Fish pathogens near the Arctic Circle: molecular, morphological and ecological evidence for unexpected diversity of Diplostomum (Digenea: diplostomidae) in Iceland

International Journal for Parasitology 44 (2014) 703–715

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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara

Fish pathogens near the Arctic Circle: molecular, morphological and ecological evidence for unexpected diversity of Diplostomum (Digenea: diplostomidae) in Iceland q Isabel Blasco-Costa a, Anna Falty´nková a, Simona Georgieva a,b, Karl Skírnisson c, Tomáš Scholz a, Aneta Kostadinova a,⇑ Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 Cˇeské Budeˇjovice, Czech Republic Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 Cˇeské Budeˇjovice, Czech Republic c Laboratory of Parasitology, Institute for Experimental Pathology, University of Iceland, Keldur, 112 Reykjavik, Iceland a

b

a r t i c l e

i n f o

Article history: Received 1 January 2014 Received in revised form 21 March 2014 Accepted 16 April 2014 Available online 11 June 2014 Keywords: Integrative taxonomy Fish pathogens Diplostomum cox1 ITS Sub-Arctic

a b s t r a c t Host–parasite systems at high latitudes are promising model systems for detecting and predicting the impact of accelerated environmental change. A major challenge is the lack of baselines for the diversity and distribution of parasites in Arctic wildlife, especially in the freshwater environment. Here we present the first known estimates of the species diversity and host associations of Diplostomum spp. in sub-Arctic freshwater ecosystems of the Palaearctic. Our analyses integrating different analytical approaches, phylogenies based on mitochondrial and nuclear DNA, estimates of genetic divergence, character-based barcoding, morphological examination, precise detection of microhabitat specialisation and host use, led to the discovery of one described and five putative new species that complete their life-cycles within a fairly narrow geographic area in Iceland. This increases the species richness of Diplostomum in Iceland by 200% and raises the number of molecularly characterised species from the Palaearctic to 17 species. Our results suggest that the diversity of Diplostomum spp. is underestimated globally in the high latitude ecosystems and call for a cautionary approach to pathogen identification in developing the much needed baselines of pathogen diversity that may help detect effects of climate change in the freshwater environment of the sub-Arctic. Ó 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The ecosystems in the Earth’s northern circumpolar regions, typically characterised as simple, low diversity systems with short trophic linkages, few pathogens and limited capacity for adaptation to environmental change (Hoberg et al., 2012), have been identified as a vital frontier for the exploration of emerging infectious diseases and the large-scale drivers that influence the distribution, host associations and evolution of pathogens in wildlife populations (Hoberg et al., 2008). Host-parasite systems at high latitudes are characterised by low diversity, structured by cycles of episodic dispersal/isolation and diversification in response to

q Nucleotide sequence data reported in this paper are available in GenBank under accession numbers KJ726508–KJ726542 (ITS1-5.8S-ITS2) and KJ726433–KJ726507 (cox1) ⇑ Corresponding author. Tel.: +420 38 5310351; fax: +420 38 5310388. E-mail addresses: [email protected], [email protected] (A. Kostadinova).

shifting climates (reviewed in Hoberg et al., 2012) and thus hold promise as model systems for detecting and predicting the impact of accelerated environmental change. This has called for an integrative approach in developing baselines for contemporary diversity and distribution of parasites and parasitic diseases in the northern wildlife (Hoberg et al., 2008, 2012). Accurate identification is vital for our understanding of the diversity of northern parasites, host-parasite associations and the detection of disease emergence. The application of molecular prospecting for taxonomic diversity and combining morphological and molecular data for accurate parasite identification appear key to the recent detection of considerable unrecognised diversity among most major groups of macroparasites, with new species and genera being discovered in well-studied host groups (Hoberg et al., 2012 and references therein). However, in contrast to the wealth of knowledge gained recently in research focused on terrestrial host-parasite systems (reviewed in Hoberg et al., 2012), data on parasite diversity and/or distribution in freshwater systems are rather limited. Recent exploration of freshwater parasite diversity

http://dx.doi.org/10.1016/j.ijpara.2014.04.009 0020-7519/Ó 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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with the application of morphological and molecular approaches has revealed that two new echinostomatid species and at least eight species of bird schistosomes complete their life-cycles in Iceland (Kostadinova and Skírnisson, 2007; Skírnisson et al., 2009; Georgieva et al., 2012, 2013a). An increasing incidence and several massive outbreaks of swimmer’s itch caused by cercariae of bird schistosomes have been observed in the past decade, thus suggesting that the latter may be considered an emerging disease in Iceland (Skírnisson et al., 2009). Another emerging disease in freshwaters is proliferative kidney disease (PKD), detected in 2008 for the first time in Icelandic populations of Arctic charr and brown trout (Kristmundsson et al., 2010). Species of the trematode genus Diplostomum von Nordmann, 1832 represent a group of macroparasites with potential impact on the wildlife populations in the northern ecosystems. These digeneans are obligate parasites of fish-eating birds and are widely distributed across the Holarctic. Species of Diplostomum utilise three-host life cycles involving freshwater lymnaeid snails and fish as intermediate hosts. The larval stages of Diplostomum spp. encysting in the eyes and brain of fish are considered major pathogens, causing mortalities and reduced host survival (Shigin, 1986; Chappell et al., 1994). The problematic identification of these larval stages is a major impediment in the assessment of their actual role in wild fish populations and advancing knowledge of the distribution ranges and evolutionary aspects of host–parasite associations of Diplostomum spp. (Georgieva et al., 2013b). Recent studies characterised molecularly 24 species-level lineages of Diplostomum including three complexes previously recognised as single species. Of these, 12 were found in fishes in northern Canada and 12 in fishes, snails and birds in central and northern Europe (Locke et al., 2010a,b; Georgieva et al., 2013b). Although limited in their geographic extent, these studies indicate a substantial unrecognised genetic diversity inferred from molecular evidence for Diplostomum spp. in both Europe and North America and provide a framework for exploring the taxonomic diversity and distribution of the species of this genus. Here, we present the first known estimates of the species diversity and host associations of Diplostomum in the sub-Arctic using the molecular framework and the recently generated genetic datasets for Nearctic and Palaearctic species of the genus. In an ongoing study on the diversity of larval digeneans in the northern freshwater ecosystems we found infections with Diplostomum spp. in populations of the freshwater snail Radix peregra and three fish species, Salmo trutta fario, Salvelinus alpinus and Gasterosteus aculeatus, in four lakes in south-western Iceland. The combination of different analytical approaches and lines of evidence (molecular, morphological and ecological) have led to the discovery of surprisingly high species richness of these fish pathogens in the limited area studied. Further, using novel and recent data for the mitochondrial (cytochrome c oxidase subunit 1 (cox1)) and nuclear (ribosomal internal transcribed spacer region ITS1-5.8S-ITS2) loci, we explore the associations between the evolutionary history of Diplostomum spp. and the patterns of microhabitat specialisation and geographic distribution.

2. Materials and methods 2.1. Parasite samples and sequence generation A total of 79 parasite isolates was morphologically and molecularly characterised from fish and snails, sampled opportunistically in 2012 from four lakes in south-western Iceland, near Reykjavik. Eye-dwelling metacercariae (70 isolates) were collected from G. aculeatus, S. trutta fario and Sa. alpinus from Lake Hafravatn (64° 70 5000 N, 21° 390 5400 W) and from G. aculeatus from Lakes Family

Park (64° 080 1500 N, 21° 520 0300 W) and Nordic House (64° 080 1900 N, 21° 560 4500 W). Infective dispersal stages of Diplostomum spp. (cercariae; nine isolates) were collected from the snail intermediate host, R. peregra (n = 434), sampled in Lakes Raudavatn (64° 050 3500 N, 21° 470 1400 W), Family Park and Nordic House (see Table 1 for a list of isolates with details on hosts, localities and microhabitats). The lakes are very closely located, the maximum distance being approximately 10 km, so that snails and fish are exposed to infection by Diplostomum spp. from a common regional species pool. Fish metacercariae were allocated to six morphotypes based on the microhabitat, host use and relative size: (i) lens (all hosts); (ii) vitreous humour (salmonids); (iii) retina (salmonids); (iv) retina ‘large’ (ex G. aculeatus); (v) retina ‘small’ (ex G. aculeatus); and (vi) brain (ex G. aculeatus). Partial fragments of cox1 in 10–28 specimens and complete ITS1-5.8S-ITS2 gene cluster (in 2–9 subsampled specimens) were sequenced for each morphotype and for all cercarial isolates ex R. peregra. Total genomic DNA was extracted by placing single ethanolfixed larvae in 200 lL of a 5% suspension of deionised water and ChelexÒ containing 0.1 mg/ml of proteinase K, followed by incubation at 56 °C for 3 h, boiling at 90 °C for 8 min and centrifugation at 16,000g for 10 min. PCR amplifications were carried out using illustra puReTaq Ready-To-Go PCR beads (GE Healthcare, UK) in a total volume of 25 ll (10 pmol of each primer) with 50 ng of genomic DNA. Partial cox1 fragments were amplified using the diplostomidspecific PCR primers designed by Moszczynska et al. (2009): PlatdiploCOX1F (forward; 50 -CGT TTR AAT TAT ACG GAT CC-30 ) and Plat-diploCOX1R (reverse; 50 -AGC ATA GTA ATM GCA GCA GC-30 ). Two newly designed primers (Plag-Dipcox1hF, forward: 50 -ACG TTG GAT CAY AAG CG-30 and Diplocox1iR, reverse: 50 -CTC AGT TAT CCC CAN GGT AAC-30 ), amplifying almost entire cox1 and partial 16 rRNA mitochondrial genes, were also used. The amplified region (approximately 2,380 bp) corresponds to positions 16– 1,533 in the cox1 gene of Fasciola hepatica (GenBank Accession No AF216697; Le et al., 2000), 1–69 in the tRNA-Thr and 1–778 in the 16S rRNA gene. Cycling conditions for cox1 were as follows: (i) diplostomid-specific primers: denaturation at 94 °C for 2 min followed by 35 cycles (94 °C for 30 s, 50 °C for 30 s, 72 °C for 60 s) and a final extension step at 72 °C for 10 min; (ii) newly designed primers: denaturation at 95 °C for 3 min followed by 35 cycles (94 °C for 30 s, 58 °C for 30 s and 72 °C for 2 min), and a final extension step at 72 °C for 5 min. The ITS1-5.8S-ITS2 cluster of the rRNA gene was amplified for a subset of isolates from each cox1derived lineage using the primers of Galazzo et al. (2002): D1 (forward; 50 -AGG AAT TCC TGG TAA GTG CAA G-30 ) and D2 (reverse; 50 -CGT TAC TGA GGG AAT CCT GGT-30 ). The following thermocycling profile was applied: denaturation at 94 °C for 2 min followed by 30 cycles (94 °C for 60 s, 56 °C for 60 s and 72 °C for 2 min) and a final extension step at 72 °C for 5 min. PCR amplicons were purified using either a QIAquick Gel Extraction Kit (Qiagen Ltd, UK) or a QIAquick PCR purification kit (Qiagen Ltd) following the manufacturer’s instructions. PCR fragments were sequenced directly with ABI BigDye chemistry (ABI Perkin-Elmer, UK), alcohol-precipitated and run on an ABI Prism 3130x1 automated sequencer. The following primers were used: (i) cox1: Plat-diploCOX1F and Plat-diploCOX1R or Plag-Dipcox1hF and Plag-Dipcox1gF (forward; 50 -GGT KCT GGN GTN GGT TG-30 ; newly designed); (ii) ITS1-5.8S-ITS2: BD1 (forward; 50 -GTC GTA ACA AGG TTT CCG TA-30 ) and BD2 (reverse; 50 -TAT GCT TAA ATT CAG CGG GT-30 ) (Luton et al., 1992). Contiguous sequences were assembled and edited using MEGA v.5 (Tamura et al., 2011). Unique haplotypes were determined using the web application FaBox (Villesen, 2007). All sequences were submitted to GenBank (accession numbers are listed in Table 1). Pairwise genetic

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Table 1 Data summary for the isolates of Diplostomum spp. from Iceland used for generation of the new cytochrome c oxidase subunit 1 (cox1) and internal transcribed spacer (ITS) sequences. Lineage

Isolate

Life-cycle stagea

Site in hostb

Host

Lake

cox1 haplotype ID

cox1 GenBank sequence No.

ITS GenBank sequence No.c

Lineage 1

GAFP1 GAFP2 GANH2 GANH3 GANH1 SAH1 GAFP3

M M M M M M M

L L L L L L L

Gasterosteus aculeatus Gasterosteus aculeatus Gasterosteus aculeatus Gasterosteus aculeatus Gasterosteus aculeatus Salvelinus alpinus Gasterosteus aculeatus

Family Park Family Park Nordic House Nordic House Nordic House Hafravatn Family Park

1 1 2 2 3 4 5

KJ726433 KJ726434 KJ726435 KJ726436 KJ726437 KJ726438 KJ726439

KJ726508

GAH8 GAH9 GANH4 STH7 STH8 STH10 STH9 STH6 GAH7 GAH2 STH5 STH1 STH2 STH3 GAH3 STH4 RPR1 GAH4 GAH5 GAH6 GAH1

M M M M M M M M M M M M M M M M C M M M M

L L L L L L L L L L L L L L L L L L L L

Gasterosteus aculeatus Gasterosteus aculeatus Gasterosteus aculeatus Salmo trutta fario Salmo trutta fario Salmo trutta fario Salmo trutta fario Salmo trutta fario Gasterosteus aculeatus Gasterosteus aculeatus Salmo trutta fario Salmo trutta fario Salmo trutta fario Salmo trutta fario Gasterosteus aculeatus Salmo trutta fario Radix peregra Gasterosteus aculeatus Gasterosteus aculeatus Gasterosteus aculeatus Gasterosteus aculeatus

Hafravatn Hafravatn Nordic House Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Raudavatn Hafravatn Hafravatn Hafravatn Hafravatn

1 1 1 1 1 1 2 3 4 5 6 7 7 7 7 7 7 7 7 8 9

KJ726440 KJ726441 KJ726442 KJ726443 KJ726444 KJ726445 KJ726446 KJ726447 KJ726448 KJ726449 KJ726450 KJ726451 KJ726452 KJ726453 KJ726454 KJ726455 KJ726456 KJ726457 KJ726458 KJ726459 KJ726460

SAH2 SAH3 SAH4 STH11 SAH5 STH12 STH13 STH14 SAH6 STH15 SAH7 STH17 STH16 STH18

M M M M M M M M M M M M M M

VH VH VH VH VH VH VH VH VH VH VH VH VH VH

Salvelinus alpinus Salvelinus alpinus Salvelinus alpinus Salmo trutta fario Salvelinus alpinus Salmo trutta fario Salmo trutta fario Salmo trutta fario Salvelinus alpinus Salmo trutta fario Salvelinus alpinus Salmo trutta fario Salmo trutta fario Salmo trutta fario

Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn

1 2 3 4 5 6 7 7 7 7 7 7 8

KJ726461 KJ726462 KJ726463 KJ726464 KJ726465 KJ726466 KJ726467 KJ726468 KJ726469 KJ726470 KJ726471 KJ726472 KJ726473

GANH7 GANH8 RPNH1 RPNH2 GAH11 GANH6 GAH10 GANH5 GANH9 GANH10 RPNH3

M M C C M M M M M M C

R R

Gasterosteus aculeatus Gasterosteus aculeatus Radix peregra Radix peregra Gasterosteus aculeatus Gasterosteus aculeatus Gasterosteus aculeatus Gasterosteus aculeatus Gasterosteus aculeatus Gasterosteus aculeatus Radix peregra

Nordic House Nordic House Nordic House Nordic House Hafravatn Nordic House Hafravatn Nordic House Nordic House Nordic House Nordic House

1 1 1 1 2 3 4 5 6 6

KJ726474 KJ726475 KJ726476 KJ726477 KJ726478 KJ726479 KJ726480 KJ726481 KJ726482 KJ726483

SAH8 STH20 STH19 SAH9 SAH12 STH21 SAH10 SAH11 STH22 STH23 STH24 STH25 SAH13

M M M M M M M M M M M M M

R R R R R R R R R R R R R

Salvelinus alpinus Salmo trutta fario Salmo trutta fario Salvelinus alpinus Salvelinus alpinus Salmo trutta fario Salvelinus alpinus Salvelinus alpinus Salmo trutta fario Salmo trutta fario Salmo trutta fario Salmo trutta fario Salvelinus alpinus

Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn Hafravatn

1 2 3 3 3 4 5 6 7 7 7 8

KJ726484 KJ726485 KJ726486 KJ726487 KJ726488 KJ726489 KJ726490 KJ726491 KJ726492 KJ726493 KJ726494 KJ726495

GANH13 RPNH1 GANH14 GANH11

M C M M

R

Gasterosteus aculeatus Radix peregra Gasterosteus aculeatus Gasterosteus aculeatus

Nordic Nordic Nordic Nordic

1 1 1 2

KJ726496 KJ726497 KJ726498 KJ726499

Lineage 2

Lineage 3

Lineage 4

Lineage 5

Lineage 6

B R B R B R

R R

House House House House

KJ726509

KJ726510

KJ726511 KJ726512 KJ726513 KJ726514

KJ726515

KJ726516

KJ726517 KJ726518

KJ726519

Used for species tree p

p

p

p p p p

p

p

p p

p

KJ726520 KJ726521 KJ726522 KJ726523 KJ726524 KJ726525 KJ726526 KJ726527

p p p p p p p

KJ726528 KJ726529 KJ726530

KJ726531 KJ726532

p p

p p

KJ726533 KJ726534 KJ726535

p p

(continued on next page)

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Table 1 (continued) Lineage

a b c

Isolate

Life-cycle stagea

Site in hostb

Host

Lake

GANH12 RPNH3 RPNH4 GANH15 GANH16 RPNH5 RPNH2 GANH17 GANH18

M C C M M C C M M

R

Gasterosteus aculeatus Radix peregra Radix peregra Gasterosteus aculeatus Gasterosteus aculeatus Radix peregra Radix peregra Gasterosteus aculeatus Gasterosteus aculeatus

Nordic Nordic Nordic Nordic Nordic Nordic Nordic Nordic Nordic

R R

R R

House House House House House House House House House

cox1 haplotype ID

cox1 GenBank sequence No.

ITS GenBank sequence No.c

3 4 5 6 7 8 8 9

KJ726500 KJ726501 KJ726502 KJ726503 KJ726504 KJ726505 KJ726506 KJ726507

KJ726536 KJ726537 KJ726538 KJ726539 KJ726540 KJ726541

Used for species tree p p p p p p

KJ726542

Life-cycle stages: a, adult; C, cercaria; M, metacercaria. Site in host: l, eye lens; VH, eye vitreous humour; R, eye retina; B, brain. ITS1-5.8S-ITS2.

distances were calculated using Kimura-2-Parameter distance model implemented in MEGA v.5. Both published and newly generated sequences were aligned together with MUSCLE implemented in MEGA v.5. 2.2. Morphological examination and morphometric analyses The morphology of the larval stages of Diplostomum spp. was studied on live and fixed material by means of light and scanning electron microscopy (SEM). Morphological re-assessment and morphometric comparisons were carried out following molecular analyses for the metacercarial and cercarial isolates of the lineages depicted by the cox1 phylogeny. Series of photomicrographs were made for each isolate with a digital camera of an Olympus BX51 microscope prior to sequencing and measurements were taken from the digital images with the aid of Quick Photo Camera 2.3 image analysis software. The structure of the secondary excretory system was reconstructed from serial microphotographs and the number of excretory concretions was counted in representative isolates subjected to sequencing. Upon examination in vivo, the metacercariae were fixed in ethanol (molecular biology grade) for DNA isolation and photographed again. Two samples of cercariae per isolate were fixed, one in molecular grade ethanol for DNA isolation and one in cold 4% formaldehyde solution for SEM examination. The latter samples were post-fixed in 2% osmium tetroxide for 2 h, washed in 0.1 M phosphate buffer, dehydrated through an acetone series, critical point-dried and sputter-coated with gold. Samples were examined using a JEOL JSM 7401-F scanning electron microscope at an accelerating voltage of 4 kV. Thirteen morphometric variables were measured from the digital images of live metacercariae prior to sequencing and analysis of principal components (PCA) was applied to reveal the multivariate relationship between the metacercariae of the six novel lineages discovered in Iceland. The data from a set of 48 specimens used for obtaining sequences were log-transformed prior to analysis. The morphometric dataset was then subjected to a general linear model multivariate analysis of covariance (MANCOVA) after controlling for the effect of body length and with post hoc pairwise contrasts with Bonferroni correction. 2.3. Alignments and phylogenetic analyses Three alignments were analysed. The first comprised 45 unique cox1 haplotypes (out of 75 novel sequences; Table 1) for isolates from Iceland plus 66 sequences for Diplostomum spp. retrieved from GenBank; three representative sequences per species/lineage identified in previous studies were used and those with the longest sequences were selected where possible (see Supplementary Table S1 for details). In cases when published sequences clustered together with those originating from Iceland, all available

sequences for these species/lineages were included in the alignment in order to check for the presence of identical haplotypes. Sequences were aligned with reference to the amino acid translation, using the echinoderm and flatworm mitochondrial code (Telford et al., 2000); the alignment contained no insertions or deletions. Overall, the cox1 alignment (407 bp; 111 sequences) represented the data currently available for seven and 12 species from Europe and North America, respectively (Moszczynska et al., 2009; Locke et al., 2010a,b; Behrmann-Godel, 2013; Georgieva et al., 2013b). The second alignment (985 bp; 70 sequences) comprised 35 newly-generated sequences for the ITS1-5.8S-ITS2 gene cluster based on a sub-sampling of the cox1derived clades (Table 1), plus 35 published sequences for European isolates of Diplostomum spathaceum and Diplostomum pseudospathaceum, and ‘Diplostomum baeri’ and ‘Diplostomum mergi’ species complexes as defined by Georgieva et al. (2013b), and for North American isolates of three identified (D. baeri, Diplostomum huronense and Diplostomum indistinctum) and six unidentified Diplostomum spp. (Galazzo et al., 2002; Locke et al., 2010a,b) (see Supplementary Table S1). The third alignment was restricted to the ITS1 rDNA region (583 nt, 103 sequences), aimed at molecular identification via matching with sequences from an early European study (Niewiadomska and Laskowski, 2002) and the species/lineages subsequently identified based on molecular evidence from Europe and North America. The alignment encompassed the available data for nine species each from Europe and North America and comprised 35 novel (Table 1) and 68 published sequences retrieved from GenBank (Supplementary Table S1). Tylodelphys spp. were used as outgroups in all analyses. Species boundaries were assessed in phylogenies inferred separately for mitochondrial and nuclear sequence datasets using Bayesian inference (BI) and maximum likelihood (ML) analyses. Prior to analyses the best fitting models of nucleotide substitution were estimated based on Akaike Information Criteria (AIC) using jModelTest 2.1.1 (Guindon and Gascuel, 2003; Darriba et al., 2012). These were the HKY + C (cox1 dataset) and the GTR + C (ITS1-5.8S-ITS2 and ITS1 datasets). BI analyses were carried out with MrBayes version 3.2 (Ronquist et al., 2012) using Markov chain Monte Carlo (MCMC) searches on two simultaneous runs of four chains for 107 generations, sampling trees every 103 generations. The first 103 trees sampled were discarded as ‘burn-in’, determined by stationarity of lnL assessed using Tracer v. 1.5 (Rambaut and Drummond, 2009) and a consensus topology and nodal support estimated as posterior probability values (Huelsenbeck et al., 2001) were calculated from the remaining trees. ML analyses were performed with PhyML 3.0 (Guindon et al., 2010) using the best result of subtree pruning and regrafting, and nearest-neighbour interchange tree search strategies starting with 10 random trees, with a non-parametric bootstrap validation based on 1,000 replicates.

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Further, species tree topology was estimated from mitochondrial and nuclear gene trees under a Bayesian multispecies coalescent model (BEAST v.1.7; Drummond et al., 2012) using a subsample of 61 isolates of 16 species/lineages in the ITS1-5.8SITS2 dataset for which data for both loci were available (indicated in Table 1 and Supplementary Table S1). Substitutions, clock and tree models were unlinked between partitions. Substitution models for each partition were applied as above and a Yule process was selected as a prior for the species tree under a piecewise linear and constant root population size model. No time calibration was included (strict clock model with rate = 1). Species traits were assigned on the basis of the distinct lineages depicted by the cox1 phylogenetic tree. Final posterior distribution was estimated from two separate MCMC analyses run for 108 generations, with parameters sampled every 104 steps. Convergence of the stationary distribution and the effective sample size of the parameters were visualised and checked in Tracer v.1.5. The first 2,000 trees of each run were discarded as ‘burn-in’ and the remainder combined to produce the maximum clade credibility species tree using TreeAnnotator v.1.7 (Drummond et al., 2012) with posterior probability set to 0 and summarising mean node heights. In addition to phylogenetic inference, a character-based DNA barcoding approach to identify diagnostic characters discriminating the novel Icelandic lineages of Diplostomum was applied with the Characteristic Attribute Organisation System (CAOS) workbench (http://boli.uvm.edu/caos-workbench; Sarkar et al., 2002a, 2008; Bergmann et al., 2009). Character searches were performed on datasets comprising cox1 (unique haplotypes only) and ITS15.8S-ITS2 sequences for the six lineages. CAOS workbench offers an improved version of the CAOS algorithm (Sarkar et al., 2002b; Rach et al., 2008) automatically following the steps described in Rach et al. (2008). First, CAOS-Analyser evaluates the diagnostic characters at each node of the tree. Then, CAOS-barcoder selects single pure and single private diagnostic characters and provides a barcode list for all isolates (Rach et al., 2008). The comparisons were limited to single pure species-specific diagnostic characters, i.e. those character states at nucleotide positions of individuals from one species that are unique to this species and differ from those in all individuals of the other species. Pure diagnostic characters with intraspecific variation were excluded in the analysis of the cox1 dataset only. The most variable sites (nucleotide positions comprising pure diagnostic character for at least two species) were chosen for the cox1 barcode. Due to the limited variation in the ITS1-5.8S-ITS2 sequences, all pure diagnostic characters identified from this region that distinguish the six lineages were included in the barcode. 2.4. Species delineation Different lines of evidence (molecular, morphological and ecological) were combined to infer the boundaries of the Diplostomum lineages discovered in Iceland: (i) support for reciprocal monophyly in the model-based phylogenies; (ii) concordance of phylogenies inferred for mitochondrial and nuclear loci and species tree estimations; (iii) pairwise divergence at cox1 above the known range of intraspecific variation for Diplostomum spp. (Georgieva et al., 2013b); (iv) presence of pure species-specific diagnostic characters inferred from character-based barcode analysis; (v) morphological differences (based on both life-cycle stages where possible); (vi) degree of morphometric differentiation; (vii) the intermediate fish host and microhabitat specialisation. 2.5. Reconstructions of ancestral microhabitat and geographic ranges Reconstructions of the possible ancestral microhabitat specialisation (i.e. site within the fish host) and geographic ranges of

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Diplostomum spp. were conducted using Statistical DispersalVicariance Analysis (S-DIVA) implemented in the programme Reconstruct Ancestral State in Phylogenies (RASP 2.0b; available at http://mnh.scu.edu.cn/soft/blog/RASP) (Yu et al., 2010) and the species tree generated by BEAST. The distribution range of the molecularly identified Diplostomum spp. was divided broadly into three areas (North America, Europe and Iceland) and four microhabitat states were considered (eye lens, vitreous humour and retina and brain) (indicated in Table 1 and Supplementary Table S1). S-DIVA analyses were run twice, on the consensus species tree and on 5,000 randomly selected species trees from the BEAST output. The option ‘use ancestral ranges’ was activated for the analysis on the consensus tree, which optimises the states on the final tree, allowing only those with a frequency > 1 in a node on the final tree to be used in the calculations using all trees. No state combinations were a priori excluded, but the maximum number of states per node was kept to 2. The state of the root was considered as ‘wide’ for both reconstructions. 3. Results All three fish species examined carried heavy eye infections with Diplostomum spp. (up to 713 worms per fish), the prevalence ranging between 37% and 100% depending on the host and/or lake (Table 2). Our integrative approach combining molecular, morphological and ecological data led to the discovery of high species diversity of Diplostomum among the 2,753 metacercariae from the eye lens, vitreous humour and retina, and the brain of the three fish species studied in Iceland. Furthermore, the screening of snail populations and molecular analyses helped establish links between different stages of the life-cycle of Diplostomum spp. 3.1. Phylogenetic analyses BI and ML analyses based on the cox1 dataset yielded similar phylogenetic hypotheses, depicting four main clades (labelled A–D in Fig. 1) of lens-infecting (clades A and D) and non-lens-infecting (clades B and C) species of Diplostomum. Clade D representing the ‘D. mergi’ species complex sensu Georgieva et al. (2013b) showed labile placement as early divergent within either clade A or as sister to clades B–C depending on the reconstruction method (see Supplementary Fig. S1). The newly sequenced isolates from Iceland clustered in six strongly supported reciprocally monophyletic lineages (Fig. 1); of these, three (Lineages 2, 5 and 6) represented only Icelandic haplotypes. Lineage 1 comprised metacercariae from the lenses of G. aculeatus and Sa. alpinus that clustered with isolates of larval and adult stages of D. spathaceum from Europe (Georgieva et al., 2013b) and thus represented the only link between larval isolates sampled in Iceland and identified adult isolates of Diplostomum spp. Lineage 2 comprised metacercariae from the lenses of G. aculeatus and S. trutta plus one cercarial isolate ex R. peregra. Lineages 3 and 4 exhibited a strongly supported sister relationship (Fig. 1) with an average sequence divergence of 7 ± 1%. Lineage 3 comprised metacercariae from the eye vitreous humour of the two salmonids with several haplotypes shared (12 unique haplotypes out of 27 isolates in total) with the European ‘trout’ lineage of the ‘D. baeri’ species complex sensu Georgieva et al. (2013b). Lineage 4 included metacercariae from the eye retina and brain of G. aculeatus and two cercarial isolates ex R. peregra that clustered together (but with no shared haplotypes; 26 isolates in total) with the European ‘perch’ lineage of the ‘D. baeri’ complex sensu Georgieva et al. (2013b). Lineage 5 comprised isolates from the eye retina of the two salmonids and exhibited a strong sister group relationship with Diplostomum sp. 6 from Canada. Lineage 6 consisted of

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metacercariae from the eye retina of G. aculeatus and four cercarial isolates from R. peregra that clustered together with two Canadian species, Diplostomum spp. 8 and 9 (clade C in Fig. 1). The phylogenetic hypotheses inferred from the ITS1-5.8S-ITS2 dataset differed from the mitochondrial hypotheses in that the main clade C was depicted either within clade A (BI, Fig. 2) or as its sister clade (ML, Supplementary Fig. S2). Both BI and ML reconstructions confirmed the identification of Lineage 1 as D. spathaceum based on linking sequences of larval and adult isolates from Europe (Georgieva et al., 2013b) and provided support for the distinct status of three Icelandic lineages (Lineages 2, 5 and 6) inferred from the mitochondrial gene. However, the isolates of Lineages 3 and 4 intermingled in a single unsupported clade (Fig. 2) which includes sequences from both ‘trout’ and ‘perch’ lineages of the ‘D. baeri’ complex from Europe (Georgieva et al., 2013b). Lineage 5, D. baeri of Galazzo et al. (2002) and Diplostomum sp. 2 of Locke et al. (2010a,b) were recovered as sister to Lineages 3 and 4, thus expanding the content of the ‘D. baeri’ species complex to all members of the main clade B (Fig. 2, see also Supplementary Fig. S2). The sequences for Lineage 6 did not match any sequence available on GenBank. The ITS1 marker alone proved to be insufficient to unequivocally discriminate amongst most of the lineages of the ‘D. baeri’ complex (as in Georgieva et al., 2013b) and most relationships were unsupported (see Supplementary Figs. S3, S4). The BEAST maximum clade credibility tree topology shown in Fig. 3 was largely congruent with the mitochondrial hypothesis for Diplostomum spp., i.e. containing all main clades identified in the cox1 reconstruction but with the ‘D. mergi’ complex as the earliest divergent. Support for the main clades was strong except for clade B (‘D. baeri’ complex as defined here); a few deeper nodes were not supported.

3.3. Reciprocal illumination Overall, there was good agreement between the morphotypes, to which we initially assigned the metacercariae based on their relative size, microhabitat and host use, and the genetically distinct lineages. Exceptions were the ‘lens’ morphotype which comprised two species (D. spathaceum and Lineage 2) and the ‘brain’ morphotype ex G. aculeatus which was found to belong to Lineage 4. With a single exception, i.e. D. spathaceum (Lineage 1), our attempts at molecular identification failed to establish links between the newly discovered lineages from Iceland and Diplostomum spp. considered distinct based on morphological and/or molecular evidence from either Europe or North America. Their novelty and genetic distinctness as revealed by the species tree hypothesis prompted a detailed examination of parasite morphology in association with distance- and character-based barcoding and ecological (host and microhabitat selection) data. Notably, the links established between larval stages (i.e. cercaria and metacercaria) identified via clustering patterns in phylogenies resulted in an increased range of larval morphological characters (Tables 3 and 4). PCA based on 13 morphometric variables for a total of 48 of the sequenced metacercariae indicated differentiation of Lineage 6 along the first axis due to its strong association with body size but the remaining lineages formed a loose cluster in the morphometric space (Supplementary Fig. S6). A general linear model MANCOVA design, in which the effect of body length was controlled for, revealed significant differences between the lineages (F(60,144) = 28.61, P < 0.0001; details from post hoc contrasts are given in Supplementary Tables S2 and S3). In addition to the morphometric variability, we herein provide data on features considered important for the taxonomy of Diplostomum spp. (Shigin, 1986): the morphology of the dispersive infective stages (cercariae) for three of the novel lineages (Table 4) that did not match any of the existing descriptions, and the structure of the secondary excretory system in the metacercariae for all lineages (Supplementary Table S2). Our interpretation of the system studied in terms of the combination of molecular, morphological and ecological data (summarised in Table 3) follows the application of the taxonomic circle sensu DeSalle et al. (2005), a framework that requires corroboration from more than one line of evidence for species delimitation i.e. ‘‘breaking out’’ of the taxonomic circle of inference. No geographic hypothesis could be formulated because the six lineages occurred in sympatry in a fairly small geographic area. In most cases all three lines of evidence used in our comparisons corroborated the distinct status of the six phylogenetic lineages of Diplostomum with only two somewhat ‘problematic’ contrasts. Lineages 1 and 2 shared the microhabitat (lens) and host (G. aculeatus) and the structure of the secondary excretory system, but

3.2. Character-based DNA barcodes Using the CAOS algorithm, a total of 57 pure diagnostic characters that distinguish the novel lineages at at least 20 nucleotide positions was identified within cox1 sequences. Character-based DNA barcodes for the six lineages of Diplostomum from Iceland at 14 selected most variable species-specific nucleotide positions, that revealed the highest number of diagnostic characters at the important nodes, are given in Supplementary Fig. S5. Based on the selected barcodes, all Icelandic lineage combinations could be distinguished by at least six diagnostic characters (range 6–14, Table 3). However, the ITS1-5.8S-ITS2 marker was less suitable to barcode Icelandic lineages. Only 16 species-specific diagnostic characters were found out of 970 nt positions (including gaps). Lineages 3 and 4 were indistinguishable; both could be differentiated from Lineage 5 by just two diagnostic characters (Table 3; see also Supplementary Fig. S5).

Table 2 Prevalence (P), mean abundance (MA) and intensity (I, range) for the six lineages of Diplostomum discovered in the three species of fish from Iceland. Host

Gasterosteus aculeatus

Lake Sample size Fish total lengtha

Hafravatn 20 39–46 (42 ± 2) P (%)

Lineage Lineage Lineage Lineage Lineage Lineage Overall a

1 (Diplostomum spathaceum) 2 3 4 5 6

Range (mean ± S.D.) (mm).

MA

Family Park 19 43–57 (51 ± 4) I

Nordic House 20 42–68 (49 ± 6)

P (%)

MA

I

P (%)

MA

I

16

0.4

1–6

0.7 0.2

1–7 1

35

1.0

1–10

30 15

30

0.8

1–5

50

3.6

1–35

85 95

6.1 10.5

2–16 1–50

60

1.8

1–10

32 37

0.4 0.8

1–2 1–7

Salmo trutta fario

Salvelinus alpinus

Hafravatn 19 190–350 (257 ± 55)

Hafravatn 4 245–260 (251)

P (%)

MA

I

P (%)

MA

I

25

0.3

1

37 84

0.6 22.9

1–3 5–52

100

45.3

29–59

79

9.2

1–48

100

422.0

288–654

90

32.7

3–64

100

467.5

345–713

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Fig. 1. Detailed phylogram from Bayesian inference analysis of the cytochrome c oxidase subunit 1 sequence alignment (407 bp, 111 sequences) for 20 species/lineages of Diplostomum from Iceland (unique haplotypes only), Europe and North America. Main clades are denoted as A–D; only posterior probability values > 0.95 are shown. Lineages discovered in Iceland are coded as in Table 1. Live photomicrographs of metacercariae in fish (shown to scale) and schematic drawings of cercariae are given at the corresponding clades. The scale bar indicates the expected number of substitutions per site. Sequence identification is as in GenBank, followed by a letter: B, Behrmann-Godel (2013); Ge, Georgieva et al. (2013b); L, Locke et al. (2010a,b); M, Moszczynska et al. (2009).

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Fig. 2. Detailed phylogram from Bayesian inference analysis of the ITS1-5.8S-ITS2 sequence alignment (985 bp, 70 sequences) for species/lineages of Diplostomum from Iceland, Europe and North America. Main clades are denoted as A - D; only posterior probability values > 0.96 are shown. Lineages discovered in Iceland are coded as in Table 1. Live photomicrographs of metacercariae in fish (shown to scale) and schematic drawings of cercariae are given at the corresponding clades. The scale bar indicates the expected number of substitutions per site. Sequence identification is as in GenBank, followed by a letter: B, Behrmann-Godel (2013); Ga, Galazzo et al. (2002); Ge, Georgieva et al. (2013b); L, Locke et al. (2010a,b).

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A

711

B

Fig. 3. Maximum clade credibility tree relating 16 species/lineages of Diplostomum based on cytochrome c oxidase subunit 1 and ITS1-5.8S-ITS2 loci for 61 isolates using Bayesian coalescence. Pie charts at nodes represent probabilities of unique or alternative ancestral sites of infection within (A) the fish host and (B) geographic ranges based on a Statistical Dispersal-Vicariance Analysis over 5,000 randomly selected species trees. Main clades are denoted as A–D; ranges of terminals are shown in parentheses. B, brain; L, eye lens; VH, eye vitreous humour; R, eye retina; E, Europe; I, Iceland; NA, North America. Stars indicate nodal support > 0.97.

could be readily distinguished using molecular and morphological lines of evidence (six diagnostic characters, Table 3). Lineages 4 and 6 shared the host (G. aculeatus) and microhabitat (retina) but showed substantial differentiation with respect to the remaining seven diagnostic characters (molecular and morphological). Lineages 3 and 4 exhibited the lowest levels of sequence divergence and a lack of barcode characters for the nuclear sequences but exhibited differential host and microhabitat selection, and could be differentiated using four other diagnostic characters (i.e. molecular, morphological and ecological evidence, Table 3). Detailed descriptions of the larval stages of the six lineages of Diplostomum will be published elsewhere. Formal naming of the new species would require the discovery of the adults in the bird definitive hosts. 3.4. Ancestral microhabitat selection and geographic distribution The reconstruction of the ancestral site of infection within the fish host of Diplostomum spp. showed a clear pattern of microhabitat specialisation. The most favoured ancestral state of the most recent common ancestor (MRCA) of Diplostomum spp. was assessed as the lens (marginal probability (mp) 64%, Fig. 3A). The ancestor of species in clade A infected exclusively lens whereas the MRCA of the species in clades B and C infected the retina (both mp 100%); a vicariant event from retina to vitreous humour occurred (Fig. 3A). The analysis suggests multiple dispersal events in nonlens-infecting species of clades B and C. The reconstruction of the

ancestral geographic ranges also depicted a clear pattern with possible ancestral ranges of Diplostomum spp. in both North America and Europe (mp 99%). Remarkably, whereas one of the descendants, the ‘D. mergi’ species complex (clade D), is restricted to Europe, the remaining descendants have widely diversified in North America with independent dispersal and vicariant events to Iceland and, in one case, to Europe (Fig. 3B). In the case of the non-lens ‘D. baeri’ complex as defined here (clade B), the reconstructions suggest North America as an ancestral area with further dispersal and vicariance within the Palaearctic. 4. Discussion To the best of our knowledge this study provides the first estimates of the species diversity of Diplostomum in sub-Arctic freshwater ecosystems. Our analyses integrating different analytical approaches, phylogenetic analyses, estimates of genetic divergence, character-based barcoding, morphological examination of live larval stages, precise detection of microhabitat specialisation and host use, led to the discovery of one described and five putative new species in a limited area in Iceland. For parasites with complex life-cycles that utilise sequentially more than one intermediate host species in their transmission to definitive hosts, an overlap of the distributions of all hosts is required for successful transmission. Thus, the distribution of digeneans in general, and of Diplostomum spp. in particular, in the relatively simple freshwater ecosystems in the Arctic would be

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Table 3 Summary of the diagnostic characters discriminating the six lineages of Diplostomum from Iceland. Contrast

DNA

Morphology

cox1

L1 vs. L2 L1 vs. L3 L1 vs. L4 L1 vs. L5 L1 vs. L6 L2 vs. L3 L2 vs. L4 L2 vs. L5 L2 vs. L6 L3 vs. L4 L3 vs. L5 L3 vs. L6 L4 vs. L5 L4 vs. L6 L5 vs. L6

Metacercariae

Cercariae

Average sequence divergence (%)

Barcode characters

ITS1-5.8S-ITS2 Average sequence divergence (%)

Barcode characters

Number of different featuresd

No. of different features

12 ± 2 15 ± 2 14 ± 2 14 ± 2 14 ± 2 15 ± 2 15 ± 2 16 ± 2 12 ± 2 7±1 11 ± 1 14 ± 2 10 ± 1 14 ± 2 14 ± 2

12 13 9 9 11 14 12 13 10 10 8 10 7 6 7

1.3 ± 0.4 2.7 ± 0.5 2.7 ± 0.5 2.8 ± 0.5 1.6 ± 0.4 2.5 ± 0.4 2.5 ± 0.4 2.6 ± 0.4 1.4 ± 0.4 0.1 ± 0.0 0.4 ± 0.2 2.8 ± 0.5 0.4 ± 0.2 2.8 ± 0.5 2.9 ± 0.5

7 4 5 7 11 3 3 5 9 0 2 8 2 8 10

5 7+e 0+e 3+e 9+e 9+e 1+e 2+e 9+e 8+e 6 6+e 2+e 9+e 7+e

13 – 12 – 13 – 16 – 14 – – – – 10 –

Microhabitata

Shared fish hostb

Taxonomic circlec

L for both L vs. VH L vs. R/B L vs. R L vs. R L vs. VH L vs. R/B L vs. R L vs. R VH vs. R/B VH vs. R VH vs. R R/B vs. R R/B vs. R R for both

Ga Sa Ga Sa Ga St Ga St Ga none St & Sa none none Ga none

DNA + Mo DNA + Mo + E(Mi) DNA + Mo(e)+E(Mi) DNA + Mo(e)+E(Mi) DNA + Mo + E(Mi) DNA + Mo + E(Mi) DNA + Mo + E(Mi) DNA + Mo(e)+E(Mi) DNA + Mo + E(Mi) DNA + Mo + E(Mi + Ho) DNA + Mo + E(Mi) DNA + Mo + E(Mi + Ho) DNA + Mo(e)+E(Ho) DNA + Mo DNA + Mo + E(Ho)

Lineages are indicated as L1 to L6; overlaps for the individual contrasts are highlighted in bold; details are given in Supplementary Tables S1 and S2, Fig. S5. a Microhabitats: L, eye lens; VH, eye vitreous humour; R, eye retina; B, brain. b Fish hosts (in Iceland only): Ga, Gasterosteus aculeatus; St, Salmo trutta fario; Sa, Salvelinus alpinus. c Lines of evidence jointly supporting the phylogenetic hypotheses of species delineation of the Icelandic Diplostomum spp. and allowing us to ‘‘break out‘‘ of the taxonomic circle of inference sensu DeSalle et al. (2005): DNA, morphology (Mo, including excretory system; Mo(e) excretory system alone) and ecology (E, microhabitat (Mi) and host specialisation (Ho)). d Number of different metric variables (Multivariate Analysis of Covariance, (MANCOVA); post hoc comparisons with Bonferroni correction) + different structure of the secondary excretory system (e).

Table 4 Comparative qualitative and meristic data for the cercarial isolates collected from Radix peregra in Iceland. Features

Lineage 1 (Diplostomum Lineage 2 spathaceum) Radix ovata, R. peregra, R. peregra R.auricularia Niewiadomska and Present study Kiseliene (1994)

Hosts Source Yellow pigment in body Relation BL-TSL-FL Relation VSW–AOW No. of pre-oral spines (median group) a No. of pre-oral spines in each lateral group a No. of post-oral rows of spines a Zone of dispersed post-oral spines a Transverse rows of spines on body a Double transverse rows a Incomplete transverse rows a Transverse rows with additional spines laterally a No. of spine rows on ventral sucker a No. of spines on ventral sucker a Penetration gland-cells

Lineage 4

Lineage 6

R. peregra

R. peregra

Present study

Present study

Present BL < TSL = FL VSW > AOW 8–16 in 3–4 rows

Absent BL < TSL < FL VSW > AOW 15

Absent BL < TSL = FL VSW < AOW 8 spines in 3 rows

Present BL < TSL = FL VSW < AOW 11

Absent

7

Absent

5

10–14 Present

10–11 Entire body surface

5–6 Present, wide

7–9 Present

9–10

Absent

9

11

Rows 1–2 ? ?

Not applicable Not applicable Not applicable

Rows 1–2 Rows 6 and 8–9 Rows 1–4

Rows 1–3, 11 latero-dorsally Rows 7–11 None

2

4–5

3

3 (a 4th row may be partly formed)

108–125 Large, do not cover ends of caeca Absent

At least 240 Small, do not cover ends of caeca Present, robust

38 per row (c.120 in total) Large, cover ends of caeca

c.25 per row (75–82 in total) Large, do not cover ends of caeca

Absent

Present

Present along nearly entire tail stem (2 bands of 3–4 scale-like spines each) Present (2 bands of 2–3 scale-like spines each)

Fin-folds on furcae No. of caudal bodies Shape of caudal bodies

Present 11–12 pairs Incised contours

Resting position

Tail stem bent at 90°

Absent 8–9 pairs Weakly-incised contours Tail stem bent at 90°

Present along entire tail (2 bands of 2 spines each) Present (2 bands of 3 small, pointed spines each) Absent 6 pairs Smooth contours Tail stem straight

Tail stem bent at 90°

Spines on tail stem Spines on furcae

a

a

a

Absent 6 pairs Smooth contours

BL, body length; TSL, tail-stem length; FL, furcae length; VSW, ventral sucker width; AOW, anterior organ width; c, circa. a Data from scanning electron microscopy.

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effectively constrained by the low diversity of the snail and fish hosts that reflects both historical colonisation processes and the limiting effects of the extreme environments. We consider these ecosystems to represent a state of ‘life-cycle bottleneck’ in relation to digenean transmission. Iceland offers a unique natural setting to study the effect of limited host availability on digenean diversity. The island is isolated from the continents on both sides of the central North Atlantic thus making colonisation from the mainland difficult. Icelandic lakes are extremely recent (less than 10,000 years old) and colonised after the last glaciation; the contemporary freshwater invertebrate fauna is mainly of Palaearctic origin with a low richness (Gislason, 2005). The snail R. peregra is the most abundant freshwater mollusc (Skírnisson et al., 2009) and the only suitable intermediate host for Diplostomum spp. Only six fish species living partly or entirely in freshwater have colonised Iceland: Salmo salar, S. trutta, Sa. alpinus, Anguilla anguilla and G. aculeatus. Due to this low richness of potential intermediate hosts, Icelandic aquatic habitats effectively represent an excellent model system and an extreme example of a ‘life-cycle bottleneck’ in the transmission of Diplostomum spp. in the high-latitude freshwaters, and previous surveys support the expectation for low parasite diversity. Only two species of Diplostomum have to date been reported in fish in Iceland: eye-dwelling metacercariae in Sa. alpinus, S. trutta and G. aculeatus have been referred to as D. spathaceum (see Natsopoulou et al., 2012) or D. baeri (see Natsopoulou et al., 2012; Karvonen et al., 2013); unidentified Diplostomum spp. metacercariae have also been recorded in Sa. alpinus and S. trutta, G. aculeatus and A. anguilla (see Kristmundsson and Helgason, 2007; Kristmundsson and Richter, 2009; Karvonen et al., 2013). Contrary to our initial expectations we found that at least six species of Diplostomum complete their life-cycles within a fairly narrow geographic area, a finding that increases the species richness of Diplostomum in Iceland by 200% and raises the number of molecularly characterised Diplostomum spp. from the Palaearctic to 17 species. The species diversity detected in Iceland, which comprises nearly a third of the Palaearctic Diplostomum diversity (27% of the species considered valid; see Georgieva et al., 2013b), was unexpected in view of the isolation, harsh climatic conditions and limited diversity of intermediate hosts in Iceland, and the restricted geographic extent and relatively low sampling effort of our study. At a local scale, comparisons with the survey in the River Ruhr drainage in Germany by Georgieva et al. (2013b), which provides taxonomic resolution consistent with our study, indicate higher richness of Diplostomum spp. in the fish species examined in Iceland than in central Europe. In Iceland S. truta and G. aculeatus hosted three and four species, respectively, versus two species each in central Europe; unfortunately no salmonid or gasterosteid hosts were studied in the large-scale sequencing survey of Locke et al. (2010a,b) in Canada. The increased taxonomic resolution achieved in our study has important implications for parasite diversity baselines in salmonid and gasterosteid hosts in the high-latitude ecosystems. Current estimates of species richness in sub-Arctic and Arctic ecosystems range from four to 18 parasite species for salmonid and coregonid hosts and from one to 11 (usually under seven) in G. aculeatus (Poulin et al., 2011; Wrona et al., 2013). Our study adds to these estimates six putative species: four in salmonids (populations in Lake Hafravatn) and two (populations in Lakes Hafravatn and Family Park) to four (the population from Lake Nordic House) in G. aculeatus. At a community diversity scale, molecular identification of Diplostomum spp. may shed light on parasite community patterns and host-parasite associations. Poulin et al. (2011) warned that a scenario of high species diversity revealed by molecular identifications, exemplified by Diplostomum spp., can invalidate community similarity analyses based on published identifications

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and Locke et al. (2013) demonstrated that increased taxonomic resolution for the Diplostomoidea reveals the important role of host phylogeny in explaining variations in parasite community similarity in freshwater fish. Phylogenetic relationships inferred from cox1 and the species tree provided evidence for lineage exclusivity for isolates of the six lineages sampled in Iceland. This was corroborated by the nuclear gene phylogeny, distance- and character-based barcodes, and by morphological and ecological evidence. In most problematic contrasts, at least two lines of evidence, molecular and morphological, supported ‘‘breaking out’’ of the taxonomic circle of inference for species delineation in the system studied. One important result from our study is the overall good agreement between the genetically distinct lineages and the morphotypes to which we initially assigned the metacercariae based on the relative size, microhabitat and host use. Of the two exceptions, only the differentiation of the lens metacercariae required additional morphometric analysis. Therefore the reciprocal illumination achieved here, following the identification of independent evolutionary lineages, provides a framework for monitoring the diversity and transmission of Diplostomum spp. in the high latitude ecosystems that counteracts the diminishing taxonomic expertise. Notably, all non-lens species found in S. trutta, Sa. alpinus and G. aculeatus in Iceland can be discriminated in the field based on examination of live material and precise detection of the site of infection; this enables rapid and practical identification. In contrast, metacercariae obtained from frozen fish (e.g. Locke et al., 2010a,b) can be identified only after sequencing, thus increasing substantially the effort and costs of large-scale or site-intensive parasite surveys. Our phylogenetic analyses indicate high rates of speciation within the ‘D. baeri’ species complex (Clade B) which appears to be the most diverse species group within the genus. Diplostomum baeri has long been considered a single widespread Holarctic species that uses percids as its main fish intermediate hosts. Georgieva et al. (2013b) first provided molecular evidence for the existence of distinct ‘trout’ and ‘perch’ lineages in Europe. Our results show unequivocally that this species complex is comprised of at least eight molecularly characterised species, five recorded in North America, two in both Europe and Iceland and one in Iceland only. These findings indicate that studies based on the assumption that parasites of the eye vitreous humour and retina of fish represent a single species (reported as D. baeri, e.g. Natsopoulou et al., 2012; Karvonen et al., 2013) should be interpreted with caution. The overall levels of infection revealed in our study are comparable with those recently recorded in the three fish hosts in Iceland (Kristmundsson and Richter, 2009; Natsopoulou et al., 2012; Karvonen et al., 2013). Of particular importance are the high prevalence and abundance of Diplostomum spp. in S. trutta and Sa. alpinus, which are similar to those observed in Lake Hafravatn and in the nearby Lake Ellidavatn (approximately 6 km apart) (Kristmundsson and Richter, 2009). Notably, a long-term monitoring program of water temperature in Lake Ellidavatn revealed a significant temperature increase (with a mean monthly rise of 2.3–2.7 °C in spring and summer) during 1988–2006, which was correlated with an increase in the monthly mean air temperatures in Reykjavík (Malmquist et al., 2009). The high abundance of Diplostomum spp. in the fishes from the lakes in and around Reykjavík observed recently may provide circumstantial evidence for possible association with increased parasite transmission rates due to the rise of the temperature in the area. The first record of the thermally linked PKD in Iceland, based on examination of the two salmonids in Lake Ellidavatn (Kristmundsson et al., 2010), is consistent with this suggestion. We found that the parasite load of the two Diplostomum spp. shared by the two salmonid hosts was >10-fold higher in Sa.

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alpinus than in S. trutta; this agrees with the data reported by Kristmundsson and Richter (2009) for fish populations in Lakes Hafravatn and Ellidavatn. These higher infection levels appear to coincide with substantial declines of the populations of Sa. alpinus in several lakes in Iceland, including Lake Ellidavatn, as opposed to the steady state of the populations of S. trutta (see Malmquist et al., 2009; Kristmundsson et al., 2010). The ecological niche of Sa. alpinus is commonly reduced in sympatry with other salmonids and it is predicted that climate-driven range expansion of S. trutta would further limit future distributions of Sa. alpinus via competition (Hein et al., 2012). Salvelinus alpinus is also more susceptible to parasitic diseases than S. trutta (see Kristmundsson and Richter, 2009; Kristmundsson et al., 2010). The differences in infection levels (Kristmundsson and Richter, 2009; Kristmundsson et al., 2010; this study) indicate that infections with pathogenic Diplostomum spp. may mediate the outcomes of the competition between the two species with consequences for patterns of potential extinctions of Sa. alpinus in the high latitude ecosystems. It is early to infer the evolutionary and biogeographic history of the genus Diplostomum in view of the potentially uncovered diversity and the insufficient knowledge of the ecology and distribution of the molecularly identified species/lineages. However, the unique dataset for the mitochondrial and nuclear loci of Diplostomum spp. examined here allows exploration of the evolutionary history of the group in the context of microhabitat selection and geography. The speciation in Diplostomum could be the result of expansion– contraction events of both the ecological niche and geographic ranges of the common ancestor, which is inferred as a lens-infecting species with a Holarctic distribution. Geographic range analysis indicates that with the exception of the species of the ‘D. mergi’ complex that diverged earlier in the Palaearctic and appear to have remained there, most European Diplostomum spp. may have originated in, and dispersed from, North America into Iceland and further into Europe. Although vicariance events in Iceland were depicted in the analysis, we believe these should be treated with caution in view of the youth of the Icelandic freshwater systems and the limitations of the currently available data, especially with respect to the geographic ranges of Diplostomum spp. We also found that microhabitat extension/segregation is associated with the evolutionary history of the Diplostomum spp. depicted in the species tree hypothesis. Strikingly, the basal clustering in the phylogeny appears to be better explained by the microhabitat specialisation, the species inhabiting lens (clades A and D) being clearly divergent from the species found in non-lens microhabitats (clades B and C). As inferred, secondary dispersal and vicariance events gave rise to the lineages infecting retina, and their descendants colonised other non-lens tissues. Although it is not possible to explore definitive host associations of Diplostomum spp. due to the lack of data for the molecularly identified species, another non-exclusive hypothesis for the diversification drivers in this group can be suggested. The general division of the clades in the phylogeny may correspond to differences in the definitive host ranges, each clade being associated with a different host group. Thus, a ‘larid clade’ can be identified in the phylogeny (clade A) based on the host records of four out of the eight species confirmed by molecular data, i.e. D. huronense, D. indistinctum, D. pseudospathaceum and D. spathaceum (see Shigin, 1993; Galazzo et al., 2002; Locke et al., 2010a; Georgieva et al., 2013b). The species of the ‘D. mergi’ complex (clade D) most likely complete their life-cycles in mergid ducks (Shigin and Kostadinova, 1985; Shigin, 1993; Georgieva et al., 2013b), whereas the species of the non-lens infecting clades B and C may have diversified in other fish-eating birds with northern Holarctic distribution. The data on the abundance and feeding habits of the species of the depauperate bird fauna of Iceland indicate that the gaviids Gavia immer and Gavia stellata, the podicipedid Podiceps

auritus, and the anatids Mergus serrator and Mergus merganser, may act as definitive hosts (Cramp and Simmons, 1986; Petersen, 1998). Molecular and morphological identification of adult Diplostomum spp. from bird hosts in Iceland and North America are required to test this hypothesis. In conclusion, our findings in the sub-Arctic lakes in Iceland strongly suggest that the biodiversity of the parasite group studied is underestimated globally in the high latitude ecosystems and call for a cautionary approach to pathogen identification in developing the much needed baselines of pathogen diversity that may help detect effects of climate change in the freshwater environment of the sub-Arctic. Acknowledgements We would like to thank two anonymous reviewers for their helpful comments and suggestions on an earlier version of the manuscript. This research was funded by the Czech Science Foundation (AF, AK, SG, TS, grants P505/10/1562 and P505/12/G112), Institute of Parasitology (AF, AK, SG, TS, RVO: 60077344), University of Iceland (KS, Research Fund of the University of Iceland) and a Marie Curie Outgoing International Fellowship within the 7th European Community Framework Programme (IB-C, grant PIOF-GA-2009-252124). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijpara.2014. 04.009. References Behrmann-Godel, J., 2013. Parasite identification, succession and infection pathways in perch fry (Perca fluviatilis): new insights through a combined morphological and genetic approach. Parasitology 140, 509–520. Bergmann, T., Hadrys, H., Breves, G., Schierwater, B., 2009. Character-based DNA barcoding: a superior tool for species classification. Berl. Munch. Tierarztl. Wochenschr. 122, 446–450. Chappell, L.H., Hardie, L.J., Secombes, C.J., 1994. Diplostomiasis: the disease and host-parasite interactions. In: Pike, A.W., Lewis, J.W. (Eds.), Parasitic Diseases of Fish. Samara Publishing Limited, Tresaith, Dyfed, UK, pp. 59–86. Cramp, S., Simmons, K.E.L., 1986. Handbook of the birds of Europe, the Middle East, and North Africa: The Birds of the Western Palearctic, Vol. 1. Oxford University Press, Oxford. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. JModelTest 2: more models, new heuristics and parallel computing. Nat. Methods 9, 772. DeSalle, R., Egan, M.G., Siddall, M., 2005. The unholy trinity: taxonomy, species delimitation and DNA barcoding. Phil. Trans. R. Soc. B 360, 1905–1916. Drummond, A.J., Suchard, M.A., Xie, D., Rambaut, A., 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973. Galazzo, D.E., Dayanandan, S., Marcogliese, D.J., McLaughlin, J.D., 2002. Molecular systematics of some North American species of Diplostomum (Digenea) based on rDNA-sequence data and comparisons with European congeners. Can. J. Zool. 80, 2207–2217. Georgieva, S., Kostadinova, A., Skírnisson, K., 2012. The life-cycle of Petasiger islandicus Kostadinova & Skirnisson, 2007 (Digenea: Echinostomatidae) elucidated with the aid of molecular data. Syst. Parasitol. 82, 177–183. Georgieva, S., Selbach, C., Falty´nková, A., Soldánová, M., Sures, B., Skírnisson, K., Kostadinova, A., 2013a. New cryptic species of the ‘revolutum’ group of Echinostoma (Digenea: Echinostomatidae) revealed by molecular and morphological data. Parasites Vectors 6, 64. Georgieva, S., Soldánová, M., Pérez-del-Olmo, A., Dangel, D.R., Sitko, J., Sures, B., Kostadinova, A., 2013b. Molecular prospecting for European Diplostomum (Digenea: Diplostomidae) reveals cryptic diversity. Int. J. Parasitol. 43, 57–72. Gislason, G.M., 2005. Origin of freshwater fauna of the North-Atlantic islands: present distribution in relation to climate and possible migration routes. Verh. Internat. Verein. Limnol. 29, 198–203. Guindon, S., Gascuel, O., 2003. A simple, fast and accurate method to estimate large phylogenies by maximum-likelihood. Syst. Biol. 52, 696–704. Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321. Hein, C.L., Öhlund, G., Englund, G., 2012. Future distribution of Arctic char Salvelinus alpinus in Sweden under climate change: Effects of temperature, lake size and species interactions. Ambio 41 (Suppl. 3), 303–312.

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