Contrasting Definitive Hosts As Determinants of The

Molecular Ecology (2015) 24, 1060–1073 doi: 10.1111/mec.13080

Contrasting deﬁnitive hosts as determinants of the genetic structure in a parasite with complex life cycle along the south-eastern Paciﬁc

Z. LOPEZ,*† L. CARDENAS,‡ F. RUNIL‡ and M. T. GONZALEZ* *Instituto de Ciencias Naturales “Alexander Von Humboldt”, Facultad de Ciencias del Mar y Recursos Biologicos, Universidad de Antofagasta, Av. Angamos 601, P.O. Box 170, Antofagasta, Chile, †Programa Magıster en Ecologıa de Sistemas Acuaticos, Universidad de Antofagasta, Antofagasta, Chile, ‡Instituto de Ciencias Ambientales & Evolutivas, Facultad de Ciencias, Universidad Austral de Chile, Independencia 641, P.O. Box 567, Valdivia, Chile

Abstract The spatial genetic structure (and gene flow) of parasites with complex life cycles, such as digeneans, has been attributed mainly to the dispersion ability of the most mobile host, which most often corresponds to the definitive host (DH). In this study, we compared the genetic structure and diversity of adult Neolebouria georgenascimentoi in two fish species (DHs) that are extensively distributed along the south-eastern Pacific (SEP). The analysis was based on the cytochrome oxidase subunit I gene sequences of parasites collected between 23°S and 45°S. In total, 202 sequences of N. georgenascimentoi in Pinguipes chilensis isolated from nine sites and 136 sequences of Prolatilus jugularis from five sites were analysed. Our results showed that N. georgenascimentoi is a species complex that includes three different parasite species; however, in this study, only lineage 1 and 2 found in P. chilensis and P. jugularis, respectively, were studied because they are widely distributed along the coastline. Lineage 1 parasites had two common haplotypes with wide distribution and unique haplotypes in northern sites. Lineage 2 had only one common haplotype with wide distribution and a large number of unique haplotypes with greater genetic diversity. Both lineages have experienced recent population expansion. Only lineage 1 exhibited a genetic structure that was mainly associated with a biogeographical break at approximately 30°S along the SEP. Our finding suggests that host access to different prey (=intermediate hosts) could affect the genetic structure of the parasite complex discovered here. Conse- quently, difference between these patterns suggests that factors other than DH dispersal are involved in the genetic structure of autogenic parasites.

Keywords: digeneans, genetic diversity, parasites, phylogeography, population genetic structure, south-eastern Paciﬁc Received 28 April 2014; revision received 8 January 2015; accepted 14 January 2015

that possess complex life cycles (using one or more Introduction intermediate hosts), such as trematodes, depend mostly The major factors affecting the genetic structure among on the potential of the hosts to disperse due to the populations of free-living organisms as well as parasites small size and limited intrinsic mobility of the infective are gene ﬂow, life history and, potentially, local adapta- stages of the parasites themselves (Blouin et al. 1995; tion within populations (Criscione 2008; Dionne et al. Criscione & Blouin 2004; Nieberding et al. 2008; Blasco- 2008; Blasco-Costa & Poulin 2013). However, parasites Costa et al. 2012). The population structures of parasites with complex life cycles are commonly determined by Correspondence: Zambra Lopez, Fax: +56-55-2637631/804; the dispersion ability of the most mobile host, which E-mail: [email protected] most likely corresponds to the deﬁnitive host (DH)

(Criscione & Blouin 2004; Criscione 2008; Keeney et al. predictors on the dispersal opportunities and genetic 2009; Louhi et al. 2010; Blasco-Costa et al. 2012; Blasco- structure patterns of autogenic marine parasites infest- Costa & Poulin 2013). The intermediate hosts of trema- ing one or several DHs. todes are generally invertebrates that possess limited The south-eastern Pacific (SEP) coast presents two dispersal abilities (Thieltges et al. 2011; Keeney et al. major biogeographical breaks (Camus 2001; Thiel et al. 2009). In contrast, the DHs are often vertebrates, which 2007). The northern break is at approximately 30°S and possess greater geographical dispersal capabilities is characterized by an important shift in the diversity, (Thieltges et al. 2011; Keeney et al. 2009). In addition, abundance and recruitment of several intertidal marine some authors have proposed that host specificity might invertebrate species (Broitman et al. 2001; Rivadeneira affect parasite diversification (Nadler 1995; Criscione et al. 2002). The southern break is located at approxi- et al. 2005) because gene flow might be facilitated or con- mately 42°S and has been recognized as a major biogeo- strained by the number of host species that a parasite graphical discontinuity (Camus 2001). At this latitude, a can use (Nadler 1995; Blasco-Costa & Poulin 2013; Falk divergence of the main oceanic currents (Humboldt and & Perkins 2013). Based on the cytochrome oxidase sub- Cape Horn current systems) occurs (Valdovinos et al. unit I gene (COI), Johnson et al. (2002) concluded that 2003). Several population genetic studies have recog- the lice species Physconelloides spp. and Columbicola spp. nized genetic barriers among marine species along the exhibited genetic structures that were in concordance SEP. Most of these studies were focused on the break at with their host specificity. Physconelloides spp. exhibited 30°S where several free-living invertebrate species high host specificity and greater genetic differentiation showed a genetic break in this area (Zakas et al. 2009; among localities than Columbicola spp., which is a more Sanchez et al. 2011; Brante et al. 2012; Varela & Haye generalist parasite (Johnson et al. 2002). Similarly, Falk & 2012; Vilches et al. 2012; Haye et al. 2014). One study Perkins (2013) (using 18S and COI) suggested that the showed that an intertidal gastropod has an additional differences in population structure between two Nema- genetic break at 42°S(Sanchez et al. 2011). However, a toda species (Spauligodon anolis and Parapharyngodon cub- number of free-living invertebrate species have not ensis) are associated with a greater number of hosts, shown a genetic break along the SEP (Cardenas et al. providing more opportunities for dispersal. 2009a; Haye et al. 2014). No previous phylogeographical Previous studies analysing population structures in studies have been performed on marine parasite spe- trematode parasites were performed mainly on small cies. However, given that parasites are closely tied to spatial scales (5–400 km) in both marine and freshwa- their host, parasites and their hosts might share similar ter systems (Keeney et al. 2008, 2009; Steinauer et al. phylogeographical patterns (Nieberding et al. 2004; Cri- 2009; Blasco-Costa et al. 2012). Only one study was scione et al. 2005; Criscione 2008). On the other hand, conducted on an extensive spatial scale (approximately the biogeographical patterns of prey are considered key 700 km, covering four rivers) with autogenic (species determinants of the endoparasite community structure that mature in fishes, sensu Esch et al. 1988) and allo- of the host (Gonzalez et al. 2006). Along the SEP, each genic (species that mature in other vertebrates) trema- biogeographical area is composed of particular commu- todes; in this study, the allogenic parasite species, nities of free-living organisms (Briggs 1974; Broitman which had a DH with high dispersal ability, did not et al. 2001; Rivadeneira et al. 2002), which provide (or exhibit a population genetic structure (Criscione & make available) different prey species (intermediate Blouin 2004). Additionally, Thieltges et al. (2011) analy- hosts) to the DH. Then, the geographical variations of sed the effect of dispersal capacity of the DH on the potential prey to the DH could affect the transmission ranges of European freshwater trematode fauna. The of parasite species and consequently the phylogeo- authors did not find differences in range sizes among graphical pattern in marine parasite species. trematode species using hosts with high (birds) and Here, we present the first study to compare the spa- limited dispersal capacity (e.g. fish), suggesting that the tial genetic diversity of one adult stage of a digenean host dispersal capacity for parasite dispersal on small Opecoelidae (Neolebouria georgenascimentoi; Bray 2002) spatial scales is diminished by other factors acting on a that parasitizes two marine fish species, Pinguipes chilen- larger scale (Thieltges et al. 2011). In a recent meta- sis and Prolatilus jugularis (Teleostei: Pinguipedidae), analysis, Blasco-Costa & Poulin (2013) concluded that which are distributed across different biogeographical the type of parasite life cycle (allogenic vs. autogenic) areas in the SEP. This digenean species was described is a better predictor of population genetic structure in by Bray (2002), who indicated that this species parasitiz- trematodes than the host geographical range. However, es the gastrointestinal tract of its two DH species. The their survey was focused on only parasites with a sin- holotype was defined as a parasite of P. chilensis, and gle DH from freshwater or terrestrial environments, the paratype was defined as a parasite of P. jugularis highlighting the necessity of testing the effects of these (Bray 2002). The life cycle of this trematode species is

© 2015 John Wiley & Sons Ltd 1062 Z. LOPEZ ET AL. almost unknown, but digeneans have complex life cycles involving at least two invertebrate hosts and hav- ing short free-living stages (Rohde 2005). The adults of Opecoelidae live in the digestive tract of marine and freshwater fishes (Jousson & Bartoli 2000), and some studies have shown that members of this family use snails (Prosobranchia) as a first intermediate host, shrimp (Crustacea) as a second intermediate host and a fish as a definitive host (Meenakshi et al. 1993; Jousson & Bartoli 2000; Yoshida & Urabe 2005). Pinguipes chilensis (Valenciennes 1833) is distributed approximately from Tumbes in Peru(3 °340S) to Magallanes in Chile (52°090S) (Oyarzun 2003), whereas Prolatilus jugularis (Valenciennes 1833) is distributed from Huacho (Peru, 11°060S) to Chiloe (Chile, 43°300S) (Chirichigno 1998), with sporadic records in Puerto Aysen (45°240S) (Oyarzun 2003). Pinguipes chilensis preferably inhabits the nearshore rocky subtidal habitat associated with Macrocystis pyrifera and Lessonia trabeculata kelps in the northern Chile (Ortiz 2008). However, other authors described this species inhabits associated with bare rock covered with noncalcareous algae and sand-intermediate microhabitats (Farina~ et al. 2005; Perez-Matus et al. 2007), and it is an active and generalist predator (Gonzalez & Oyarzun 2003; Medina et al. 2004). On the other hand, P. jugularis preferably inhabits the nearshore rocky and sandy subtidal habitat (Angel & Ojeda 2001; Cortes et al. 2012), and it is a second- or third- order consumer in the food web of coastal environments (Cortes et al. 2012). Although no information about the potential adult mobility is available, the degree of home fidelity of both fish species and data from other species of the same family suggests limited adult mobility and high home fidelity (e.g. Cole et al. 2000; Venerus et al. 2013). Thus, the P. chilensis–P. jugularis–N. georgenascimentoi system is an excellent model of a host–parasite system to evaluate the effect of the geographical range of the DHs on the spatial pattern of the genetic diversity in marine autogenic parasites. We expect that the parasite Fig. 1 Localities sampled along the south-eastern Pacific. The genetic diversity pattern of each DH reflects the geo- main biogeographical breaks are shown (30°S and 42°S). graphical barriers to gene flow as described above for some free-living organisms along the SEP. However, if the host ecological differences that allow the host access coastline. Between November 2011 and June 2013, sam- to different prey (=intermediate hosts) have an effect on ples of Pinguipes chilensis and Prolatilus jugularis were genetic structure, we expect to find genetic differentia- obtained from local fishermen using autonomous (scuba) tion between parasites from both DHs. or apnoea diving, and samples were immediately frozen at -20°C. Fishes and their parasites were collected from 9 sites (Fig. 1 and Table 1). The fish were subsequently Materials and methods thawed and dissected, and the parasites were recovered according to a standardized protocol. We recovered all Study area, fish sampling and parasite collection specimens of N. georgenascimentoi, but retained an aver- 0 0 Samples were obtained between 23°03 S and 70°30 W age of three parasites per fish for analysis, except in one 0 0 and 44°44 S-72°41 W (Fig. 1), along 2300 km of marine host from site 2, from which nine parasite specimens

Table 1 Population genetics summary statistics for both lineages (1 and 2) of Neolebouria georgenascimentoi from Pinguipes chilensis and Prolatilus jugularis at each site and total

– p Site Coordinates (S W) P (%) MI F N Nhap SHe Tajima’s D Fu’s FS

N. georgenascimentoi lineage 1 1a 23°030–70°300 60 3.6 6 8 6 10 0.929 (0.08) 0.004 (0.002) 1.51 1.88 1b 23°210–70°360 75 2.7 8 12 8 13 0.849 (0.10) 0.004 (0.003) 1.33 2.62 1c 23°290–70°310 60 3.8 8 13 10 17 0.923 (0.07) 0.005 (0.003) 1.44 4.29* 229°570–71°200 81 3.6 18 30 22 30 0.945 (0.03) 0.033 (0.020) 2.34** 21.68** 336°430–73°060 54.4 6.7 17 32 20 26 0.929 (0.03) 0.026 (0.017) 2.35* 18.81** 439°480–73°140 34.5 2.5 14 24 15 16 0.920 (0.04) 0.024 (0.015) 1.94* 12.32** 541°300–72°480 88.9 31.5 8 16 6 7 0.733 (0.10) 0.020 (0.015) 0.61 1.06 642°380–73°450 60 4.2 8 11 5 5 0.618 (0.16) 0.011 (0.009) 1.79* 2.31* 744°440–72°410 27.9 2.3 8 11 5 7 0.709 (0.14) 0.019 (0.014) 1.46 1.03 Total 51.7 5.5 95 157 81 85 0.918 (0.02) 0.032 (0.019) 2.6** 26.72** N. georgenascimentoi lineage 2 1b 23°210–70°360 21.7 4.6 4 5 5 19 1.000 (0.13) 0.098 (0.063) 1.07 0.68 229°570–71°200 90.5 4.1 26 37 30 47 0.982 (0.01) 0.054 (0.030) 2.17* 25.64** 336°430–73°060 71.4 4 16 30 25 39 0.966 (0.03) 0.052 (0.029) 2.07* 22.27** 439°480–73°140 68.8 5.1 15 36 31 47 0.983 (0.02) 0.059 (0.033) 2.06* 25.57** Total 68.8 4.3 61 108 83 88 0.977 (0.01) 0.058 (0.031) 2.34** 25.56**

P (%), prevalence; MI, mean intensity; F, number of ﬁsh used for the sequences obtained; N, number of sequences analysed; Nhap, p number of different haplotypes; S, number of polymorphic sites; He, haplotype diversity (standard deviation); , nucleotide diversity (standard deviation); Tajima’s D test (Tajima 1989) and Fu’s FS test (Fu 1997). *Signiﬁcant P-values (0.05). **P-values<0.001.

were used. The sampled parasites were stored in 95% ofeachprimer,0.6XBSA,3.5lL of DNA concentrate, 0.025 ethanol for subsequent DNA extraction. units of GoTaqâ DNA polymerase (Promega) and sufficient l The prevalence (number of fish infested with one or H2Otoreachthefinal35 L volume. For the V4 region, the more individuals of a determined parasite species thermocycling programme included an initial denaturation divided by the number of examined fish, expressed as step (94 °C for 5 min), 35 cycles of amplification (94 °Cfor percentage) and mean intensity (mean number of a par- 30 s, 45 °C for 30 s and 72 °C for 3 min) and a final exten- ticular parasite species per fish, considering only sion step (72 °C for 10 min) (Hall et al. 1999). The COI gene infested fish) were calculated (Bush et al. 1997). was amplified using the following thermocycling profile: an initial denaturation step (95 °C for 2 min), 40 cycles of amplification (95 °C for 30 s, 48 °C for 40 s and 72 °Cfor DNA extraction, amplification, sequencing and 1 min) and a final extension step (72 °Cfor10min)(Leung alignment et al. 2009). â The DNA of each individual was isolated following a The PCR products were purified using E.Z.N.A. modified protocol based on Miller et al. (1988) involving Cycle Pure PCR Purification Kit (Omega Bio-tek). The treatment with sodium dodecyl sulphate, digestion with purified PCR products were sequenced at Macrogen Proteinase K, NaCl protein precipitation and subsequent Inc. Company, South Korea (www.macrogen.com) ethanol precipitation of the DNA. using an ABI Prism 3730xl automated sequencer. Com- TheV4regionoftheSSUrRNA(V4region)wasampli- plementary sequences were assembled and edited using 0 fied using the primers SB3a (5 -GGAGGGCAAG PROSEQ v2.9 (Filatov 2002). The fragments obtained were 0 0 TCTGGTGC-3 ) and A27a (5 -CCATACAAATGCCCCCG aligned using the CLUSTAL 2 software package (Larkin TCTG-30) as described by Hall et al. (1999). The COI gene et al. 2007). was amplified using JB3 (50-TTTTTTGGGCATCCTG AGGTTTAT-30)(Bowleset al. 1993) as the forward primer 0 0 Distribution of Neolebouria georgenascimentoi and trem.cox1.rrnl (5 -AATCATGATGCAAAAGGTA-3 )of taxonomic units Kralov a-Hromadov a et al. (2001) as the reverse primer. All PCRs were performed in a final volume of 35 lL containing The V4 region was chosen due to the availability of

1X PCR buffer, 3 mM MgCl2,0.2mM of each dNTP, 0.4 pM primers that universally amplify trematode DNA (Hall

~ et al. 1999; Valdivia et al. 2010; Munoz et al. 2013); the (Tajima 1989) and Fu’s FS (Fu 1997) statistics were cal- V4 region is commonly used to study trematode phy- culated to assess the consistency of the observed genetic logeny and to identify operational taxonomic units and variation based on a neutral model of evolution for species (Hall et al. 1999; Valdivia et al. 2010). Addition- each sampling site and over all sites combined for each ally, the GenBank database contains many V4 region host with 1000 permutations using ARLEQUIN v3.1. Signif- sequences for several Digenea species, allowing further icant deviations from neutrality can be a consequence comparisons and analyses. The COI gene has also been of selection (as well as population expansions or bottle- used to determine taxonomic units and differentiation necks) or demographic fluctuations. Fu’s FS statistic is at the species level, but this gene is always compared caused by selection and population expansions and is with other, more conserved genes (Criscione & Blouin highly sensitive to demographic expansions, which pro- 2004; Miura et al. 2005). duce large negative values (Fu 1997). To determine the distribution of the taxonomic units Genetic population structures were examined for each in N. georgenascimentoi, data sets regarding the V4 lineage (host) using an hierarchical analysis of molecu- region were analysed using maximum likelihood (ML), lar variance (AMOVA) as implemented in ARLEQUIN v3.1. Φ neighbour-joining (NJ) and Bayesian inference (BI) Genetic structure ( ST estimate) was examined among Φ methods. ML and NJ analyses were performed using all sites. Genetic structure ( CT estimate) was also the software package Mega v6 (Tamura et al. 2013), and examined among three regions separated by two bio- BI was performed using the software package Mr. geographical breaks (Camus 2001); these regions were Bayes (Huelsenbeck & Ronquist 2001). To determine the the ‘Peruvian Province (PP)’ (sites 1a, 1b and 1c), the nodal support in ML and NJ, a 1000 bootstrap analysis ‘Intermediate Area (IA)’ (sites 2, 3 and 4) and the ‘Mag- was used. For the ML and NJ analyses, the TN93 evolu- ellanic Province (MG)’ (sites 5, 6 and 7). Genetic struc- Φ tion model was used, and for the BI analyses, the ture ( SC estimate) was examined among sites within HKY+G model was used. Both models were chosen these regions. The AIC of JMODELTEST version 3.7 (Posada according to the Akaike information criterion (AIC) as & Crandall 1998) was used to select the most appropri- implemented in Modeltest 3.7 (Posada & Crandall ate model of sequence evolution according to each DH 1998). To estimate BI inference, posterior probabilities species. Based on this method, TPM2uf+I was the most were estimated over 50 000 000 generations via one run appropriate model for sequences from the host P. chil- of four simultaneous Markov chain Monte Carlo chains ensis, and TIM2 + I+G was the most appropriate model with every 1000th tree saved. The first 5 000 000 genera- for sequences from the host P. jugularis. However, tions (10% burn-in) were discarded as suggested by because these models are not implemented in ARLEQUIN Felsenstein (1985). Peracreadium idoneum (GenBank v3.1, the Tamura & Nei (1993) model with gamma dis- Accession no AJ287558.1) was used as an outgroup spe- tribution (a = 0.115 for P. chilensis and a = 0.016 for cies and, Macvicaria macassarensis (AJ287533.1) was used P. jugularis) was used. The significance of genetic struc- as a sister group (Olson et al. 2003). ture was determined based on 10 000 permutations The COI gene was used to aid in determining the (Excoffier et al. 1992). Additionally, patterns of genetic number of species using the approximation of delinea- divergence were investigated using the spatial AMOVA tion of species boundaries in the automatic barcode gap procedure and SAMOVA v.1.0 (Dupanloup et al. 2002) to discovery method (ABGD) (Puillandre et al. 2012). define the number of groups along the SEP populations These methods deliver species circumscriptions based that are geographically and genetically homogeneous on patterns of pairwise genetic distances (ABGD), pro- and maximally differentiated from each population. viding estimates of a maximum limit for intraspecific This method is based on a simulated annealing proce- genetic divergence and using this limit to group dure that aims to maximize the proportion of total sequences belonging to the same species (with lesser genetic variance due to differences among groups of Φ divergences) from sequences belonging to different spe- populations. Finally, the fixation index ( ST) was calcu- cies (with greater divergences) (Puillandre et al. 2012). lated for pairwise comparisons between all collection sites. A haplotype network was constructed using HAPLO- Genetic structure analysis using the COI gene VIEWER (available at http://www.cibiv.at/~greg/haplo-

The number of unique haplotypes (Nhap), the number viewer) and a neighbour-joining tree reconstructed with of polymorphic sites (S), haplotype diversity (He) and MEGA v6 for trematodes in both DHs. To distinguish his- nucleotide diversity (p) were calculated for both lin- torical growth events and population declines, a mis- eages (from each host species) at each sampling site match distribution analysis was performed at each and over all sites (all individuals treated as one sample) sampling site according to the studied DH and over using ARLEQUIN v3.1 (Excofﬁer et al. 2005). Tajima’s D all sites with 1000 permutations; the analysis was

© 2015 John Wiley & Sons Ltd GENETIC STRUCTURE OF DIGENEANS IN HOST FISHES 1065 performed using ARLEQUIN v3.1. To estimate the time elapsed since the expansion, we used s = 2lt, where t = time (in generations) and l = mutation rate/generation. The s parameter is an estimate of the time elapsed after expansion in mutational units. If the divergence rate per nucleotide and year (s = 2l, where l is the substitution rate per lineage) and the number of nucleo- tides of the fragment analysed (1) are known, it is possible to calculate the time when the expansion occurred using the expression s = llt, as modiﬁed from Harpending et al. (1993) and obtained by Cardenas et al. (2009a). Isolation by distance was tested using the relationship between genetic (FST⁄1-FST) (Rousset 1997) and geographical distance along SEP among all sites for each lineage using a Mantel test (Mantel 1967) as implemented in the Isolation by DISTANCE WEB SERVICE version 3.15 (Bohonak 2002; Jensen et al. 2005).

Results

DNA sequencing and identification of taxonomic units of Neolebouria georgenascimentoi Fig. 2 Phylogenetic tree of 34 specimens of Neolebouria george- A total of 68 sequences (forward and reverse) of the V4 nascimentoi obtained from the definitive hosts Pinguipes chilensis region from 34 specimens of N. georgenascimentoi were (Pc) and Prolatilus jugularis (Pj) from sites 1a to 7 (s1a—s7) examined: 16 individuals were obtained from P. chilen- based on maximum-likelihood analyses of the V4 region. Num- sis and 18 were obtained from P. jugularis (the bers along the branches indicate the percentages of support sequences were submitted to GenBank under access values resulting from the different analyses in the order ML/ numbers KJ527643–KJ527676, Table S1, Supporting in- NJ/BI. Values lower than 50% are indicated by dashes or are not indicated. The model for the ML and NJ trees (TN93) had fromation). The total length of the analysed V4 region an -lnL score of 623.5784 and an Akaike information criterion sequences was 392 bp. The analysis to determine the (AIC) of 1397.1567. distribution of taxonomic units and/or number of species revealed that N. georgenascimentoi could be classified into three lineages (Fig. 2). The first lineage NCBI database with access numbers KJ527677 to included specimens from P. chilensis found at sites 1a to KJ528014 (Table S1, Supporting infromation). The 7 (lineage 1). The second lineage incorporates parasites ABGD analysis showed a tri-modal pairwise genetic from P. jugularis found at sites 1b, 2, 3 and 4 (lineage distance (K2P) distribution with a clear and wide bar- 2). Finally, a third lineage was identified that included code gap located between 3 and 8% of genetic distance parasites collected from P. jugularis at sites 1b, 2 and 5 and a second gap located between 11 and 14% of (lineage 3) (Fig. 2). The genetic distance between lin- genetic distance (Fig. 3a). Furthermore, the method eages 1 and 2 was 0.3%, the distance between lineages used detected three stable candidate species with esti- 1 and 3 was 0.5%, and the distance between lineages 2 mated prior maximum divergences of intraspecific and 3 was 0.8%. Within each lineage, no mutations diversity (P) as large as 6% (Fig. 3b) (one-tail 95% confi- were detected; therefore, the genetic distance was 0%. dence interval). Notably, this result was consistent with Similar tree topologies were obtained using the three the three N. georgenascimentoi lineages found using the different methods (Fig. 2). Lineage 1 was closely related phylogenetic analysis (Fig. 2). to lineage 2 with a node support of 66% to ML and 61% to NJ and a posterior probability of 0.95 to BI Genetic structure analysis (Fig. 2). DNA sequences comprising 739 bp of the COI gene In the population analysis, we incorporated sample lin- were analysed for 338 individuals of N. georgenascimen- eages 1 and 2 as shown in Fig. 2 because they are toi collected from P. chilensis (202 parasites) and P. jugu- widely distributed along the sampled hosts and coast- laris (136 parasites). Sequences were deposited at the line. Lineage 3 was restricted to a few sites (1b, 2 and

(a) suggesting that selection, population expansion or bot- tlenecks might be affecting the current genetic diversity. Hierarchical AMOVA analysis revealed significant genetic differentiation among lineage 1 (ΦCT = 0.17; P-values 0.05), and 17.08% of the genetic variance was explained by the ‘PP’, ‘IA’ and ‘MP’ groupings (Table 2). In contrast, hierarchical AMOVA analysis did not reveal significant genetic differentiation among lineage 2 (ΦCT = 0.05; P-values > 0.2) (see Table 2). The data from lineage 1 are best explained using SAM- OVA by assuming three groups of populations (/CT = 0.256, P-values = 0.0068) (Table 2). For lineage 2, SAMOVA analysis did not reveal an optimized aggregation. The pairwise Φ of lineage 1 exhibited significant differ- (b) ST ences among 25 out of 36 comparisons (P-values < 0.05), and the sequences obtained from sites 1a, 1b and 1c were significantly different from the sequences obtained from sites 2 to 7 (Table S2, Supporting infor- Φ mation). The pairwise ST for lineage 2 exhibited significant differences in 2 of 6 comparisons (P-values < 0.05) (Table S2, Supporting information). The haplotype network for lineage 1 (Fig. 4a) revealed two common haplotypes, occurring at frequen- cies of 27% (H23) and 11% (H25) along sites 2 to 7. The haplotype H23 is located in the centre of the network, suggesting that it could be the ancestral haplotype. Additionally, one common haplotype (H2) was con- Fig. 3 Distribution of pairwise distances for the COI gene and nected by one mutation at the central haplotype (H23) automatic barcode gap discovery (ABGD). a) Frequency distribution of K2P distances between haplotype pairs for the COI at sites 1a, 1b and 1c (Fig. 4a). The following five addi- gene. b) ABGD results showing the number of lineages tional haplotypes were shared between two sites: H5 obtained for a range of prior maximum divergences of intra- (sites 1b and 2), H13 (sites 1a and 1c), H31 (sites 2 and specific diversity. Dashed lines (a and b) indicate the upper 5), H45 (sites 3 and 4) and H74 (sites 5 and 6). All hapl- bound of estimated maximum limits for intraspecific genetic otypes were connected by a maximum of eight muta- divergences that resulted in two stable candidate species. tions (usually fewer). The haplotype network for lineage 2 exhibited only one common haplotype (H5) with a frequency of 15% (Fig. 4b) and a high number of 5); at site 1b and 5, individuals of lineage 3 were abun- unique haplotypes. In fact, the genetic diversity was dant, but at site 2, only one individual was present. highest in lineage 2 (P-values < 0.01). The following five Therefore, we decided to exclude this lineage from the additional shared haplotypes were detected between following analysis. two sites: H6 (sites 2 and 4), H43 (sites 3 and 4), H48 To analyse the genetic diversity and structure at the (sites 3 and 4), H55 (sites 3 and 4) and H58 (sites 3 and component population level (i.e. all of the individuals 4). All haplotypes were connected by seven or fewer of a specified life-history phase at a particular place mutations. The haplotype network of each lineage and time, according Bush et al. 1997), we excluded exhibited a star-like structure with one central haplo- those sequences that were similar within an individual type, suggesting that each lineage of trematode para- fish from the analysis. Thus, 157 individuals from line- sites had most likely undergone a recent population age 1 and 108 individuals from lineage 2 were incorpo- expansion (Fig. 4). The mismatch distribution analysis rated. Lineage 1 exhibited 85 polymorphic sites (Fig. 5) exhibited a unimodal distribution of pairwise segregating 81 different haplotypes, whereas lineage 2 differences for lineage 1 and lineage 2, which is consis- contained 88 polymorphic sites segregating 83 haplo- tent with a sudden population expansion model types (Table 1). Neutrality tests yielded nonsignificant (Fig. 5a). Based on a mutation rate of 2.5 e 8 per site results for sites 1a, 1b, 1c, 5 and 7 in lineage 1 and for (Attwood et al. 2008) and assuming a generation time of site 1b in lineage 2. Neutrality tests were significant 1 year, the onset of the most recent demographic expan- for the entire data sets in lineages 1 and 2 (Table 1), sion in lineages 1 and 2 was estimated. The estimate of

Table 2 Result of AMOVA and SAMOVA for N. georgenascimentoi lineage 1 and lineage 2

Structure tested % Variation among group F statistic P-values

Neolebouria georgenascimentoi lineage 1 AMOVA 3 (Site 1a, Site 1b, Site 1c) 17.08 ΦSC = 0.01554 0.10020 (Site 2, Site 3, Site 4) (Site 5, Site 6, Site 7) ΦST = 0.18362 <0.00001 ΦCT = 0.17073 0.00426 3 (Site 1a, Site 1b, Site 1c) 18.49 ΦSC = 0.01487 0.09030 (Site 2, Site 3, Site 4, Site 6) (Site 5, Site 7) ΦST = 0.19703 <0.00001 ΦCT = 0.18491 0.00158 SAMOVA 2 (Site 1a, Site 1b, Site 1c) 25.56 ΦSC = 0.02436 0.04106 (Site 2, Site 3, Site 4, Site 5, Site 6, Site 7) ΦST = 0.27376 <0.00001 ΦCT = 0.25563 0.01466 3 (Site 1a) (Site 1b, Site 1c) 25.59 ΦSC = 0.01862 <0.00001 (Site 2, Site 3, Site 4, Site 5, Site 6, Site 7) ΦST = 0.26977 <0.00001 ΦCT = 0.25591 0.00684 4 (Site 1a) (Site 1b, Site 1c) 21.92 ΦSC = 0.0013 0.02737 (Site 2, Site 3, Site 4, Site 6, Site 7) (Site 5) ΦST = 0.22020 <0.00001 ΦCT = 0.21919 0.00098 5 (Site 1a) (Site 1b, Site 1c) 20.03 ΦSC = -0.00559 <0.00001 (Site 2, Site 3, Site 4, Site 6) (Site 5) (Site 7) ΦST = 0.19582 <0.00001 ΦCT = 0.20029 0.00196 Neolebouria georgenascimentoi lineage 2 AMOVA 2 (Site 1b) (Site 2, Site 3, Site 4) 5.52 ΦSC = 0.00293 0.21505 ΦST = 0.05799 0.08407 ΦCT = 0.05522 0.24927 SAMOVA 2 (Site 1b) (Site 2, Site 3, Site 4) 5.57 ΦSC = 0.0029 0.21114 ΦST = 0.05843 0.07722 ΦCT = 0.05570 0.24242 3 (Site 1b) (Site 2) (Site 3, Site 4) 3.40 ΦSC = -0.01520 0.99707 ΦST = 0.01935 0.05670 ΦCT = 0.03404 0.17595

s for the entire data set corresponded to an onset of with Pinguipes chilensis and, showed a genetic break at expansion of 127 000 (95% confidence interval = 98 000– approximately 30°S, coincident with those breaks 150 000) years before present (bp) for lineage 1, whereas described for free-living organisms along the SEP. In for lineage 2, the expansion was calculated at contrast, lineage 2 that parasitized only Prolatilus jugu- 235 000 bp (95% confidence interval = 140 000–362 000). laris did not show evidence of genetic breaks along a Mantel tests revealed a significant correlation similar geographical area. Our finding suggests that a between genetic and geographical distances for lineage host’s access to different prey (=intermediate hosts) 1(r= 0.7742; P-values = 0.003) but not for lineage 2 could affect the genetic structure of the parasite com- (r = 0.2997; P-values = 0.292) (Fig. 6). plex discovered here. Records of cryptic species are becoming more common as more studies utilize molecular markers (Crisci- Discussion one & Blouin 2004; Criscione et al. 2005, 2011; Miura Here, we describe the population genetic structure of et al. 2005; Falk & Perkins 2013). We demonstrated that the digenean Neolebouria Neolebouria georgenascimentoi N. georgenascimentoi corresponds to a species complex that parasitizes two fish species distributed across the that includes at least three Neolebouria spp. (see Figs 2 SEP. Our results showed the occurrence of a species and 3). Bray (2002) described N. georgenascimentoi para- complex with particular genetic lineages associated with sitization of Prolatilus jugularis and Pinguipes chilensis; each DH (here, we reported the results for the two the author reported little morphological variation in main lineages: lineages 1 and 2). Lineage 1 associated this parasite between both hosts apart from finding a

(a) (b)

Fig. 4 Median-joining haplotype networks for a) Neolebouria georgenascimentoi lineage 1 from Pinguipes chilensis and b) N. georgenascimentoi lineage 2 from Prolatilus jugularis. Each circle represents a haplotype, and the circled area represents haplotype frequency. Small blue circle inserts in the branches represent inferred haplotypes that are not observed in the data or median vectors; all connec- tions represent a single mutational step.

distinctly greater number of ovarian follicles (lobes) in species are not available to date. However, the existing parasites obtained from P. jugularis. However, parasites works reveal that P. chilensis is a generalist species that from P. chilensis are longer and show higher fecundity preys on several species, such as crustacean, fishes, than those parasites collected from P. jugularis annelids, mollusks, echinoderms and others (Moreno & (Gonzalez et al. 2013). Currently, N. georgenascimentoi Flores 2002; Gonzalez & Oyarzun 2003; Perez-Matus has been recorded in only these two host species along et al. 2012; Cornejo-Acevedo et al. 2014). In the northern the Chilean coast (Munoz~ & Olmos 2008; Gonzalez & range of Chile (between 21°S and 30°S), the primary Oliva 2009), but our data strongly suggest the occur- prey is crustaceans, principally Pilumnoides perlatus, rence of cryptic (morphologically similar but genetically Petrolisthes sp. and Rhynchocinetes typus (Perez-Matus distinct) species. Genetic subdivision among parasites et al. 2012). At around 30°S, Moreno & Flores (2002) in different host species could arise through extrinsic or described as the primary prey crustaceans specifically intrinsic mechanisms (McCoy 2003). Here, the transmis- Rhynchocinetes typus and Petrolisthes violaceus, whereas sion and dispersal of parasites are important factors to in the southern range (at approximately 38 °S), the pri- be considered (Criscione et al. 2005). In trematodes, ces- mary prey is the crustaceans Neomysis sp., followed by todes and nematodes, these two processes occur pas- unidentified crustaceans and Synalpheus spinifrons sively by ingestion of an intermediate host, indicating (Gonzalez & Oyarzun 2003), and recently Cornejo-Acev- that not all local DH types will necessarily be available edo et al. (2014) showed results from southern range (at to parasite individuals. If an infected sympatric DH approximately 39°S) indicating that the primary prey uses a different ecological niche (such as space and also is crustaceans mainly Homalaspis plana followed by food), separate parasite propagule pools that infect dif- Taliepus dentatus. ferent intermediate hosts could form, resulting in The published evidence shows that P. jugularis preys genetic isolation. In this case, the physical barrier is the on a lower number of species, such as crustaceans, distance between the hosts, and this result could be annelids, platyhelminthes and nemertines; the primary considered allopatric speciation (McCoy 2003). prey is the crustacean species Pagurus sp., followed by Few studies have analysed diet in these fish species, the platyhelminth Thyttosoceros inca (Moreno & Flores and therefore, studies comparing diets between the 2002). Both fish species showed a minimum overlap of

(a) (a)

(b)

Fig. 6 Isolation by distance analysis. Relationship between pairwise geographical distance and genetic distance (ΦST/1- ΦST) between sites for a) Neolebouria georgenascimentoi lineage 1 and b) N. georgenascimentoi lineage 2. Darker dots indicate the Fig. 5 Pairwise mismatch distribution analysis of a) N. georgenasci- relationship between sites 1a, 1b and 1c and the other sites. mentoi lineage 1 and b) Neolebouria georgenascimentoi lineage 2. prey, and they have minimized trophic competition consistent with the observed phylogeographical breaks using different substrata; P chilensis preys on species along the SEP for free-living organisms (Tellier et al. inhabiting rocky substrata, whereas P. jugularis preys 2009; Sanchez et al. 2011; Brante et al. 2012; Haye et al. on species in sandy substrata (Moreno & Flores 2002). 2014). Further analysis including more sites might be In addition, with respect to the space utilization, P. chil- necessary to clarify the current influence of biogeo- ensis inhabits rocky and sand-intermediate environ- graphical barriers on the genetic structure of this trema- ments with high home fidelity (Cole et al. 2000; Ortiz tode species and/or the intermediate or DH. 2008; Venerus et al. 2013), whereas P. jugularis inhabits Additionally, lineage 1 exhibited a clear pattern of isola- the nearshore rocky and sandy subtidal habitat (Angel tion by distance (see Fig. 6). This result supports the & Ojeda 2001; Cortes et al. 2012). However, P. jugularis existence of an oceanographic barrier across latitudes 23 is captured as ‘bycatch’ of the demersal fisheries that and 30°S that could influence the genetic structure of use trawl nets (Melo et al. 2007), suggesting that its the host species, thus preventing parasite dispersion home fidelity could be less than P. chilensis, like was among these sites (Criscione & Blouin 2007). Similar reported for other Pinguipidae, the blue cod Parapercis biogeographical and phylogeographical patterns colias by Dıaz-Guisado (2014); therefore, P. jugularis can between DHs and their hosts have been observed for be classified as a species with moderate mobility. Con- some parasite–host relationships (Wickstrom€ et al. 2003; sequently, lineage 1 and lineage 2 N. georgenascimentoi Meinila et al. 2004; Criscione & Blouin 2007). Therefore, may be transmitted by different prey (=intermediate the oceanographic barrier that affects the gene flow of hosts). parasite transmission northward to 30°S most likely also The population structure of trematodes has been affects the genetic structure of the DH. However, this attributed to the dispersion ability of the most mobile hypothesis should be tested by conducting genetic host, which most likely corresponds to the DH (Crisci- analyses of this host species. one & Blouin 2004; Keeney et al. 2009; Blasco-Costa Unlike lineage 1, lineage 2 did not show any genetics et al. 2012). In this study, lineage 1 exhibited a genetic breaks along the SEP; lineage 2 recorded a higher hap- break at approximately 30°S (between sites 1a–c and 2) lotype and nucleotide diversity than lineage 1, and only and approximately 42 S (sites 5 and 7); this finding was one haplotype (H5) was widely shared (Fig. 4b). This

© 2015 John Wiley & Sons Ltd 1070 Z. LOPEZ ET AL. lack of a genetic break pattern has also been observed population along the SEP. The difference between these for some free-living organisms that inhabit this region patterns suggests that factors other than DH dispersal (Cardenas et al. 2009a,b; Ibanez~ et al. 2011). A lack of (e.g. wider range of intermediate hosts) are involved in genetic structure has also been recorded in endopara- the genetic structure of the autogenic parasites. sites of freshwater organisms such as Cestoda (Ligula intestinalis), which are present in cyprinid fish (the Acknowledgements intermediate hosts) (Stefka et al. 2009), and allogenic trematodes (Nanophyetus salmincola), which are present We are grateful to Felipe Docmac to provide some fish samples in salmonids (the intermediate hosts) (Oncorhynchus for this study. The authors express their thanks to E. Poulin, mykiss, O. clarki, O. kisutch and O. tshawytscha) (Crisci- the editor and the anonymous referees for their constructive suggestions, which considerably improved the quality of the one & Blouin 2004). The DHs of these species are terres- paper. LC acknowledgement to the Millennium Nucleus trial birds and mammals with high dispersion ability. Center for the Study of Multiple drivers on Marine Socio-Eco- Considering that P. jugularis and P. chilensis present logical Systems (MUSELS) by MINECON Project NC120086. similar geographical ranges (and most likely dispersal This research was partially supported by projects INNOVA patterns), lineage 2 may parasitize a widespread sec- CORFO 09CNN14-5829, FONDECYT 11090149 and FONDE- ondary intermediate host’s range, thus favouring its CYT 1130629 granted to MTG. genetic flow and the absence of parasite genetic structure in its host (Nadler 1995; Johnson et al. 2002; Falk & References Perkins 2013). Species inhabiting the same biogeographical area can Angel A, Ojeda FP (2001) Structure and trophic organization of subtidal fish assemblages on the northern Chilean coast: the present different and independent evolutionary histo- effect of habitat complexity. Marine Ecology Progress Series, ries (Poulin 2007). In this study, both lineages revealed 217,81–91. a star-like network in which the most common and cen- Attwood SW, Fatih FA, Upatham ES (2008) DNA-Sequence tral haplotype, and therefore the most probable ances- variation among Schistosoma mekongi populations and related tral haplotype (Avise 2000; Hewitt 2000), is connected taxa; phylogeography and the current distribution of asian by a few mutation steps to many haplotypes of lower schistosomiasis. Plos neglected tropical diseases, 2, e200. frequency. However, lineage 2 showed a larger number doi:10.1371/journal.pntd.0000200. Avise JC (2000) Phylogeography: the History and Formation of Spe- of mutation steps in the network. Network and mis- cies. Harvard University Press, Cambridge, MA. match distribution are consistent with a demographic Blasco-Costa I, Poulin R (2013) Host traits explain the genetic expansion that is associated with the colonization of structure of parasites: a meta-analysis. Parasitology, 140, new geographical regions (Excoffier 2004) in both lin- 1316–1322. eages. According to coalescence theory (Slatkin & Hud- Blasco-Costa I, Waters JM, Poulin R (2012) Swimming against son 1991), the present expansion pattern suggests an the current: genetic structure, host mobility and the drift par- – expansion of the population from a limited number of adox in trematode parasites. Molecular Ecology, 21, 207 217. Blouin MS, Yowell CA, Courtney CH, Dame JB (1995) Host founders. The results of the coalescence-based demo- movement and the genetic structure of populations of para- graphic analysis are consistent with an expansion sitic nematodes. Genetics, 141, 1007–1014. growth model, and calculations reveal the onset of the Bohonak AJ (2002) IBD (Isolation by Distance): a program for expansion of lineage 1 at approximately 120 000 years analyses of isolation by distance. Journal of Heredity, 93, 153– (at the beginning of the Taratian Pleistocene) and the 154. onset of the expansion of lineage 2 at approximately Bowles J, Hope M, Tiu WU, Xushian L, McManus DP (1993) 230 000 years (at the end of the Ionian Pleistocene). Nuclear and mitochondrial genetic markers highly conserved between Chinese and Philippine Schistosoma japonicum. Acta In summary, in this first study of the spatial genetic Tropica, 55, 217–229. diversity patterns of a marine parasite on a large bio- Brante A, Fernandez M, Viard F (2012) Phylogeography and geographical scale (along the SEP), we found that biogeography concordance in the marine gastropod Crepipa- N. georgenascimentoi corresponds to a species complex tella dilatata (Calyptraeidae) along the southeastern Pacific that includes three species. N. georgenascimentoi lineage coast. Journal of Heredity, 103, 630–637. 1 and lineage 2 revealed a recent population expansion, Bray R (2002) Three species of plagioporine opecoelids (Dige- given that they exhibited star-like structures and uni- nea), including a new genus and two new species, from marine fishes from off the coast of Chile. Systematic Parasitology, modal mismatch distributions. Only lineage 1 exhibited 51, 227–236. a genetic structure that was mainly associated with a Briggs JC (1974) Marine Zoogeography. McGraw-Hill Co., New ° biogeographical break at approximately 30 S (Camus York. 2001), suggesting the existence of several populations Broitman BR, Navarrete SA, Smith F, Gaines SD (2001) Geo- along the SEP. The lack of a genetic structure in lineage graphic variation of southeastern Pacific intertidal communi- 2 suggests that this species comprises a single large ties. Marine Ecology Progress Series, 224,21–34.

Bush AO, Lafferty KD, Lotz JM, Shostak AW (1997) Parasitol- Excoffier L (2004) Patterns of DNA sequence diversity and ogy meets ecology on its own terms: Margolis et al. revisited. genetic structure after a range expansion: lessons from the Journal of Parasitology, 83, 575–583. infinite-island model. Molecular Ecology, 13, 853–864. Camus PA (2001) Biogeografıa marina de Chile continental. Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecu- Revista Chilena de Historia Natural, 74, 587–617. lar variance inferred from metric distances among DNA Cardenas L, Castilla JC, Viard F (2009a) A phylogeographical haplotypes: application to human mitochondrial DNA analysis across three biogeographical provinces of the South- restriction data. Genetics, 131, 479–491. Eastern Pacific: the case of the marine gastropod Concholepas Excoffier L, Laval G, Schneider S (2005) ARLEQUIN ver.3.0: an concholepas. Journal of biogeography, 36, 969–981. integrated software package for population genetics data Cardenas L, Silva A, Magoulas A, Cabezas J, Poulin E, Ojeda analysis. Evolutionary Bioinformatics Online, 1,47–50. FP (2009b) Genetic population structure in the Chilean Jack Falk BG, Perkins SL (2013) Host specificity shapes population mackerel, Trachurus murphyi (Nichols) across the South-east- structure of pinworm parasites in Caribbean reptiles. Molecu- ern Pacific Ocean. Fisheries Research, 100, 109–115. lar Ecology, 22, 4576–4590. Chirichigno NF (1998) Clave para identificar los peces marinos del Farinã JM, Palma AT, Ojeda FPA (2005) Subtidal Kelp- Peru, 2nd edn. Instituto del Mar del Peru, Publicacion Espe- Associated Communities off the Temperate Chilean Coast. In: cial, Callao, Peru´. 496 pp. Food Webs and the Dynamics of Marine Reefs (eds McClanahan Cole RG, Villouta E, Davidson RJ (2000) Direct evidence of lim- TR, Branch GM), pp. 75–102. Oxford University Press, UK. ited dispersal of the reef fish Parapercis colias (Pinguipedidae) Felsenstein J (1985) Confidence limits on phylogenies: an within a marine reserve and adjacent fished areas. Aquatic approach using the bootstrap. Evolution, 39, 783–791. Conservation: Marine and Freshwater Ecosystems, 10, 421–436. Filatov DA (2002) PROSEQ: software for preparation and evolu- Cornejo-Acevedo MF, Fierro P, Bertran C, Vargas-Chacoff L tionary analysis of DNA sequence datasets. Molecular Ecology (2014) Dietary composition and overlap of Pinguipes chilensis Notes, 2, 621–624. (Perciformes: Pinguipedidae), Cheilodactylus variegatus (Perci- Fu YX (1997) Statistical tests of neutrality of mutations against formes: Cheilodactylidae) and Aplodactylus punctatus (Perci- population growth, hitchhiking and background selection. formes: Aplodactylidae) in the Valdivian coast, Chile. Genetics, 147, 915–925. Gayana, 78,98–108. Gonzalez MT, Oliva ME (2009) Is the nestedness of the parasite Cortes N, Ibanez~ C, Garcıa C (2012) Peces de Chile, principales assemblages of marine fishes from southeastern Pacific a peces marinos de importancia comercial de la zona Centro- generalized pattern associated with the geographic distribu- Sur de Chile. http://www2.udec.cl/~coyarzun/catalogo/ tional range of the host? Parasitology, 136, 401–409. Catalogo1.htm Gonzalez P, Oyarzun C (2003) Diet of the Chilean sandperch, Criscione CD (2008) Parasite co-structure: broad and local scale Pinguipes chilensis (Peciformes, Pinguipedidae) in southern approaches. Parasite, 15, 439–443. Chile. Journal of Applied Ichthyology, 19, 371–375. Criscione CD, Blouin MS (2004) Life cycles shape parasite evo- Gonzalez MT, Barrientos C, Moreno CA (2006) Biogeographical lution: comparative population genetics of salmon trema- patterns in endoparasite communities of a marine fish (Se- todes. Evolution, 58, 198–202. bastes capensis) with extended range in the Southern Hemi- Criscione CD, Blouin MS (2007) Parasite phylogeographical sphere. Journal of Biogeography, 33, 1086–1095. congruence with salmon host evolutionarily significant units: Gonzalez MT, Henrıquez V, Lopez Z (2013) Variations in the implications for salmon conservation. Molecular Ecology, 16, fecundity and body size of digenean (Opecoelidae) species 993–1005. parasitizing fishes from Northern Chile. Revista de Biologıa Criscione CD, Poulin R, Blouin MS (2005) Molecular ecology of Marina y Oceanografıa, 48, 421–429. parasites: elucidating ecological and microevolutionary pro- Hall K, Cribb T, Barker S (1999) V4 region of small subunit cesses. Molecular Ecology, 14, 2247–2257. rDNA indicates polyphyly of the Fellodistomidae (Digenea) Criscione CD, Vilas R, Paniagua E, Blouin MS (2011) More than which is supported by morphology and life-cycle data. meets the eye: detecting cryptic microgeographic population Systematic Parasitology, 43,81–92. structure in a parasite with a complex life cycle. Molecular Harpending HC, Sherry ST, Rogers AR, Stoneking M (1993) Ecology, 20, 2510–2524. The genetic structure of ancient human populations. Current Dıaz-Guisado D (2014) Effects of Marine Reserve Protection on Anthropology, 34, 483–496. Adjacent Non-protected Populations in New Zealand. PhD Haye PA, Segovia NI, Munoz-Herrera~ NC et al. (2014) Phyloge- Thesis, Victoria University of Wellington, Wellington, New ographic structure in benthic marine invertebrates of the Zealand, 222 pp. Southeast Pacific Coast of Chile with differing dispersal Dionne M, Caron F, Dodson JJ, Bernatchez L (2008) Landscape potential. PLoS ONE, 9, e88613. genetics and hierarchical genetic structure in Atlantic sal- Hewitt GM (2000) The genetic legacy of the Quaternary ice mon: the interaction of gene flow and local adaptation. ages. Nature, 405, 907–913. Molecular Ecology, 17, 2382–2396. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference Dupanloup I, Schneider S, Excoffier L (2002) A simulated of phylogenetic trees. Bioinformatics, 17, 754–755. annealing approach to define the genetic structure of popula- Ibanez~ CM, Cubillos LA, Tafur R, Arguelles€ J, Yamashiro C, Poulin tions. Molecular Ecology, 11, 2571–2581. E (2011) Genetic diversity and demographic history of Dosidicus Esch GW, Kennedy CR, Bush AO, Aho JM (1988) Patterns in gigas (Cephalopoda: Ommastrephidae) in the Humboldt Current helminth communities in freshwater fish in Great Brit- System. Marine Ecology Progress Series, 431,163–171. ain: alternative strategies for colonization. Parasitology, 96, Jensen JL, Bohonak AJ, Kelley ST (2005) Isolation by distance, 519–532. web service. BMC Genetics, 6, 13.

Johnson KP, Williams BL, Drown DM, Adams RJ, Clayton DH ennes 1833 y Prolatilus jugularis Valenciennes 1833 en bahıa (2002) The population genetics of host specificity: genetic de la Herradura, Coquimbo, durante primavera del 2001. differentiation in dove lice (Insecta: Phthiraptera). Molecular Gayana, 66, 213–217. Ecology, 11,25–38. Munoz~ G, Olmos G (2008) Bibliographic revision of endopara- Jousson O, Bartoli P (2000) The life cycle of Opecoeloides colum- site and host species from aquatic systems of Chile. Revista bellae (Pagenstecher, 1863) n. comb. (Digenea, opecoelidae): de Biologıa Marina y Oceanografıa, 43, 173–245. evidence from molecules and morphology. International Jour- Munoz~ G, Lopez Z, Cardenas L (2013) Morphological and nal for Parasitology, 30, 747–760. molecular analyses of larval trematodes in the intertidal Keeney DB, Bryan-Walker K, King TM, Poulin R (2008) Local bivalve Perumytilus purpuratus of central Chile. Journal of variation of within-host clonal diversity coupled with genetic Helminthology, 87, 356–363. homogeneity in a marine trematode. Marine Biology, 154, Nadler SA (1995) Microevolution and the genetic structure of 183–190. parasite populations. Journal of Parasitology, 81, 395–403. Keeney DB, King TM, Rowe DL, Poulin R (2009) Contrasting Nieberding CM, Morand S, Libois R, Michaux JR (2004) A par- mtDNA diversity and population structure in a direct-devel- asite reveals cryptic phylogeographic history of its host. Pro- oping marine gastropod and its trematode parasites. Molecu- ceedings of the Royal Society of London. Series B: Biological lar Ecology, 18, 4591–4603. Sciences, 271, 2559–2568. Kralov a-Hromadov aI, Spakulova M, HorackovaE et al. (2001) Nieberding CM, Durette-Desset M-C, Vanderpoorten A et al. Sequence analysis of ribosomal and mitochondrial genes of (2008) Geography and host biogeography matter for under- the giant liver fluke Fascioloides magna (Trematoda: Fascioli- standing the phylogeography of a parasite. Molecular Phyloge- dae): intraspecific variation and differentiation from Fasciola netics and Evolution, 47, 538–554. hepatica. Journal of Parasitology, 94,58–67. Olson PD, Cribb TH, Tkach VV, Bray RA, Littlewood DTJ (2003) Larkin MA, Blackshields G, Brown NP et al. (2007) CLUSTAL W Phylogeny and classification of the Digenea (Platyhelminthes: and CLUSTAL X version 2.0. Bioinformatics, 23, 2947–2948. Trematoda). International Journal for Parasitology, 33, 733–755. Leung T, Donald K, Keeney D, Koehler A, Peoples R, Poulin R Ortiz M (2008) Mass balanced and dynamic simulations of tro- (2009) Trematode parasites of Otago Harbour (New Zealand) phic models of kelp ecosystems near the Mejillones Penin- soft-sediment intertidal ecosystems: life cycles, ecological sula of northern Chile (SE Pacific): comparative network roles and DNA barcodes. New Zealand Journal of Marine & structure and assessment of harvest strategies. Ecological Freshwater Research, 43, 857–865. Modelling, 216,31–46. Louhi KR, Karvonen A, Rellstab C, Jokela J (2010) Is the popu- Oyarzun C (2003) Catalogo de los peces presentes en el sistema lation genetic structure of complex life cycle parasites deter- de corrientes de Humboldt frente a Chile Centro-Sur. De- mined by the geographic range of the most motile host? partamento de Oceanografıa seccion Pesquerıas, UdeC. Infection, Genetics and Evolution, 10, 1271–1277. http://www2.udec.cl/~coyarzun/catalogo/Catalogo1.htm Mantel N (1967) The detection of disease clustering and Perez-Matus A, Ferry-Graham L, Cea A, Vasquez J (2007) a generalized regression approach. Cancer Research, 27, Community structure of temperate reef fishes in kelp-domi- 209–220. nated subtidal habitats of northern Chile. Marine and Fresh- McCoy KD (2003) Sympatric speciation in parasites - what is water Research, 58, 1069–1085. sympatry? Trends in Parasitology, 19, 400–404. Perez-Matus A, Pledger S, Dıaz FJ, Ferry LA, Vasquez JA Medina M, Araya M, Vega C (2004) Alimentacion y relaciones (2012) Plasticity in feeding selectivity and trophic structure troficas de peces costeros de la zona norte de Chile. Investi- of kelp forest associated fishes from northern Chile. Revista gaciones marinas, 32,33–47. Chilena de Historia Natural, 85,29–48. Meenakshi M, Madhavi R, Swarnakumari VGM (1993) The life- Posada D, Crandall KA (1998) MODELTEST: testing the model of cycle of Helicometra gibsoni n. sp. (Digenea: Opecoelidae). DNA substitution. Bioinformatics Applications Note, 14, 817–818. Systematic Parasitology, 25,63–72. Poulin R (2007) Evolutionary Ecology of Parasites, 2nd edn. Meinila M, Kuusela J, Zietara MS, Lumme J (2004) Initial Princeton University Press, Princeton, NJ. 332 pp. steps of speciation by geographic isolation and host switch Puillandre N, Lambert A, Brouillet S, Achaz G (2012) ABGD, in salmonid pathogen Gyrodactylus salaris (Monogenea: Automatic Barcode Gap Discovery for primary species Gyrodactylidae). International Journal of Parasitology, 34, delimitation. Molecular Ecology, 21, 1864–1877. 515–526. Rivadeneira MM, Fernandez M, Navarrete SA (2002) Latitudi- Melo T, Silva N, Munoz~ P et al. (2007) Caracterizacion del nal trends of species diversity in rocky intertidal herbivore fondo marino entre la III y X Regiones. Informe Final Proy- assemblages: spatial scale and the relationship between local ecto FIP N° 2005-61. Estud. Doc. N° 22/2007. 287 p. and regional species richness. Marine Ecology Progress Series, Miller SA, Dykes DD, Polesky HF (1988) A simple salting out 245, 123–131. procedure for extracting DNA from human nucleated cells. Rohde K (2005) Marine Parasitology. CSIRO PUBLISHING, Aus- Nucleic Acids Research, 16, 1215. tralia. 565 pp. Miura O, Kuris AM, Torchin ME, Hechinger RF, Dunham EJ, Rousset F (1997) Genetic differentiation and estimation of gene Chiba S (2005) Molecular-genetic analyses reveal cryptic flow from F-statistics under isolation by distance. Genetics, species of trematodes in the intertidal gastropod, Batillaria 145, 1219–1228. cumingi (Crosse). International Journal for Parasitology, 35, Sanchez R, Sepulveda RD, Cardenas L (2011) Spatial pattern of 793–801. genetic and morphological diversity in the direct developer Moreno M, Flores H (2002) Contenido estomacal de Cheilodacty- Acanthina monodon (Gastropoda: Mollusca). Marine Ecology lus variegatus Valenciennes 1833, Pinguipes chilensis Valenci- Progress Series, 434, 121–131.

Slatkin M, Hudson RR (1991) Pairwise comparisons of mito- anoplocephala arctica species complex (Cestoda: Anoplocepha- chondrial DNA sequences in stable and exponentially grow- lidae) parasitizing collared lemmings (Dicrostonyx spp.). ing populations. Genetics, 129, 555–562. Molecular Ecology, 12, 3359–3371. Stefka J, Hypsa V, Scholz T (2009) Interplay of host specificity Yoshida R, Urabe M (2005) Life cycle of Coitocoecum plagiorchis and biogeography in the population structure of a cosmopol- (Trematoda: Digenea: Opecoelidae). Parasitology International, itan endoparasite: microsatellite study of Ligula intestinalis 54, 237–242. (Cestoda). Molecular Ecology, 18, 1187–1206. Zakas C, Binford J, Navarrete SA, Wares JP (2009) Restricted Steinauer ML, Hanelt B, Agola LE, Mkoji GM, Loker ES (2009) gene flow in Chilean barnacles reflects an oceanographic and Genetic structure of Schistosoma mansoni in western Kenya: biogeographic transition zone. Marine Ecology Progress Series, the effects of geography and host sharing. International Jour- 394, 165–177. nal for Parasitology, 39, 1353–1362. Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics, 123, 585–595. Z.L. performed the research and molecular analyses Tamura K, Nei M (1993) Estimation of the number of nucleo- and wrote an earlier manuscript. L.C. and F.R. assisted tide substitutions in the control region of mitochondrial with the analytical tools. M.T.G. contributed to the DNA in humans and chimpanzees. Molecular Biology and research design and assisted in data collection. L.C. and – Evolution, 10, 512 526. M.T.G. revised and finalized the manuscript. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729. Tellier F, Meynard AP, Correa JA, Faugeron S, Valero M (2009) Phylogeographic analyses of the 30° S south-east Pacific bi- Data accessibility ogeographic transition zone establish the occurrence of a - DNA sequences with V4 region: GenBank Accession sharp genetic discontinuity in the kelp Lessonia nigrescens: Vicariance or parapatry? Molecular Phylogenetics and Evolu- nos KJ527643 - KJ527676. tion, 53, 679–693. - DNA sequences with COI: GenBank Accession nos Thiel M, Macaya EC, Acuna~ E et al. (2007) The Humboldt Cur- KJ527677 - KJ528014. rent System of northern and central Chile. Oceanography and Final DNA sequence assembly uploaded as online Marine Biology: An Annual Review, 45, 195–344. supplemental material, Neolebouria-georgenascimentoi Thieltges DW, Hof C, Borregaard MK et al. (2011) Range size V4 and Neolebouria-georgenascimentoi COI data input patterns in European freshwater trematodes. Ecography, 34, files FASTA format: Dryad doi:10.5061/dryad.4 ft57 982–989. Valdivia I, Cardenas L, Gonzalez K et al. (2010) Molecular evi- Phylogenetic trees resultant for construction Figure 2: dence confirms that Proctoeces humboldti and Proctoeces chilen- Neolebouria sp ML-TN93, Neolebouria sp NJ and Neol- sis (Digenea: Fellodistomidae) are the same species. Journal of ebouria sp BI assembly uploaded as online supplemen- Helminthology, 84, 341–347. tal material Newik format: Dryad doi:10.5061/ Valdovinos C, Navarrete SA, Marquet PA (2003) Mollusk spe- dryad.4 ft57. Data available from the Dryad Digital cies diversity in the Southeastern Pacific: why are there more Repository: http://doi.org/10.5061/dryad.NNNNN. species towards the pole? Ecography, 26, 139–144. Varela AI, Haye PA (2012) The marine brooder Excirolana bra- ziliensis (Crustacea: Isopoda) is also a complex of cryptic species on the coast of Chile. Revista Chilena de Historia Natural, Supporting information 85, 495–502. Venerus LA, Irigoyen AJ, Galvan DE, Parma AM (2013) Spatial Additional supporting information may be found in the online ver- dynamics of the Argentine sandperch, Pseudopercis semifasci- sion of this article. ata (Pinguipedidae), in temperate rocky reef from northern Patagonia, Argentina. Marine and Freshwater Research, 65, Table S1. Access numbers of sequences deposited in the NCBI 39–49. database, according at the lineage classified of Neolebouria spp., Vilches C, Pradenas J, Quinones~ A, Brante A (2012) Association host, localities, years, number of fish, number of the individual of Crepidula coquimbensis with Pagurus edwardsi: effect on dis- and sequence name. persal potential and population genetic structure. Revista de Table S2. Pairwise analysis of molecular variance estimations Biologıa Marina y Oceanografıa, 47, 327–331. (ΦST estimates/ P-value) between sites for lineage 1 in the Wickstrom€ LM, Haukisalmi V, Varis S, Hantula J, Fedorov VB, lower matrix and lineage 2 in the upper matrix. Henttonen H (2003) Phylogeography of the circumpolar Par-