyses were repeated usinggroupings of traditional species, monophyletic lineages,and genetic clusters in turn. Genetic distances were calculatedas uncorrected p-distances. Geographical distances werespecified as the matrix of all paired kilometric distances,estimated from angular distances between paired coor-dinates. First, we calculated each cos(d°) = cos(90° – ϕ1) *cos(90° – ϕ2) + sin(90° – ϕ1) * sin(90° – ϕ2) * cos(λ1 – λ2), whereϕ1 and ϕ2 are the latitudes of sites 1 and 2, and λ1 and λ2their longitudes; then, we obtained the kilometric distance(d), as d = d°* 111.13 km/°, where 111.13 km is the sphericalapproximation at the equator for 1°.

To test for correlation between genetic and geographicaldistances, we applied one-tailed Mantel tests with 2000replicates using the Pearson correlation method in r 2.5 (RDevelopment Core Team 2007), package vegan (http://vegan.r-forge.r-project.org/). The test was one tailed as onlya positive correlation is expected comparing geographicaland genetic distances; significant positive relationshipsbetween genetic and geographical distances representdistance–decay scenarios, which would oppose the EisE.On the contrary, the absence of such relations would provideevidence that the probability of sampling two closely relatedindividuals is independent of the distance between thesampling locations, which will support the EisE. To avoid

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possible artefacts of sampling close relatives in a givensample, we repeated Mantel tests by including only singlerepresentative of each haplotype found in a given sample.

Results

Morphology: supporting EisE

We sampled 88 individuals of Adineta and 164 individualsof Rotaria. The latter include 77 individuals sampled byFontaneto et al. (2007b) and Barraclough et al. (2007), and87 new samples.

Only 59 strains of Adineta could be ascribed to one of sixdescribed species, while all the strains of Rotaria could beascribed to one of 10 described species (Fig. 1, Fig. S1,Supplementary material). Two species previously thoughtto be endemics, Adineta grandis in Antarctica and Adineta ricciaein Australia were confirmed as endemics, and not foundanywhere else. All other traditional taxonomic specieswere widely distributed. Range sizes expressed as the lineardistance between the most distant individuals were com-monly over 1000 and even 10 000 km (Table 1, Fig. 2). Evenin Australia and Antarctica, the places where we foundendemic species, other widespread and cosmopolitanspecies were present (e.g. Adineta gracilis and Rotaria sordida).

Species reliability: morphology vs. phylogeny

Phylogenetic analysis of the mitochondrial DNA (mtDNA)provided robust support for a number of distinct cladeswithin both genera (Fig. 1). In Adineta, the clustering analysisidentified 19 genetic clusters (Fig. 1, a1–a19; Fig. S1) and17 singlets. However, none of the morphospecies with morethan one animal sampled was supported as a mono-phyletic lineage by the mtDNA phylogenetic analysis.The most surprising result was the extreme diversitywithin the genus: many animals could not be ascribedunambiguously to traditional species based on morphology.They presented either unknown traits, or intermediatemorphologies, providing evidence of still not describedmorphological diversity. Moreover, many individualswere identified as Adineta vaga according to morphology,but this taxon derives from taxonomic ‘lumping’ of manyclades revealed as not monophyletic by the mtDNA data:at least 17 distinct genetic entities (10 clusters plus 7 singlets)were hidden under a similar morphology (Fig. 1).

The clustering analysis for Rotaria identified 25 geneticclusters (Fig. 1, r1–r25; Fig. S2, Supplementary material) plus29 singlets, which comprised 20 taxonomic monophyleticlineages (Fig. 1, RA-RO plus five singlets). Quite surprisinglyfor such minute and understudied animals, morphologymatched well with the phylogeny: six morphospecies (Rotariacitrina, R. macrura, R. magnacalcarata, R. neptunia, R. socialis,and R. tardigrada) were monophyletic with high support

values; R. sordida was monophyletic without strong support,and only Rotaria rotatoria was not monophyletic, with atleast nine unrelated monophyletic clades. However, specieswith more than two individuals, except R. socialis, includedtwo or more groups of independent clusters.

Geographical distribution

In both genera, range size declines as resolution increasesfrom morphospecies to monophyletic taxonomic lineagesto genetic clusters (Fig. 2). The permutation test shows that thedecline in range size at lower levels is not simply an artefactof sampling fewer individuals at lower levels (Fig. 2). How-ever, despite the overall trend, the maximum values for rangesize in Adineta were similar at all analysed levels (Fig. 2).

Mantel test would not be reliable with sample size lessthan five. Nevertheless, almost all groups with at least sixindividuals gave evidence of distance–decay relationship,with significant relationships at all levels, traditionalspecies, monophyletic lineage, and independent cluster(Table 1). One possible bias is that a single sample mighttend to include identical sequences because animals arerelated, that is, sisters or cousins. Therefore, we repeated theanalyses retaining just a single sequence for each haplotype.The results remain the same for 16 out of 22 groups (Table 1).

Looking in detail at the relationships between geograph-ical and genetic distances in Rotaria, high genetic distanceswithin groups can be found at very short or very longgeographical distances, even in the same sample. However,closely related individuals (< 5% sequence divergence) areonly found at geographical scales of less than 2000 km. Wefound no Rotaria cluster with range extent above 2000 km,while monophyletic lineages and traditional species easilyreached 10 000 km. In Adineta, close relatives can be foundat any of the scales considered, although less frequently atbroader scales, and consequently, the maximum rangeextent is similar whether traditional species, monophyleticlineages or genetic clusters are considered.

Notwithstanding the overall positive relationship betweengeographical and genetic distances, some evidence ofeffective long-distance dispersal is present: in A. vaga,cluster a18, very similar sequences have been repeatedlyfound in the UK, Tanzania and New Zealand, with geneticdistances completely unrelated to geographical distances.In R. macrura, two clusters are present (Fig. 1, r17, r18), butthey are not spatially isolated: one cluster is commonbetween Italy and the UK, but two Italian samples(R.macr.IT.1 and R.macr.IT.2), only a few hundred metresfar apart, fall in the two different clusters, showing a scenarioof high genetic and short geographical distances.

Other taxa produced evidence of groups limited to distinctgeographical areas. Rotaria magnacalcarata is split in twodifferent clusters, one with animals from different localitiesin UK and Poland (Fig. 1, r7), and one with only Italian

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Table 1 Number of individuals (n) and range in kilometres for every taxon identified at all levels, traditional species, monophyletictaxonomic lineage and independent cluster, both for Adineta (a) and for Rotaria (b). The results of Mantel correlation test (r) between geneticand geographical distances, with one tailed P value (P) are given. Results with sequences collapsed in haplotypes for each sample are alsoprovided; only results of previously significant tests are reported(a)

Taxon n Range r P ncollapsed rcollapsed Pcollapsed

A. gracilis 9 13084 0.7074 0.0035 5 0.4275 0.06575A. grandis 2 0 NA NA NA NA NAA. oculata 1 0 NA NA NA NA NAA. ricciae 2 0 NA NA NA NA NAA. steineri 1 0 NA NA NA NA NAA. vaga 48 19319 0.1035 0.01175 37 0.1506 0.011AA 5 12773 0.9948 0.09725 NA NA NAAB 4 0 NA NA NA NA NAAC 4 93 0.9426 0.13175 NA NA NAAD 2 0 NA NA NA NA NAAE 4 985 0.7912 0.04625 3 –0.5097 0.41425AF 4 971 0.1322 0.2085 NA NA NAAG 2 0 NA NA NA NA NAAH 2 577 NA NA NA NA NAAI 2 0 NA NA NA NA NAAJ 2 0 NA NA NA NA NAAK 15 17746 0.6516 < 0.001 11 0.6376 0.0015AL 12 18472 0.01265 0.1265 NA NA NAa1 4 0 NA NA NA NA NAa2 4 65 –0.6441 0.4995 NA NA NAa3 4 0 NA NA NA NA NAa4 2 0 NA NA NA NA NAa5 4 93 0.9426 0.12 NA NA NAa6 2 0 NA NA NA NA NAa7 4 985 0.7912 0.0435 3 –0.5097 0.41425a8 2 0 NA NA NA NA NAa9 4 971 0.1322 0.206 NA NA NAa10 2 0 NA NA NA NA NAa11 2 578 NA NA NA NA NAa12 2 0 NA NA NA NA NAa13 2 0 NA NA NA NA NAa14 2 0 NA NA NA NA NAa15 5 245 0.8134 0.092 NA NA NAa16 5 0 NA NA NA NA NAa17 2 0 NA NA NA NA NAa18 11 18472 0.5496 0.00725 10 0.5792 0.028a19 8 1146 –0.1702 0.3445 NA NA NA

Fig. 1 Phylogenetic relationships in the genera Adineta and Rotaria. The consensus of 40 000 sampled trees from Bayesian analysis of COX1mtDNA data sets is shown under a GTR + inv + gamma substitution model. Posterior probabilities above 0.80 are shown above eachbranch. White diamonds indicate monophyletic taxonomic species; closed circles indicate independent clusters. The boxes in the firstcolumn represent the country of origin, which colour code refers to the maps at the bottom of the picture; European countries arerepresented by plain colours, countries from other areas are represented by compound motives. The second column has vertical boxes withlight colour grouping individuals in clusters (indicated by closed circles on the tree), which are named from r1 to r25 for Rotaria and froma1 to a19 for Adineta. The third column has vertical boxes with dark colour grouping individuals in monophyletic taxonomic lineages(indicated by open diamonds on the tree), which are named from RA to RO for Rotaria and from AA to AL for Adineta. The forth columnhad vertical boxes indicating traditional species; some names are abbreviated: cit, citrina; nep, neptunia; noi, neptunoida; rot, rotatoria (forRotaria); gra, grandis; ocu, oculata, ric, ricciae; ste, steineri (for Adineta).

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(b)

Taxon n Range r P ncollapsed rcollapsed Pcollapsed

R. citrina 2 0 NA NA NA NA NAR. macrura 8 852 –0.07051 0.28775 NA NA NAR. macroceros 2 0 NA NA NA NA NAR. magnacalcarata 21 1361 0.7897 < 0.001 10 0.6309 0.00425R. neptunia 3 1325 0.7975 0.071 NA NA NAR. neptunoida 1 0 NA NA NA NA NAR. rotatoria 63 9837 0.1437 0.00675 47 0.1372 0.022R. socialis 33 1132 0.3051 < 0.001 20 0.2583 0.00125R. sordida 19 12050 0.3026 0.02025 14 0.2566 0.0665R. tardigrada 12 5194 0.2301 0.031 10 0.1432 0.04975RA 2 0 NA NA NA NA NARB 7 860 0.4464 0.00875 6 0.1508 0.158RC 13 97 0.5081 0.00825 9 0.3831 0.02475RD 9 0 NA NA NA NA NARE 21 1361 0.7897 0.0005 10 0.6309 0.00425RF 6 769 0.5603 0.03925 5 0.4065 0.04875RG 33 1132 0.3051 < 0.001 20 0.2583 0.00125RH 18 1133 –0.08349 0.426 NA NA NARI 5 838 –0.1995 0.1195 NA NA NARJ 3 7669 0.9815 0.1645 NA NA NARK 8 852 –0.07051 0.28425 NA NA NARL 3 1325 0.7975 0.08475 NA NA NARM 12 5194 0.2301 0.02675 10 0.1432 0.04975RN 17 12050 0.4485 < 0.001 NA NA NARO 2 4 NA NA NA NA NAr1 2 0 NA NA NA NA NAr2 2 0 NA NA NA NA NAr3 3 301 –0.189 0.33725 NA NA NAr4 13 97 0.5081 0.0075 9 0.3831 0.02475r5 7 0 NA NA NA NA NAr6 2 0 NA NA NA NA NAr7 4 228 0.8665 0.03925 3 0.9884 0.085r8 17 1361 0.6741 0.0035 7 0.8095 0.0265r9 6 769 0.5603 0.03775 5 0.4065 0.04875r10 33 1132 0.3051 < 0.001 20 0.2583 0.00125r11 3 804 –0.4958 0.33 NA NA NAr12 2 0 NA NA NA NA NAr13 3 338 0.9998 0.17 NA NA NAr14 4 1131 0.9977 0.16825 NA NA NAr15 3 861 0.9936 0.1585 NA NA NAr16 4 838 0.8645 0.1285 NA NA NAr17 4 766 0.373 0.1685 NA NA NAr18 3 101 0.8292 0.1665 NA NA NAr19 2 182 NA NA NA NA NAr20 3 1928 0.668 0.166 NA NA NAr21 4 28 0.6252 0.09375 NA NA NAr22 2 0 NA NA NA NA NAr23 3 0 NA NA NA NA NAr24 2 259 NA NA NA NA NAr25 3 0 NA NA NA NA NA

Table 1 Continued

samples (Fig. 1, r8). Rotaria socialis has a similar pattern ofBritish–Italian disjunction, although falling into just onecluster. Rotaria sordida was split in two distinct lineages(Fig. 1, RN, RO), but individuals from both lineages co-existed

in the same moss sample in UK. Adineta vaga lineage AKcomprises animals from distant countries, as Australia,Tanzania, Spain, and UK, but most of its subclades arelimited to single areas (Fig. 1, AK).

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Discussion

Many traditional bdelloid species were found commonlyin almost every place we sampled, supporting the idea ofcosmopolitan distribution. Nevertheless, the phylogeneticanalysis discovered considerable cryptic diversity. Mosttraditional species of Rotaria were found to be monophyleticbut contained significantly distinct genetic clusters. Theexceptions were Rotaria rotatoria, which is not monophyleticand subsumes an enormous degree of hidden geneticdiversity, and, in contrast, Rotaria socialis, which is mono-phyletic and comprises a single genetic cluster. In Adineta,morphological species provide a very poor match to patternsof genetic diversity and clearly considerable taxonomic

work is needed to establish a robust taxonomy for thisgenus. Therefore, the results from the morphospeciesapproach, classically supporting EisE, may be biased byour inability to describe bdelloid true diversity usingmorphological identification.

As expected, if EisE results in part from poorly resolvedtaxonomy of microscopic organisms, the range sizes ofgroups declined from traditional species, to monophyletictaxonomic lineages to clusters identified by the phylogeneticanalysis. While these results could still be affected byundersampling, the randomization test indicates thatthe decline is greater than expected simply by differentsampling efforts at the different levels of resolution. Similarevidence of persistent genetic signature of colonization hasbeen recently discovered in monogonont rotifers (Gómezet al. 2007; Mills et al. 2007), and in bacteria and protists(Bass et al. 2007; Rusch et al. 2007). Moreover, several tradi-tional species of bdelloids are only known from limitedareas (Ricci et al. 2001, 2003; Fontaneto & Melone 2003;Segers & Shiel 2005). One possibility is that the pattern ofgeographical restriction is mediated by habitat restriction.Almost no data are available on habitat preferences inbdelloids, except for macroscopic differences (e.g. lichenvs. moss, wet vs. dry samples), and we sampled similarhabitats everywhere (e.g. moss on barks, lichen on soil).Moreover, even genetic clusters can group together animalscollected in different habitats, like Adineta vaga clusters a7,a9, and a18, comprising animals from water samples, drysediments and terrestrial mosses.

Despite evidence for geographical restriction, mostbdelloid taxa do have very widespread distributions, frompan-European to cosmopolitan range size, even at the levelof evolutionarily independent clusters. The most strikingexample is cluster a18 of A. vaga, found in Europe, Africaand New Zealand.

This size of bdelloid ranges is consistent with hugepopulation sizes and high dispersal rates. High gene flowamong sites requires both successful dispersal and successfulcolonization of new sites (Kellogg & Griffin 2006). Successfuldispersal may be achieved by bdelloid dormant propagules,which are known to disperse efficiently in the environment(Cáceres & Soluk 2002; Bohonak & Jenkins 2003; Cohen &Shurin 2003). Successful colonization is not easy to measure,but bdelloids are well known for their ability to survive awide range of environmental stresses in an adult dormantstate (Ricci & Caprioli 2005; Ricci & Perletti 2006; Ricci et al.2007), and as obligate asexuals do not suffer from the needto find mating partners relevant for sexual organisms. Thisability might help bdelloids to colonize sites where broadlyfavourable conditions are present.

To conclude, we find evidence for restricted distributionsand geographical isolation by distance within bdelloidtaxa. It is very unlikely that all species of bdelloids ‘couldeventually be discovered in one small pond’ (Finlay &

Fig. 2 Box plot of the distribution of range size in kilometres forall three levels of analysis: traditional species, monophyletictaxonomic lineages, and independent clusters, both for Adinetaand for Rotaria. The dashed line represent the expected medianvalue of range extent across taxa when localities are permutedamong individuals; the associated P value is the probability ofobtaining less than or equal to the observed medium range extentunder the null model of random localities.

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Esteban 1998). Nevertheless, the range size distribution ofbdelloids is clearly greatly shifted compared to those ofmacroscopic organisms, in which mean range sizes of bothtraditional species and those identified by genetic datatend to be well within continental scales (e.g. Brambillaet al. 2006; Orme et al. 2006; Pons et al. 2006). Bdelloidsqualitatively fall in the general trend of small passivelydispersing organisms, with high dispersal ability, largerange size and small body size (Jenkins et al. 2007). Theresults also have important implications for understandinghow bdelloid rotifers have been able to survive for longevolutionary periods despite an apparent absence of sexualreproduction (Birky 2004). Natural selection is widely heldto operate more effectively in large, intermixed populations.We cannot estimate actual population sizes or dispersalrates here, because of the lack of a calibration in units ofgeneration time or years. However, the broad distributionof coherent genetic clusters is consistent with mixed popu-lations that number many millions of individuals. Broaddispersal and large population sizes could be two factorslessening the evolutionary costs of asexuality in bdelloids.

Acknowledgements

This research was supported by Natural Environment ResearchCouncil (NERC) UK grant NER/A/S/2001/01133. Further supportcame from a Marie Curie Intra European Fellowship to D.F., a RoyalSociety University Research Fellowship to T.G.B., a Royal SocietyInternational Joint Project grant to T.G.B and C.R., and a DorothyHodgkin Royal Society Research Fellowship to E.A.H. We thankK. Linse and P. Convey (BIOPEARL project, British AntarcticSurvey) for contributing Antarctic specimens for this study.

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Diego Fontaneto and Elizabeth A. Herniou are research fellowsinterested respectively in macroecology of rotifers and in genomeevolution. Timothy G. Barraclough is a reader interested in theevolution of diversity. Claudia Ricci is a professor dealing withecology and bdelloid rotifers. Kimberly Chen was a mastersstudent involved in the project.

Supplementary material

The following supplementary material is available for this article:

Fig. S1 Phylogenetic relationships in the genus Adineta. Theconsensus of 40 000 sampled trees from Bayesian analysis of COX1mtDNA data sets is shown under a GTR + inv + gamma substitutionmodel. Posterior probabilities above 0.80 are shown above eachbranch. White diamonds indicate monophyletic taxonomic species;closed circles indicate independent clusters. Name of each indi-vidual refers to the species, the country, the number of site withinthat country for that species and the number of individual fromthat site if several were isolated, for example, Adineta vaga. UK.5.2refers to the second sample from site 5 in UK for A. vaga.

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Fig. S2 Phylogenetic relationships in the genus Rotaria. Theconsensus of 40 000 sampled trees from Bayesian analysis of COX1mtDNA data sets is shown under a GTR + inv + gamma substitutionmodel. Posterior probabilities above 0.80 are shown above eachbranch. White diamonds indicate monophyletic taxonomic species;closed circles indicate independent clusters. Name of each indi-vidual refers to the species, the country, the number of site withinthat country for that species and the number of individual fromthat site if several were isolated, for example, R.macr.IT.1.1 refersto the first sample from site 1 in Italy for Rotaria macrura.

Table S1 List of analysed individuals, with identification of tradi-tional species, GenBank Accession no., sample name (refer toFontaneto et al. 2007a for more details), country of origin, andapproximate geographical coordinates

This material is available as part of the online article from:http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-294X.2008.03806.x(This link will take you to the article abstract).

Please note: Blackwell Publishing are not responsible for the contentor functionality of any supplementary materials supplied by theauthors. Any queries (other than missing material) should bedirected to the corresponding author for the article.