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Molecular taxonomy and phylogenetic affinities of two groundwater amphipods,Crangonyx islandicus and Crymostygius thingvallensis, endemic to Iceland

Etienne Kornobis a,⇑, Snæbjörn Pálsson a, Dmitry A. Sidorov b, John R. Holsinger c, Bjarni K. Kristjánsson d

a Department of Biology, University of Iceland, Askja, Sturlugata 7, 101 Reykjavik, Icelandb Institute of Biology and Soil Science, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690022, Russiac Department of Biological Sciences, Old Dominion University, Norfolk, VA, USAd Department of Aquaculture and Fish Biology, Hólar University College, Háeyri 1, 550 Sauðárkrókur, Iceland

a r t i c l e i n f o

Article history:Received 20 October 2010Revised 17 December 2010Accepted 20 December 2010Available online 30 December 2010

Keywords:rDNACrustaceaAmphipodaCrangonyctoideaMolecular phylogenyAlignment methods

a b s t r a c t

The amphipod superfamily Crangonyctoidea is distributed exclusively in freshwater habitats worldwideand is characteristic of subterranean habitats. Two members of the family, Crangonyx islandicus andCrymostygius thingvallensis, are endemic to Iceland and were recently discovered in groundwater under-neath lava fields. Crangonyx islandicus belongs to a well-known genus with representatives both in NorthAmerica and in Eurasia. Crymostygius thingvallensis defines a new family, Crymostygidae. Considering theincongruences observed recently between molecular and morphological taxonomy within subterraneanspecies, we aim to assess the taxonomical status of the two species using molecular data. Additionally,the study contributes to the phylogenetic relationships among several crangonyctoidean species and spe-cifically among species from four genera of the family Crangonyctidae. Given the available data we con-sider how the two Icelandic species could have colonized Iceland, by comparing geographical origin of thespecies with the phylogeny.

Regions of two nuclear (18S and 28S rRNA) and two mitochondrial genes (16S rRNA and COI) for 20different species of three families of the Crangonyctoidea were sequenced. Four different methods wereused to align the RNA gene sequences and phylogenetic trees were constructed using bayesian and max-imum likelihood analysis. The Crangonyctidae monophyly is supported. Crangonyx islandicus appearedmore closely related to species from the Nearctic region. Crymostygius thingvallensis is clearly divergentfrom the other species of Crangonyctoidea. Crangonyx and Synurella genera are clearly polyphyletic andshowed a geographical association, being split into a Nearctic and a Palearctic group.

This research confirms that the studied species of Crangonyctidae share a common ancestor, which wasprobably widespread in the Northern hemisphere well before the break up of Laurasia. The Icelandic spe-cies are of particular interest since Iceland emerged after the separation of Eurasia and North America, isgeographically isolated and has repeatedly been covered by glaciers during the Ice Age. The close relationbetween Crangonyx islandicus and North American species supports the hypothesis of the Trans-Atlanticland bridge between Greenland and Iceland which might have persisted until 6 million years ago. Thestatus of the family Crymostygidae is supported, whereas Crangonyx islandicus might represent a newgenus. As commonly observed in subterranean animals, molecular and morphological taxonomy led todifferent conclusions, probably due to convergent evolution of morphological traits. Our molecular anal-ysis suggests that the family Crangonyctidae needs taxonomic revisions.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Two endemic species of the exclusively freshwater superfamilyCrangonyctoidea, characteristic of subterranean habitats, were re-cently discovered in Iceland and described using morphological

and meristic criteria. The two species are Crangonyx islandicus, anew species within a known genus in the family Crangonyctidae(Svavarsson and Kristjánsson, 2006), and Crymostygius thingvallen-sis, a new monotypic family (Crymostygidae) (Kristjánsson andSvavarsson, 2004). According to morphological taxonomy, Crang-onyx islandicus belongs to the genus Crangonyx, distributed inNorth America and Eurasia. The discovery of these two new speciesin Iceland, which was repeatedly covered by glaciers during thecold periods of the Ice Age (Pleistocene, from 2.59 Myr to12,000 years ago), has raised questions about when and how they

1055-7903/$ - see front matter � 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2010.12.010

⇑ Corresponding author. Fax: +354 525 4632.E-mail addresses: [email protected] (E. Kornobis), [email protected] (S. Pálsson), sidorov

@biosoil.ru (D.A. Sidorov), [email protected] (J.R. Holsinger), [email protected](B.K. Kristjánsson).

Molecular Phylogenetics and Evolution 58 (2011) 527–539

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colonized Iceland. A recent study has found strong evidence forsurvival of Crangonyx islandicus in sub-glacial refugia in Iceland.Using mtDNA divergence among monophyletic and geographicallyisolated populations, Kornobis et al. (2010) showed that the ob-served divergence occurred within Iceland, during the Ice Ageand even before its onset.

The existence of Iceland has been traced to a geological hotspotwhich migrated southeastward about 40 Mya from the East Green-land coast to its current location at the boundary of the Atlanticridge and the Greenland–Scotland Transverse Ridge (Lawver andMüller, 1994; Lundin and Doré, 2002). Geological and biologicalevidence support that the Greenland–Scotland Ridge, submergedat great depths at present, was above sea level from early Cenozoicto late Miocene, first as a continuous land bridge and later as achain of islands (McKenna, 1983; Eldholm et al., 1994; Grímssonet al., 2007; Poore, 2008; Denk et al., 2010). Fossil records showthat plants migrated along this land bridge between Scotland andproto-Iceland until 24 Mya and until 15 Mya or even 6 Mya be-tween Greenland and proto-Iceland (Grímsson et al., 2007; Denket al., 2010). Because Crangonyx islandicus and Crymostygius thingv-allensis belong to a superfamily of exclusively freshwater amphi-pods, it is possible that their ancestors colonized Iceland throughthis land connection rather than by marine ancestors.

The taxonomy of subterranean organisms based on morpholog-ical information faces several problems. The morphological traitsare generally characterized both by progressive (e.g. elongationof the trunk and/or sensory appendages) and regressive evolution(loss of eyes and pigmentation) (Porter, 2007; Väinölä et al.,2008). These troglomorphic traits are apparently shaped by similarselective pressures (absence of light, low nutrients, low oxygen)encountered in subterranean systems worldwide, which have ledto convergent evolution of morphological traits among differentspecies. The abundance of such traits may hinder the identificationof morphologically informative characters for phylogenetic recon-struction (Englisch et al., 2003; Wiens et al., 2003). Taxonomicrevision using molecular markers is thus especially important forsubterranean species (Fišer et al., 2008). Molecular taxonomy ofgroundwater amphipods has been widely used in the past decade(Englisch et al., 2003; Fišer et al., 2008) and has indicated thatmorphological markers are to a great extent homoplasic. In addi-tion, molecular analysis has recently revealed cryptic diversity(Lefébure et al., 2006a; Kornobis et al., 2010) and taxonomic incon-sistencies among various groundwater amphipod species (Lefébureet al., 2006a).

The aim of this study is to evaluate the taxonomic status of thetwo endemic species Crymostygius thingvallensis and Crangonyxislandicus and to contribute to the phylogenic classification of thecrangonyctoidean species, with a special focus on the family Cran-gonyctidae. In addition we consider the phylogenetic relationshipswith respect to the species distribution (Palearctic or Nearctic) toinfer putative colonization routes, focusing on the colonization ofIceland. The study is based on DNA sequence variation amongthree families (Crymostygidae, Crangonyctidae, Pseud-ocrangonyctidae) of the superfamily Crangonyctoidea and amongfive genera (Crangonyx, Amurocrangonyx, Bactrurus, Synurella andStygobromus) in the family Crangonyctidae for two nuclear genes(18S and 28S ribosomal RNA genes), and two mitochondrial genes(Cytochrome oxydase subunit I COI and 16S ribosomal RNA).

The subunits, 18S and 28S, of ribosomal genes have been exten-sively used as markers for phylogenies at the family level, e.g.within amphipods (Englisch et al., 2003; MacDonald et al., 2005;Lefébure et al., 2006a) and even for deeper phylogenies withincrustaceans (Mallatt and Giribet, 2004; Page et al., 2008). Althoughthe use of RNA genes for phylogenetic purposes has been success-ful, some methodological problems exist (Edgar and Batzoglou,2006). Like the tree building methods (Morrison, 2008), alignment

methods can have profound effects on phylogenies and are of spe-cial concern for the ribosomal genes where insertions and/or dele-tions are commonly observed. Numerous sequence alignmentmethods have been developed recently (Edgar and Batzoglou,2006; Novák et al., 2008; Bradley et al., 2009). We apply differentalignment techniques and weighting schemes to evaluate the im-pact of the alignment and variation along the sequence.

2. Material and methods

2.1. Samples

Twelve amphipod species were collected from 15 locations inNorth America, Europe and Asia (Table 1) and were preserved in96% ethanol. For the construction of phylogenetic trees, sequencesof 18S RNA (14 species) and 28S RNA (7 sp.) genes together withthe mtDNA 16S (4 sp.) and COI (5 sp.) genes (Table A1) from 17species in total belonging to the suborder Gammaridea were ob-tained from Genbank (http://www.ncbi.nlm.nih.gov/genbank/),13 of them belonging to the superfamily Crangonyctoidea. Thetotal sample resulted in 20 different species of the crangonyctoidsin five genera. The number of species sequenced for each genevaried (Table A1). To evaluate the divergence among species andgenera of the Crangonyctidae, comparisons were made with corre-sponding divergence between 18S sequences from the amphipodsuperfamilies Gammaroidea (33 species), Lysianassoidea (3),Talitroidea (10) and Eusiroidea (7), and 28S sequences fromGammaroidea (54), Lysianassoidea (18) and Talitroidea (4),obtained from Genbank.

2.2. Molecular work

DNA was extracted using 6% Chelex 100 (Biorad) from wholespecimens or only a pereopod from bigger specimens. Two regionsof the nuclear genome, the 18S RNA and 28S RNA genes, and two ofthe mitochondrial genome, COI and 16S RNA genes, were amplifiedusing primers summarized in Table 2. PCR amplifications were per-formed in 10 ll, containing 0.15 mM dNTPs, 0.1% Tween, 0.5 lg/llof BSA, 1X Taq buffer, 0.35 lM primers, 0.5 unit of Taq (New Eng-land Biolabs) and 10–100 ng of template. Amplifications were ob-tained with the PCR conditions: 94 �C 4 min, followed by 39cycles of 94 �C 30 s, annealing temperature ranging from 45 to55 �C (depending on species and genes) for 45 s, 72 �C for 1.5 min(1 min for both mt genes), with a final elongation step at 72 �Cfor 6 min. The mtDNA fragments were purified and sequenced di-rectly as in Kornobis et al. (2010). The 18S and 28S PCR productswere cut out of the gel and purified using the Nucleospin ExtractII Kit (Macherey–Nagel), ligated to TOPO vector (TOPO TA CloningKit, Invitrogen) and cloned in chemically competent cells DH5a™-T1R. Plasmids purified using the Nucleospin Plasmid Kit (Mache-rey–Nagel) and Exosap purified PCR products were sequenced inboth directions and with internal primers (Table 2) using ABI Big-Dye Terminator v3.1 (Applied Biosystems). Three to five clones perindividual of the nuclear genes were sequenced and run on an ABIPRISM™ 3100 Genetic Analyzer. Raw sequences were checked andedited using BioEdit 7.0.9.0. (Hall, 1999). Sequences have been sub-mitted to GenBank under accession numbers from HQ286000 toHQ286020 and from HQ286022 to HQ286037.

2.3. Alignment

Four different methods were used to align the RNA sequences:(1) ClustalW2 with default parameters (Larkin et al., 2007). Clu-stalW has been extensively used, but newly developed algorithmsare considered to be more accurate (Morrison, 2009). We chose

528 E. Kornobis et al. / Molecular Phylogenetics and Evolution 58 (2011) 527–539


this method for comparison with other alignment techniques. (2)MAFFT 6 (Katoh et al., 2009), with Q-INS-i strategy in order to takeinto account the secondary structure of the RNA. This method waschosen for the recent implementation of secondary structuresearch (Katoh and Toh, 2008) and its good ratio accuracy/computa-tion time (Edgar and Batzoglou, 2006). (3) FSA 1.15.2, a statisticalalignment software with a limited runtime and which avoids effi-ciently false/positive alignments (Bradley et al., 2009), was usedwith a gap factor of 1 and the Tamura Nei model. (4) RNAsalsa0.8.1 (Stocsits et al., 2009), a recent software which uses secondarystructure information for adjusting and refining the sequencealignment, was used with default parameters and the consensussecondary structure of preliminary alignments of the RNA se-quences as a structural constraint. The COI sequences were alignedby eye using BioEdit.

The mean sequence identity was estimated for each alignmentmethod and for each RNA gene, using the APE package (Paradiset al., 2004) in R (R Development Core Team, 2010). According toHall (2008) and following the results of Kumar and Filipski(2007), an alignment of non-coding DNA that presents a mean se-quence identity of 66% ensures about 50% of alignment accuracy.Variable alignment accuracies higher than 50% are considered tohave little effect on the phylogenetic reconstruction, both forBayesian and Maximum likelihood methods (Ogden and Rosen-berg, 2006).

The optimal model of evolution for each dataset was chosenaccording to the maximum likelihood tree with respect to substitu-tions, transition/transversion ratio, proportion of invariant sitesand the gamma distribution parameters selected with PhyML(Guindon and Gascuel, 2003) and implemented in the R-package

Table 1Species included in the phylogenetic analyses with geographic origin and sampling date. References are given for specimens obtained from genbank. Species from theGammaridae and Megaluropidae families were used as outgroups. ‘‘NA’’ stands for not available.

Family Species Locality Latitude Longitude Reference or year of sampling

Crangonyctidae Amurocrangonyx arsenjevi Khabarovsk, Russia 47.902 135.340 This study (2005)Crangonyctidae Bactrurus brachycaudus St. Louis Co., Missouri, USA NA NA Englisch and Koenemann (2001)Crangonyctidae Bactrurus mucronatus Saline Co., Illinois, USA 37.680 �88.420 Englisch and Koenemann (2001)Crangonyctidae Bactruruspseudo mucronatus Oregon Co., Missouri, USA 36.815 �91.181 Englisch and Koenemann (2001)Crangonyctidae Crangonyx chlebnikovi Perm, Russia 57.446 57.017 This study (2005)Crangonyctidae Crangonyx chlebnikovi Perm, Russia 57.061 57.528 This study (2003)Crangonyctidae Crangonyx floridanus Gainsville, Florida, USA �81.657 30.275 Slothouber Galbreath et al. (2009)Crangonyctidae Crangonyx forbesi St. Louis Co., Missouri, USA 38.616 �90.701 Englisch AND Koenemann (2001)Crangonyctidae Crangonyx islandicus Thingvallavatn, Iceland 64.241 �21.053 This study (2007)Crangonyctidae Crangonyx islandicus Svartarvatn, Iceland 65.469 �17.233 This study (2007)Crangonyctidae Crangonyx islandicus Klapparos Kopasker, Iceland 66.361 �16.400 This study (2008)Crangonyctidae Crangonyx pseudogracilis Lake Charles, Louisiana, USA 30.262 �93.221 Slothouber Galbreath et al. (2009)Crangonyctidae Crangonyx serratus Virginia, USA NA NA MacDonald et al. (2005)Crangonyctidae Crangonyx sp. Barnishee Slough, Tenessee, USA 35.579 �89.963 Slothouber Galbreath et al. (2009)Crangonyctidae Crangonyx subterraneus Biberach, Schwarzwald, Germany 48.326 8.126 Fišer et al. (2008)Crangonyctidae Stygobromus gracilipes (H3970) West Virginia, USA 39.302 �77.850 This study (2002)Crangonyctidae Stygobromus gracilipes (H4070) Virginia, USA 39.014 �78.276 This study (2000)Crangonyctidae Stygobromus mackini USA NA NA Englisch and Koenemann (2001)Crangonyctidae Stygobromus stegerorum Virginia, USA 38.301 �79.255 This study (2000)Crangonyctidae Synurella ambulans Ljubljana, Slovenia 46.050 14.500 This study (2010)Crangonyctidae Synurella dentata Preble Co., Ohio, USA 39.772 �84.722 Englisch et al. (2003)Crangonyctidae Synurella sp. Lake Charles, Louisiana, USA 30.262 �93.221 Slothouber Galbreath et al. (2010)Crymostygiidae Crymostygius thingvallensis Thingvallavatn, Iceland 64.241 �21.053 This study (2007)Gammaridae Gammarus abstrusus Lushan, Sichuan, China 30.280 102.970 Hou et al. (2007)Megaluropidae Megaluropus longimerus Curacao, Karibische See NA NA Englisch et al. (2003)Niphargidae Niphargus fontanus Grundwasser am Ruhrufer, Germany NA NA Englisch and Koenemann (2001)Niphargidae Niphargus kochianus Grundwasser am Ruhrufer, Germany NA NA Englisch et al. (2003)Pseudocrangonyctidae Procrangonyx primoryensis Primory, Russia 47.185 138.743 This study (2003)Pseudocrangonyctidae Procrangonyx primoryensis Primory, Russia 47.256 138.800 This study (2002)Pseudocrangonyctidae Pseudocrangonyx korkishkoorum Primory, Russia 43.100 131.547 This study (2006)

Table 2Oligonucleotides used for PCR and sequencing of the four different genes.

Genes Primer name Primer sequence (50–30) References

18S 18SF CCTAYCTGGTTGATCCTGCCAGT Englisch et al. (2003)18S 700R CGCGGCTGCTGGCACCAGAC Englisch et al. (2003)18S 1500R CATCTAGGGCATCACAGACC Englisch et al. (2003)18S R TAATGATCCTTCCGCAGGTT Englisch et al. (2003)18S F700+ AATTCCAGCTTCAGCAGCAT This study

28S 28SF TTAGTAGGGGCGACCGAACAGGGAT Hou et al. (2007)28S700F AAGACGCGATAACCAGCCCACCA Hou et al. (2007)28S1000R GACCGATGGGCTTGGACTTTACACC Hou et al. (2007)28SR GTCTTTCGCCCCTATGCCCAACTG Hou et al. (2007)28Sa TTGGCGACCCGCAATTTAAGCAT Cristescu and Hebert (2005)28Sb CCTGAGGGAAACTTCGGAGGGAAC Cristescu and Hebert (2005)

COI LCO1490 GGTCAACAAATCATAAAGATATTGG Folmer et al. (1994)HCO2198 TAAACTTCAGGGTGACCAAAAAATCA Folmer et al. (1994)

16S 16Stf GGTAWHYTRACYGTGCTAAG Macdonald et al. (2005)16Sbr CCGGTTTGAACTCAGATCATGT Palumbi et al. (1991)

E. Kornobis et al. / Molecular Phylogenetics and Evolution 58 (2011) 527–539 529


APE. The Akaike Information Criterion (AIC) (Akaike, 1974) wasused to detect the model which best fitted the data, as recom-mended by Posada and Buckley (2004).

Nuclear datasets were partitioned in stem and loop regionsaccording to the consensus structure obtained in RNAalifold(Bernhart et al., 2008) for each alignment method. Variation,including the number of phylogenetically informative sites andgaps at different regions, outside hairpins, in stems and at internaland terminal loops, was scored for the four different methods.Mountain plots were used to compare the secondary structuresobtained by RNAalifold.

2.4. Phylogenetic analysis

Phylogenetic trees were constructed separately for each genefragment as the available number of taxa for the different genesvaried, and also based on combined datasets. The analysis basedon different genes may provide a stronger support for the observedclusters of species, or indicate some gene-specific evolutionarydivergence.

Search for optimal trees was carried out with MrBayes v3.1.2(Ronquist and Huelsenbeck, 2003) and PhyML (Guindon andGascuel, 2003) using the pre-specified evolutionary model (withthe lowest AIC value). MrBayes is particularly suited for our analy-sis since it allows partitioning of the data according to secondarystructure and implementation of indels as morphological charac-ters. In MrBayes, the parameters of the selected model were opti-mized during searches as recommended by Ronquist andHuelsenbeck (2003), running two independent MCMC with onecold and three hot chain searches during 2 � 106 generations forthe 16S and 4 � 106 generations for the other genes, sampled every100 generations. In most cases a 10% ‘‘burn-in’’ was estimated suf-ficient after checking graphically for convergence to stable �ln Lscores with Tracer (Rambaut and Drummond, 2003). We comparedposterior probabilities of the splits between runs, as well as duringruns using default parameters in the program AWTY (Nylanderet al., 2008). High correlation was observed between posteriorprobabilities of the splits between runs (Pearson correlation test:r > 0.98, p < 2.2 � 10�16) and no particular trend diagnosing lackof convergence was observed (see Supplementary materialFig. A1). The secondary structure and the indels were taken intoaccount for the nuclear genes with commands implemented inMrBayes. The doublet model of evolution was applied to the stemsin order to take into account compensatory mutations. Two differ-ent partitions were used in order to test the sensitivity of the treetopology to the partitioning: runs were performed considering fourdifferent partitions (outside hairpins, stems, internal and terminalloops) and for just two partitions (stems and loops). Indels werecoded as binary characters and included as a morphological data-set. PhyML does not provide these options. Branch supports wereassessed by using the approximate likelihood ratio test (aLRT) inPhyML, applying the non-parametric method based on a Shimoda-ira–Hasegawa-like procedure (Anisimova and Gascuel, 2006). BothMrBayes and PhyML trees were rooted by the most closely relatedtaxa available in Genbank outside the superfamily: the 18S withMegaluropus longimerus and the 28S with Gammarus abstrusus(see Table 1) (Englisch et al., 2003).

The secondary structure of the 16S fragment was not taken intoaccount in the phylogenetic reconstruction, due to the short se-quences. A codon model of evolution was applied to the completeCOI dataset in MrBayes, which allows for different rates for synon-ymous and non-synonymous substitutions as well as on 0-folddegenerated codon positions in order to avoid phylogenetic signalsaturation. The COI and 16S phylogenies were rooted by G. abstru-sus (see Table 1), as used in previous amphipod phylogenies (Eng-lisch et al., 2003). In addition to the phylogenies computed for each

gene, we reconstructed trees following the same procedure as de-scribed above, separately with two combined datasets: one withthe nuclear genes (18S and 28S), and another with the mitochon-drial genes (COI and 16S).

The results from the different alignment methods were com-pared by considering the variation in number of trees obtainedwith MrBayes (Ronquist and Huelsenbeck, 2003) and their likeli-hoods using Tracer (Rambaut and Drummond, 2003), then summa-rized with the mean values and high posterior density interval(HPD). As the overall likelihood value depends on the number ofbases compared, the best alignment method was selected afteradjusting the log likelihoods of the trees using ANCOVA, with thenumber of bases in the alignment as a covariate.

2.5. Variation within Crangonyctoidea compared with other amphipodsuperfamilies

Pairwise evolutionary distances between 18S and 28S se-quences, aligned with ClustalW2, were calculated using the APEpackage with the evolutionary model selected from the PhyMLanalysis. The distances were grouped at different taxonomic levels(within and between genera, within and between families) in orderto assess both the taxonomic status of the Icelandic species and toevaluate the classification within the family Crangonyctidae. Forcomparing divergence within this family with divergence withinother amphipod families, we computed distances at the same tax-onomic levels for four other amphipod superfamilies available forthe 18S in Genbank (Gammaroidea, Lysianassoidea, Talitroidea,Eusiroidea) and three superfamilies for the 28S (Gammaroidea,Lysianassoidea and Talitroidea).

3. Results

3.1. Sequences

Sequences of the 18S gene ranged in length from 2310 bp to2440 bp (Genebank accession numbers: HQ286012–HQ286018)while the 28S sequences (HQ286019–HQ286020 and HQ286022–HQ286024) ranged from 850 to 1440 bp. The length of the se-quences retrieved from Genbank varied from 1150 to 2500 bp forthe 18S and from 820 to 1290 bp for the 28S sequences. Crymosty-gius thingvallensis had the longest fragments among the Crang-onyctoidea sequences for both 18S and 28S genes, reflecting itsunique phylogenetic status within the superfamily. The variationin length is due to several indels, ranging from 1 to 53 bases. Thegenetic distances observed between clones from the same individ-ual never exceeded 0.9%, irrespective of the alignment method andthe nuclear gene. We therefore chose a single clone from each indi-vidual as representative of each species. The 16S sequences rangedfrom 362 to 411 bp (Genebank accession numbers: HQ286000–HQ286011). No indels and no stop codons were observed in the582 bp long COI alignment (HQ286025–HQ286037).

3.2. Alignment and secondary structure

Different alignment methods resulted in different alignments ofsequences as reflected by the overall lengths and the number ofphylogenetic informative sites for each RNA gene dataset (Table 3).The number of phylogenetically informative sites and sites wheregaps were introduced varied among the alignment methods. Thenumber of gaps introduced was smallest with ClustalW2 and larg-est with FSA, for 18S: 532 (ClustalW2), 724 (MAFFT), 1277 (FSA)and 664 (RNAsalsa), and for 28S: 446 (ClustalW2), 474 (MAFFT),1094 (FSA) and 554 (RNAsalsa). Variation among species dependson the regions of secondary structure. Stems were generally least



variable: in 18S, the proportion of variable sites ranged from 0.055to 0.072, and the largest differences were found among the loops(0.116–0.175). The difference in number of informative sites be-tween four regions (outside hairpins, stems, internal and terminalloops) was significant, independent of the alignment (Fisher exacttests: P < 3.44 � 10�6), for both 18S and 28S datasets.

The mean sequence identity for all nuclear gene alignmentswas high, ranging from 71% to 95%, and is substantially higherthan the percentage needed to ensure the alignment accuracyrequired for phylogenetic reconstruction (66%, see Hall, 2008;Kumar and Filipski, 2007). The 16S mean sequence identity val-ues are more marginal and ranged from 60% to 70%; the max-imum value was obtained with the FSA alignment. Based on theAIC, the best-fit model, independently chosen for all genes, wasthe GTR + I + G model (Lanave et al., 1984). The TN93 model(Tamura and Nei, 1993), the most similar substitution modelto GTR and implemented in R, was used for pairwise distancecalculations.

Similar consensus secondary structures were obtained with thedifferent alignment methods of the 18S and 28S genes as shown bythe mountain plots (Fig. 1), except for the first part of the RNAsalsaalignment of the 18S gene which exhibits much shorter stem struc-tures than those observed with other alignment methods. The FSAalignments induced a consensus secondary structure with rela-tively longer loops for both genes, along with shorter stem struc-tures for the first part of the 18S. No difference was observedbetween phylogenetic trees constructed considering the four parti-tions based on the secondary structure (outside hairpins, stems,internal and terminal loops) or two partitions (stems and loops)in MrBayes.

The sample of most probable trees after ‘‘burn-in’’ in Bayesiananalysis varied considerably in size between the datasets alignedwith different methods. Samples of trees based on FSA and Clu-stalW2 were dominated by few, highly probable, different topol-ogies whereas MAFFT-aligned datasets produced a sample ofnumerous different topologies with low posterior probabilities.RNAsalsa showed intermediate numbers of most probable trees.The log likelihood of the trees based on the different alignmentsshowed clear patterns which depended on the length of the se-quence alignments (ANCOVA, adjusted R2 = 0.998) (Fig. 2). TheFSA alignments produced significantly the most likely trees inall cases (Table 3 and Fig. 2). FSA is a conservative method, avoid-ing stringently false homologies, and consequently shows align-ments with less informative sites (Table 3). For these reasonswe chose to present the trees based on FSA alignment techniquesand discuss the differences obtained with the other alignmentmethods.

The COI trees reconstructed with the different datasets weresimilar in topology. To avoid a misleading phylogenetic signaldue to saturation, we choose the conservative strategy to presentthe tree based on 0-fold degenerated sites.

3.3. Phylogenetic relationships among species of Crangonyctoidea,based on nuclear genes

Species belonging to the Crangonyctidae form a well-supportedmonophyletic group for both 18S and 28S genes (Fig. 3), indepen-dently of the alignment method (posterior probability: pp > 0.95).Crymostygius thingvallensis is clearly differentiated from both Nip-hargidae and Crangonyctidae based on both the 18S and 28S phy-logenies (Fig. 3). The 28S dataset aligned with ClustalW2 and the18S dataset aligned with RNAsalsa are the only datasets supportingan early divergence of the Crymostygidae from Niphargidae andCrangonyctidae, which cluster together (Table 4). All other phylog-enies clustered Crymostygius thingvallensis together with the Cran-gonyctidae. The family Crangonyctidae is composed of twomonophyletic groups (Fig. 3 and Table 4). One group is formedby the genera Bactrurus and Stygobromus clustering together withthe European Synurella ambulans and species of Crangonyx fromEurasia (i.e. Crangonyx chlebnikovi for the 18S and both C. chlebnik-ovi and Crangonyx subterraneus for the 28S). The other group iscomposed of species of Crangonyx from North America and Iceland(in both phylogenies) and the species of Synurella from NorthAmerica (18S phylogeny only). Independent from the alignmentmethod and the gene studied, all phylogenies support that Crang-onyx is clearly polyphyletic (Fig. 3 and Table 4). For example, spe-cies of Crangonyx from Eurasia (C. chlebnikovi and C. subterraneus)are more closely related to Stygobromus and Bactrurus (pp > 0.95)than to species of Crangonyx from North America and Iceland(Fig. 3a). Similarly, S. ambulans from Europe is more closely relatedto species of Stygobromus and Bacturus (pp > 0.95) than to speciesof Synurella species from North America (Synurella sp. and Synurelladentata), based on the 18S dataset. This dataset also supports agroup formed by Amurocrangonyx arsenjevi, Bactrurus, Stygobromus,and the species of Crangonyx and Synurella from Eurasia (pp > 0.95,see Fig. 3a).

The 18S phylogenies also show an early divergence of Crangonyxislandicus from other Crangonyx from North America, which aremore closely related to Synurella sp. and S. dentata (Fig. 3a). This pat-tern is observed, though less frequently, for 28S phylogenies andcombined datasets, depending on the alignment and the phyloge-netic methods used (Table 4). Moreover, the divergence observedbetween Crangonyx islandicus and other Crangonyx greatly exceedthe one observed between species of the genera Stygobromus andBactrurus or even between those species and A. arsenjevi (Fig. 3a).Conversely, phylogenies based on the 28S dataset, not encompassingspecies of Synurella from North America, support the monophyly ofthe group formed by species of Crangonyx from North America andCrangonyx islandicus (pp > 0.95, see Fig. 3b). The species of Crangonyxfrom North America (Crangonyx forbesi, Crangonyx sp., Crangonyxpseudogracilis and Crangonyx floridanus) only clustered together inthe phylogeny based on MAFFT alignment for the 18S gene. None-theless, the early divergence of C. forbesi from the other species isonly supported by informative indels. All 18S phylogenies con-structed without indels as morphological characters strongly sup-ported the monophyly of the North American species of Crangonyx(pp > 0.95, data not shown).

The different alignment methods result in slight differences intopology, which are mostly in the external nodes of the trees,grouping together species of Stygobromus, S. ambulans, C. chlebnik-ovi and C. subterraneus. These changes appeared even for nodeswhich were highly supported in the phylogeny based on FSAalignment. The monophyly of the genus Bactrurus, based on 18S,

Table 3Comparison of the likelihoods between genes and alignment methods. The tablepresents the results from the Bayesian analysis for the four alignment methods. ln L:the log likelihoods of the Bayesian trees, HPD: high posterior density interval of the�log likelihoods, bp: number of base pair in the alignment, IS: number ofphylogenetically informative sites.

Methods Genes -lnL 95%HPD bp IS

18S 12020 12010-12040 2629 439ClustalW 28S 9859 9849-9870 1561 518

16S 4592 4582-4602 424 26218S 12140 12130-12150 3224 352

FSA 28S 9984 9974-9995 2115 42716S 4459 4440-4471 689 24918S 11350 11340-11360 2840 426

MAFFT 28S 9588 9578-9598 1660 48116S 4553 4543-4564 431 25518S 11910 11900-11920 2663 345

RNAsalsa 28S 10040 10030-10050 1595 46616S 4299 4290-4309 455 260



is supported by all alignments whereas Stygobromus is only mono-phyletic in the tree based on ClustalW2 alignment (Table 4). Themonophyly of Stygobromus was never supported by the 28S phy-logenies, independent of the alignment (Table 4).

The tree based on the 18S–28S combined datasets shows a sim-ilar topology. The changes observed are due to the positioning ofCrangonyx islandicus, C. forbesi, C. chlebnikovi and S. ambulans, as

observed above with different alignment techniques (Fig. A2).Trees based on the combined dataset and aligned with MAFFTand FSA showed the same topology, different from the one basedon ClustalW2 alignment. They highly support an early divergenceof Crangonyx islandicus from C. forbesi and C. pseudogracilis(pp = 0.81 for FSA and pp = 1 for MAFFT). The species of Stygobro-mus clustered together and C. chlebnikovi clustered with S. ambu-lans, but both with less support (0.51 < pp < 0.92).

3.4. Phylogenetic relationships among species of Crangonyctoidea,based on mitochondrial genes

The phylogenies based on the mtDNA fragments of COI and16S genes are less well supported, probably due to the short se-quence lengths and signal saturation (Fig. 4). Nonetheless, COIand 16S phylogenies support the polyphyly of the genus Crang-onyx. The phylogenetic relationships at a deeper level are lessclear, due to the faster rate of evolution of these two genes.Due to the particularly weak resolution of the COI gene for thesephylogenetic relationships, we choose not to present it in Table 4.In the 16S phylogeny, the cluster with Crymostygius thingvallensisand the two species of Pseudocrangonyctidae (Pseudocrangonyxkorkishkoorum and Procrangonyx primoryensis) from Eastern Asiais relatively well supported (supported by all alignment tech-niques with 0.61 < pp < 1). This cluster could not be verified bythe nuclear gene phylogenies due to difficulties in amplifying se-quences for P. korkishkoorum and P. primoryensis with the prim-ers used. The 16S dataset supports, though weakly (pp = 0.77),the clustering of Crangonyx islandicus with the North Americanspecies Crangonyx serratus. As obtained with the 18S, 28S and16S datasets, C. chlebnikovi and S. ambulans appeared more clo-sely related to species of Stygobromus than to other species ofCrangonyx from North America (Figs. 3 and 4a).

The COI phylogeny (Fig. 4b) shows poor resolution for theinternal nodes of the tree. The species of Crangonyx from NorthAmerica (C. pseudogracilis, Crangonyx sp. and C. floridanus) clus-ter together (pp = 1) but no conclusion can be drawn from this

0 500 1000 1500 2000 2500

020

4060

position (bp)

heig

ht

ClustalW2FSAMAFFTRNAsalsa

0 500 1000 1500

020

40

position (bp)

heig

ht

ClustalW2FSAMAFFTRNAsalsa

(a)

(b)

Fig. 1. Mountain plots of the secondary structure obtained with the four alignment methods, for the 18S genes (a) and 28S genes (b). The height of the lines reflects thesecondary structure: the higher the peaks, the longer the stem. A horizontal line presents loop regions.

6.0 6.5 7.0 7.5 8.0

−120

00−1

0000

−800

0−6

000

−400

0

Log of number of bases

Mea

n lo

g lik

elih

ood

ClustalW2FSAMafftRNAsalsa

Fig. 2. Evaluation of the likelihoods for trees based on the different genes andalignment methods. Mean log likelihoods of the trees obtained with MrBayes arepresented with respect to the logarithm of the length of the aligned sequences inbase pairs. Lines present the prediction from analysis of covariance (ANCOVA)(adjusted R2 = 0.998). Both linear coefficients (intercept and slope) for the FSAalignment were significantly different from the estimates based on the otheralignment methods (p < 0.008).



dataset about their phylogenetic relationship with Crangonyxislandicus. Synurella sp., an inhabitant from North America, isclosely related to A. arsenjevi and the North American speciesof Crangonyx (pp = 0.86). Crymostygius thingvallensis did not clus-ter with any of the species integrated in the COI phylogeny. C.chlebnikovi from the Ural Mountains clustered with P. primory-ensis (pp = 1) from Eurasia, which belongs to the familyPseudocrangonyctidae.

The tree based on 16S-COI combined dataset shows an earlydivergence of Crangonyx islandicus and C. pseudogracilis from thecluster formed by the rest of the species (Fig. A3). Stygobromus gra-cilipes and S. ambulans are clustered together (pp = 0.91). Crymosty-gius thingvallensis is more closely related (pp = 0.98) to the clusterformed by C. chlebnikovi and P. primoryensis (pp = 1).

3.5. Molecular divergence

The genetic divergence observed for both 18S and 28S geneswithin the crangonyctoid genera is exceptionally high comparedto available data from other amphipod genera (Fig. 5). This diver-gence is considerably reduced when Crangonyx islandicus, C. chleb-nikovi, C. subterraneus and S. ambulans are omitted from thecomparison, in which case the divergence observed betweenCrangonyx islandicus and other species of Crangonyx is more similarto the comparison between genera than within. The variation be-tween Crymostygius thingvallensis and species from other familiesof Crangonyctoidea is comparable to the one observed betweenfamilies (Fig. 5).

Divergence among Crangonyx islandicus populations for both18S and 28S sequences was much less than observed for themtDNA genes, COI and 16S, as reported by Kornobis et al. (2010)(data not shown). Interestingly, the only three phylogeneticallyinformative sites found along an 800 bp fragment of the 28S sup-port an earlier divergence of the populations in northern Icelandfrom populations in southern Iceland.

4. Discussion

The different methods of alignment and phylogenetic recon-struction applied in this study allow several main conclusionsto be made: (1) The taxonomic status of Crymostygius thingvall-ensis as a new family is confirmed. (2) Crangonyx islandicusmay represent a new genus. (3) As commonly observed amongsubterranean species, discrepancies appear between molecularand morphological taxonomy, which may have resulted fromconvergent evolution in morphological traits. Several species ofCrangonyctidae need taxonomical revision. (4) Phylogeographicpatterns indicate colonization of Iceland from the Nearctic viaGreenland.

4.1. Molecular and morphological taxonomy

The tree topologies varied less among phylogenetic reconstruc-tion methods than among alignment methods, which showed onlyminor differences, particularly in external branches, even for well-supported nodes. This emphasizes the need in future studies onRNA phylogenies to consider different alignment methods, as com-monly done when results from different tree reconstruction meth-ods are compared.

The apparent monophyly of species in the family Cran-gonyctidae is strongly supported by the 18S and 28S nucleargenes. The classification of Crymostygidae as a monotypic familywithin the superfamily Crangonyctoidea (Kristjánsson andSvavarsson, 2004) is further supported by the phylogeniespresented in this study and it appears as a sister family to theCrangonyctidae. Its status is also supported by the genetic dis-tances observed between Crymostygius thingvallensis and thetwo other families of the Crangonyctoidea, which are similar togenetic distances observed between families among otheramphipod superfamilies. A further investigation is needed toassess their phylogenetic relationships with species of

(a) (b)

Fig. 3. Bayesian phylogenetic tree of the crangonyctid species, together with Crymostygius thingvallensis and two Niphargus species, based on 18S (a) and 28S (b) sequencesaligned with FSA. Posterior probability values are displayed in the box. The tree is rooted by Megaluropus longimerus for the 18S and by Gammarus abstrusus for the 28S;outgroups are not shown. Stars and squares correspond to Palearctic and Nearctic species respectively. Branches are drawn to scale, with the bar indicating 0.05 expectedchanges per site.



Tabl

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phyl

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ular

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pute

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num

ber

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nttr

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esis

Clu

stal

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FSA

MA

FFT

RN

Asa

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Tota

l%

#tr

ees

18S

28S

16S

28S–

18S

18S

28S

16S

28S–

18S

18S

28S

16S

28S–

18S

18S

28S

16S

28S–

18S

MrB

ayes

Cran

gony

xm

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y0

00

00

00

00

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6316

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00

11

10

11

10

10

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110

6316

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NA

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100

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14

2516

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5616

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016

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410

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4



(a) (b)

Fig. 4. Bayesian phylogenetic tree of the crangonyctid species, together with Crymostygius thingvallensis, based on 16S sequences aligned with FSA including two Niphargusspecies (a) and based on COI sequences (b). Posterior probability values are displayed in the box. The trees are rooted by Gammarus abstrusus (not shown on the figure). Starsand squares correspond to Palearctic and Nearctic species respectively. Branches are drawn to scale, with the bar indicating 0.05 expected changes per site.

1 2 3 4 5 6 7

0.00

0.05

0.10

0.15

(a)

1 2 3 4 5 6 7

0.1

0.2

0.3

0.4

(b)

Fig. 5. Comparison of genetic distances within Crangonyctoidea with distances within and among other amphipod families. Genetic distances from ClustalW2 aligned datasetcompared among various taxonomic groups for 18S (a) and 28S (b). #1: within genera, #2: within genera without Crangonyx islandicus, S. ambulans, Crangonyx chlebnikovi, #3:between genera, #4: between genera without Crangonyx islandicus, S. ambulans, Crangonyx chlebnikovi, #5: between Crangonyx islandicus other Crangonyx, #6: betweenfamilies without Crymostygius thingvallensis, #7: between Crymostygius thingvallensis and other families. Horizontal lines represent the maximum genetic distance observedfor other amphipod families: broken line: between species within genera, dotted line: between genera, mixed: between families.



Pseudocrangonyctidae, which is described as a closely alliedsister family to the Crangonyctidae (Holsinger, 1994). A furtherstudy is needed to assess the monophyly and the phylogeneticrelationships among families of the superfamily Crangonyctoidea,including for instance other species of Crangonyctidae, (e.g.Crangonyx africanus) and members of the families Sternophysin-gidae, Paramelitidae and Neoniphargidae from the SouthernHemisphere.

The observed phylogenies in this study suggest that speciesof Crangonyctidae need taxonomic revisions in order to restorethe monophyly of their respective genera. Phylogenies based onall genes, except for some 28S phylogenies, cluster Crangonyxislandicus separately from the genus Crangonyx. Svavarsson andKristjánsson (2006) hesitated to describe Crangonyx islandicusas a new genus based on morphological differences. The ab-sence of sternal gills, lateral lobe of head truncated, pereopod7 longest, uropod 3 short with inner ramus naked, and thepresence of only a single strong spine on the inner plate ofthe maxilliped are a unique combination of morphological traitswhich distinguishes Crangonyx islandicus from the other speciesof the genus Crangonyx (Svavarsson and Kristjánsson, 2006). Ourmolecular data supports that these characters might be givenmore weight in the phylogenetic classification and leads us toenvisage Crangonyx islandicus as a new genus. The similar ge-netic distances observed between genera within Crangonyctidaeand between Crangonyx islandicus and other Crangonyx also sup-port the new generic status of Crangonyx islandicus. Phylogeniesthat include more species of Crangonyx and Synurella fromNorth America may assist in lending more support to this tax-onomic revision.

Mitochondrial DNA variation in Crangonyx islandicus popula-tions revealed extensive diversity, suggesting a cryptic speciescomplex. Maximum divergence was observed for a populationin northeastern Iceland which had diverged from the other pop-ulations in Iceland about 5 millions years ago (Kornobis et al.,2010). Much less variation is observed with the conservativeRNA genes. The few segregating sites in the 28S gene groupnortheastern populations with populations from northernIceland.

Our molecular study supports the monophyly of the genusBactrurus. In accordance with previous classification based onmorphological data, C. floridanus and C. pseudogracilis appearedclosely related to each other, based on the 18S and COI phylog-enies (Zhang and Holsinger, 2003). The 18S marker supports anearly divergence of C. forbesi from those two species, as de-scribed by the morphological analysis of Zhang and Holsinger(2003).

Both Crangonyx and Synurella are polyphyletic. The monophylyof the genus Stygobromus is poorly supported by the moleculardata. Consequently, revision of the classification within the Cran-gonyctidae is needed as well as the morphological and meristictaxonomic criteria previously used, which appear to be homopla-sic. Genetic distances between genera within Crangonyctidae arelarger than distances between genera in several other families ofamphipods, suggesting that morphological differences among taxaof the Crangonyctidae are subtle.

The classification of C. chlebnikovi based on morphology is un-der revision (Sidorov et al., unpublished). The presence of a laterallobe on the head, which is rather prominent and narrowlyrounded anteriorly with inferior antennal sinus, is a morphologi-cal characteristic common to the genus Bactrurus but not to thegenus Crangonyx (Sidorov et al., 2010). P. primoryensis and P. kor-kishkoorum, in the family Pseudocrangonyctidae, are supported asclosely related species by the 16S gene phylogenies. Nonetheless,more data is necessary to reach a conclusion about their phyloge-netic relationships with the Crangonyctidae, especially if Proc-

rangonyx and Pseudocrangonyx are included within theCrangonyctidae (Holsinger, 1994). As described by Sidorov andHolsinger (2007), Amurocrangonyx is a apparent sister genus ofCrangonyx; this phylogenetic relation is partially supported bythe 18S tree in our analysis.

Holsinger (1977) presented two opposing theories to explainthe evolution of the crangonyctid genera: (1) One lineage evolvedinto Crangonyx and Synurella and another into Stygobromus andBactrurus. (2) One lineage evolved into Bactrurus and Crangonyxand another into Synurella and Stygobromus. The 18S phylogeniessupport, though weakly, an early divergence of Synurella andCrangonyx from the Bactrurus and Stygobromus groups. The samephylogenetic relationships between these four genera have alreadybeen observed by Holsinger (1994), based on morphological syna-pomorphies. Nonetheless, the polyphyly observed for both Crang-onyx and Synurella encourage a revision of the classification ofCrangonyx, Synurella and Stygobromus before reaching a final con-clusion on the evolution of the crangonyctid genera. More specifi-cally, Crangonyx islandicus, C. chlebnikovi, S. ambulans and S. dentataneed taxonomic revision in order to restore the monophyly of thegenera.

The group formed by Stygobromus, Bactrurus, Amurocrangonyxand the species of Crangonyx and Synurella from Eurasia includedin this study are almost exclusively eyeless (Holsinger, 1977;Sidorov and Holsinger, 2007). The exception is the presence ofvestigial eyes composed of a cluster of ommatidia in S. ambulans(see Karaman, 1974; Arbaciauskas, 2008). Conversely, most spe-cies of Crangonyx from North America (Holsinger, 1977), S. den-tata (Hubricht, 1943) and Crangonyx islandicus (Svavarsson andKristjánsson, 2006) possess vestigial eyes. The two groups arereciprocally monophyletic and as vestigial eyes are found inthe more distantly related Crymostygius thingvallensis (Kristjánssonand Svavarsson, 2004), it suggests that vestigial eyes is an ances-tral trait. The phylogeny reflects that the evolutionary trend to-ward the loss of the eyes might have appeared more rapidly inthe group formed by Crangonyx and Synurella from Eurasia andin Bacturus and Stygobromus from North America than in Crang-onyx and Synurella from North America. A further study of thephylogeographical patterns among species of Crangonyctidae isneeded to get a better understanding of regression of the eyestructure.

4.2. Geographical patterns and the colonization of Iceland

Species both from Crangonyx and Synurella showed genetic clus-tering that did not support the previous morphological taxonomybut which can instead be explained by their geographical distribu-tion. C. chlebnikovi and C. subterraneus, both found in the Palearcticregion, showed clear divergence from species of Crangonyx fromthe Nearctic region. Synurella exhibits the same geographical pat-tern, with S. ambulans in Europe being more closely related to spe-cies of Crangonyx in the Palearctic than to species of Synurella fromthe Nearctic. This phylogeographic pattern and the monophyly ofthe crangonyctid species tend to support a hypothesis that sug-gests the occurrence of an ancestor to the family that was wide-spread in Laurasia before its breakup in Late Paleocene (about60 Mya).

Considering that the closest relatives of Crangonyx islandicuswere found to be Nearctic species, as well as the high diversitywithin the Nearctic species of Crangonyctidae and the geologicalage of Iceland, we conclude that Crangonyx islandicus colonizedIceland from Greenland as hypothesized by Kristjánsson andSvavarsson (2007). Their ancestor either followed proto-Icelandas it departed from Greenland or colonized Iceland via theGreenland–Iceland land bridge. An alternative colonization routealong the Scotland-Faeroe-Iceland ridge is not supported. Since



Crangonyx islandicus and Crymostygius thingvallensis belong to thefreshwater superfamily Crangonyctoidea, their respective ances-tors are more likely to have inhabited freshwater as well. Wecan therefore argue that freshwater contacts occurred betweenIceland and Greenland. A tolerance to limited salinity (about 1/100 of sea salinity) of a related species, C. pseudogracilis (seeSlothouber Galbreath et al., 2010) indicates that there mightthough have been a possible route of colonization when the landbridge was reduced to a chain of islands. Two Synurella species(S. jakutana and S. levanidovorum) from the coast of the OkhotskSea are known to inhabit brackish waters (Sidorov, unpublished).Resistance of the Icelandic species to salinity has not beentested.

Past colonization events from Greenland to Iceland throughthe Greenland–Iceland land bridge have been inferred from fos-sil records for mammals (McKenna, 1983) and plant species(Grímsson et al., 2007; Denk et al., 2010). As the ice sheet cov-ered Iceland repeatedly during the glacial period of the Ice Age,most species which colonized Iceland before the Ice Age be-came extinct. The only species which might have survived inrefugia below the glacier were the groundwater species, suchas amphipods, but Crangonyx islandicus is the only known spe-cies which has survived glaciation in Iceland (Kornobis et al.,2010). Crangonyx islandicus and Crymostygius thingvallensis arethus probably the oldest known inhabitants of Iceland and theirearly colonization may predate the oldest rock formation in Ice-land, 16 Mya (Moorbath et al., 1968). The oldest rock forma-tions of the Iceland hotspot date back to 40 Mya and arefound in the Faeroe Islands and East Greenland. As Icelanddrifted apart at the tectonic boundaries, only the youngest partof Iceland remained above sea level around the hotspot beneathIceland. Terrestrial organisms present on Iceland would need tocolonize the younger parts from the older ones before being

submerged by the sea. Similar examples of organisms predatingthe age of their habitat are known from the Galapagos archipel-ago, e.g. among iguanas (Rassmann, 1997) and from the Hawai-ian islands, where organisms such as Drosophila and Hawaiianhoney creepers colonized new islands as the older islands dis-appeared by erosion and subduction due to plate tectonics(see Fleischer et al., 1998).

The records of freshwater amphipods from other formerly glaci-ated areas are rare and no such species or fossil has been describedfrom Greenland. In cases where freshwater amphipods have beenfound in formerly glaciated areas, post-glacial colonization cannotbe ruled out, e.g. in the Alps (Lefébure et al., 2006b). Fissure areasand geothermal energy have been hypothesized as providing suit-able refugia during the Ice Age for subterranean species in Iceland(Kornobis et al., 2010). The absence of such geological regions else-where in formerly glaciated areas of the Northern Hemispheremight explain the limited distribution of groundwater amphipodsin these regions.

Acknowledgments

We acknowledge Cene Fišer for providing valuable samplesof Synurella ambulans. We want to thank Virginia Escuderoand Jaume Cuart Castell for laboratory assistance. We aregrateful to the University of Iceland Research Fund and theIcelandic Research Council (Rannís) for financial support. Twoanonymous reviewers made comments that improved themanuscript.

Appendix A

See Table A1.

Table A1Genbank accession numbers of the sequences for each gene used for tree construction.

This Study 18S 28S 16S COI

Amurocrangonyx arsenjevi HQ286015 HQ286007 HQ286025Crangonyx chlebnikovi HQ286017 HQ286023 HQ286001 HQ286026Crangonyx islandicus F HQ286006 HQ286030Crangonyx islandicus F HQ286005 HQ286029Crangonyx islandicus S HQ286013 HQ286020 HQ286004 HQ286027Crymostygius thingvallensis HQ286012 HQ286019 HQ286009 HQ286032Procrangonyx primoryensis HQ286011 HQ286033Pseudocrangonyx korkishkoorum HQ286010Stygobromus gracilipes 1 HQ286016 HQ286022 HQ286002 HQ286034Stygobromus gracilipes 2 HQ286003 HQ286035Stygobromus stegerorum HQ286014 HQ286024 HQ286008 HQ286036Synurella ambulans HQ286018 HQ286000 HQ286037

From GenbankBactrurus brachycaudus AF202979Bactrurus mucronatus AF202978Bactrurus pseudomucronatus AF202985Crangonyx floridanus AJ966709 AJ968911Crangonyx forbesi AF202980 EU693287Crangonyx pseudogracilis AJ966705 EF582940 EF582845 AJ968900Crangonyx serratus AY926703Crangonyx sp. AJ966706 AY529053Crangonyx subterraneus EU693288Gammarus abstrusus EF582903 EF582950 EF582855 EF570304Megaluropus longimerus DQ378035Niphargus fontanus AF202981 EF617304 EF028464Niphargus kochianus AF419221 EU693308Stygobromus mackini DQ377995Synurella ambulans EF617236Synurella dentata AF419233Synurella sp. AJ966706 AJ968913



Appendix B. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2010.12.010.

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