Molecular Phylogeny and Dating Reveal a Terrestrial Origin in the Early Carboniferous for Ascaridoid Nematodes

Molecular Phylogeny and Dating Reveal a Terrestrial Origin in the Early Carboniferous for... Abstract Ascaridoids are among the commonest groups of zooparasitic nematodes (roundworms) and occur in the alimentary canal of all major vertebrate groups, including humans. They have an extremely high diversity and are of major socio-economic importance. However, their evolutionary history remains poorly known. Herein, we performed a comprehensive phylogenetic analysis of the Ascaridoidea. Our results divided the Ascaridoidea into six monophyletic major clades, i.e., the Heterocheilidae, Acanthocheilidae, Anisakidae, Ascarididae, Toxocaridae, and Raphidascarididae, among which the Heterocheilidae, rather than the Acanthocheilidae, represents the sister clade to the remaining ascaridoids. The phylogeny was calibrated using an approach that involves time priors from fossils of the co-evolving hosts, and dates the common ancestor of the Ascaridoidea back to the Early Carboniferous (approximately 360.47–325.27 Ma). The divergence dates and ancestral host types indicated by our study suggest that members of the Ascaridoidea first parasitized terrestrial tetrapods, and subsequently, extended their host range to elasmobranchs and teleosts. We also propose that the fundamental terrestrial-aquatic switches of these nematodes were affected by changes in sea-level during the Triassic to the Early Cretaceous. Host switching, molecular dating, parasite–host association, phylogeny, sea-level change, terrestrial origin The Ascaridoidea is a superfamily of parasitic nematodes (roundworms) containing more than 800 described species that parasitize all major lineages of vertebrates (Hartwich 1974; Hodda 2011), and as adults usually inhabit the alimentary canal. Ascaridoids are of veterinary, medical and economic importance, causing disease in domestic animals, wildlife, and humans (e.g., ascariasis, toxocariasis, baylisascariasis, and anisakiasis) (Mozgovoi 1953; Anderson 2000; Despommier 2003; Hochberg and Hamer 2010; Bauer 2013). Current classifications of the Ascaridoidea have been proposed based mainly on morphological characters, including the form of the excretory system (Hartwich 1954, 1957, 1974; Chabaud 1965; Gibson 1983), the structure of the esophago-intestinal junction (Baylis 1920; Mozgovoi 1953; Yamaguti 1961), the labial structure (Osche 1958) and the number and arrangement of caudal papillae (Fagerholm 1991). The different weighting of these morphological characters by individual investigators has led to conflicting classifications of this superfamily. The origin and the early evolutionary history of the Ascaridoidea have long been uncertain and contentious. Some authors, through strict application of “parasitophyletic rules” [e.g., Szidat’s Rule (hosts resembling the ancestral state are more likely to harbor parasites resembling the ancestral state); Fahrenholz’s Rule (host and parasite phylogenies tend to mirror one another) (MacIntosh and Frias 2017)], hypothesized that ascaridoids originated in an aquatic (marine) environment and that members of the Acanthocheilidae, parasites of elasmobranch fishes, must represent the earliest subgroup (Mozgovoi 1953; Osche 1958, 1963; Sprent 1962; Gibson 1983). Others have disagreed with this hypothesis and speculated that ascaridoids originally parasitized early terrestrial tetrapods with a secondary radiation to aquatic hosts (Chitwood and Chitwood 1950; Dougherty 1951; Chabaud 1955; Sprent 1983; Anderson 1984). These varying hypotheses result from different interpretations of observations of life history patterns and/or morphological traits. To understand the early evolution of the Ascaridoidea, a reliable and representative phylogeny is essential. Recently, some authors have made efforts to establish the phylogenetic relationships of certain taxa and to solve particular problems using nuclear rDNA sequence data and mitochondrial genes (Nadler 1992; Nadler and Hudspeth 1998, 2000; Nadler et al. 2000, 2005; Zhu et al. 2000; Mattiucci and Nascetti 2006, 2008; Mattiucci et al. 2008; Li et al. 2012, 2017; Mohandas et al. 2014; Liu et al. 2015, 2016). However, due to the scarcity and inaccessibility of suitable material for some taxa (e.g., the Acanthocheilidae, Heterocheilidae, and Raphidascarididae), these molecular phylogenetic studies have included only a very small representation of ascaridoid diversity. Hence, our knowledge of the phylogeny of the Ascaridoidea is still far from comprehensive. Estimating the age of the ascaridoid clades has also been difficult. By scaling divergence events to geological time, one can determine the timing of key innovations during the history of the group. Unfortunately, fossils of ascaridoids are extremely rare due to their soft body (Silva et al. 2014). To date, only three species represented by fossilised eggs (all tentatively assigned to the Ascarididae: i.e. † Ascarites) have been found in the coprolites of cynodonts or dinosaurs (Poinar and Boucot 2006; Poinar 2011; Silva et al. 2014), but the systematic placements of these eggs are questionable (superficially they resemble the Ascarididae, Toxocaridae and Heterocheilidae). This hinders efforts to use calibration information from ascaridoid evolutionary history along with molecular sequence data to estimate divergence times using standard molecular clock approaches. Two studies have made such attempts by estimation using eukaryotes (Douzery et al. 2004) or based on molecular evolutionary rates of other groups of organisms (Vanfleteren et al. 1994). Their results are largely in conflict. Douzery et al. (2004) estimated the divergence of the orders Spirurida and Ascaridida at $$\sim$$150 Ma, whereas Vanfleteren et al. (1994) estimated the divergence of the Anisakidae and Ascaridae to be 250–150 Ma. In this study, we performed phylogenetic analyses of ascaridoid nematodes based on five nuclear and three mitochondrial genes. These molecular phylogenetic analyses included the most comprehensive taxon sampling of the Ascaridoidea to date, yielding a robust evolutionary hypothesis for the superfamily. The phylogeny was calibrated using an approach that involves time priors from hosts rather than ascaridoid calibrations per se. We then estimated the host types of the ancestor of the ascaridoids and some of its major clades with the aim of establishing an original host-switching model for the group. Materials and Methods Taxon Sampling The in-group samples include 65 ascaridoid species belonging to 26 genera, including all of the major families (Acanthocheilidae, Anisakidae, Ascarididae, Heterocheilidae, Raphidascarididae, Toxocaridae) according to the generally accepted classifications of the Ascaridoidea (Hartwich 1974; Fagerholm 1991) (see Supplementary Table S1 available on Dryad at http://doi.org/10.5061/dryad.28g16). Representatives of the family Crossophoridae (containing only the genera Crossophorus Ehrenberg 1828 and Dartevellenia Ezzat 1954, parasites of hyracoids) were unavailable for inclusion in the analysis. Many previous phylogenetic studies based on various loci strongly support the Heterakoidea and Cosmocercoidea as being groups closely related to the Ascaridoidea, but not part of this clade (Smythe et al. 2006; Holterman et al. 2006; Nadler et al. 2007; Ross et al. 2010; Cernotíková et al. 2011; Kim et al. 2014; Liu et al. 2016; Choudhury and Nadler 2016). Consequently, three species of the Heterakoidea (Ascaridia galli) and Cosmocercoidea (Cruzia americana and Falcaustra sinensis) were used as outgroups to root the trees (see Supplementary Table S1 available on Dryad). DNA Extraction, Amplification, and Sequencing To study the evolutionary history of the Ascaridoidea, we constructed a database, including nuclear sequences [partial sequences of small ribosomal subunit (18S), large ribosomal subunit (28S), internal transcribed spacer 1 (ITS-1), 5.8S and internal transcribed spacer 2 (ITS-2)] plus mitochondrial data [partial cytochrome $$c$$ oxidase subunit 1 (cox1), cytochrome $$c$$ oxidase subunit 2 (cox2), and 12S small subunit ribosomal RNA gene] from 68 taxa, including 123 new sequences (see Supplementary Table S2 available on Dryad). Sequence data were also obtained from GenBank. Genomic DNA from an individual nematode was extracted using a Column Genomic DNA Isolation Kit (Shanghai Sangon, China) according to the manufacturer’s instructions and then stored at $$-$$20℃ until the molecular analysis. For amplifying these target sequences, the following published primers were used: the near-complete 18S rDNA by the primers 18SF and 18SR (Floyd et al. 2005), the partial ITS region by the primers A and B (D’Amelio et al. 2000), the partial 28S rDNA by the primers 28SF and 28SR (Nadler and Hudspeth 1998), the partial cox1 by the primers CO1F and CO1R (Lazarova et al. 2006), the partial cox2 by the primers CO2F and CO2R (Nadler and Hudspeth 2000), and the partial 12S by the primers 12SF and 12SR (Li et al. 2012). All polymerase chain reactions (PCRs) were performed in 50 $$\mu$$L of PCR reaction buffer with 10 mM Tris HCl at pH 8.4, 50 mM KCl, 3.0 mM MgCl$$_{{2}}$$, 250 $$\mu $$M of each dNTP, 50 pmol of each primer, and 1.5 U of Taq polymerase (Takara) in a thermocycler (2720, Applied Biosystems) under the following conditions: 94° C, 5 min (initial denaturation), followed by 30 cycles of 94° C, 30 s (denaturation), 55° C, 30 s (annealing), 72° C, 70 s (extension), and a final extension of 72° C for 7 min. PCR products were checked on GoldView-stained 1.5% agarose gel and purified by the Column PCR Product Purification Kit (Shanghai Sangon, China). Sequencing for each sample was completed for both strands using a Dideoxy Terminator Cycle Sequencing Kit (v.2, Applied Biosystems, CA, USA) and an automated sequencer (ABI-PRISM 377). All the DNA sequences obtained herein (18S, ITS-1, 5.8S, ITS-2, 28S, 12S, cox1, and cox2) are deposited in the GenBank database (http://www.ncbi.nlm. nih.gov) (Table S1). Sequence Alignment Molecular sequences of each gene (nucleotides for non-coding genes and amino acids for coding genes) were initially aligned using the E-INS-i algorithm in the online version of MAFFT v7.3 (Katoh et al. 2002; Katoh and Standley 2013) with adjustment to the direction of the first sequence, gappy regions retained and other alignment parameters left as default. The nucleotides of coding genes were aligned to the alignments of amino acids. The program MEGA v7.0.14 (Kumar et al. 2016) was used to refine the primary single-gene alignment manually. Alignment positions with unique or few ($$\leqslant $$3) indels were deleted; the ends were trimmed; the third position of coding genes was removed if indicated by the DAMBE mutational saturation test (Xia et al. 2003). Multiple genes were concatenated into one merged matrix using Mesquite v3.04 (Maddison and Maddison 2016). Data Partitions, Model Selection, and Phylogenetic Analyses We used PartitionFinder v2.0.0 (Lanfear et al. 2017) to select both a data-partitioning scheme and a best-fitting substitution model for each partition using the greedy algorithm (Lanfear et al. 2012) with the Bayesian information criterion (Schwarz 1978). The partitioning schemes and the optimal model selected for each combination of partition and tree inference program are detailed in Supplementary Table S2 available on Dryad. We performed the maximum likelihood (ML) inference using RAxML v8.2.11 (Stamatakis 2014). The best-scoring tree was found with the rapid hill-climbing algorithm (Stamatakis et al. 2007) and bootstrap support (BS) was estimated using the rapid bootstrap option (Stamatakis et al. 2008) and 1000 replicates (GTR$$+$$Gamma model, partitioned for the multigene matrices). The bipartition values for ML BS were mapped to the best-scoring tree(s). ML BS values $$\geqslant $$80 were considered to constitute strong nodal support, whereas BS values $$\geqslant $$50 and $$<$$80 were considered to constitute moderate nodal support. We performed a Bayesian inference analysis for each matrix using MrBayes v3.2.6 (Huelsenbeck and Ronquist 2001; Ronquist et al. 2012) executed on the CIPRES Science Gateway cluster (Miller et al. 2010) using partitions and models defined by PartitionFinder (see above). We made two simultaneous runs of Markov Chain Monte Carlo (MCMC) sampling, each with four chains, 20 million generations, and sampled every 1000 generations. The standard deviation of split frequencies converged at 0.003818. We used the program Tracer v1.6 (Rambaut and Drummond 2013) to diagnose convergence, determine an appropriate burn-in and verify the effective sample size (ESS) of every parameter >200 for the combined MCMC samples of each matrix. Accordingly, the first 25% of the samples of each run were dropped when generating the 50% consensus trees and estimating nodal Bayesian posterior probabilities (BPP). BPP $$\geqslant $$0.98 were considered to constitute strong nodal support, whereas BPP values $$\geqslant $$0.95 and $$<$$0.98 were considered to constitute moderate nodal support. The resulting trees were rooted with the outgroups. Phylogeny of Hosts To build the host phylogeny, we sampled a total of 89 genera of vertebrates (without outgroups), which include all the genus-level hosts of the Ascaridoidea known to us in this study (see Supplementary Table S3 available on Dryad). It was necessary to estimate such a phylogeny because a tree with these particular vertebrate genera was not available in the published literature. We used six genes, including the nuclear recombination activating gene 1 (RAG1, intron excluded) and mitochondrial cytochrome b (Cyt b), cox1, cox2, 12S, and 16S rDNA. All these sequences were mined from the GenBank database (see Supplementary Table S4 available on Dryad). We used RAxML v8.2.11 (Stamatakis 2014) to build the ML phylogenetic tree of the hosts in the same way as described for the parasites, and with a mixed matrix concatenated from protein sequences (RAG1, Cyt b, cox1, cox2) and nucleotide sequences (12S, 16S) (Table S2). The divergence times of the hosts were estimated using BEAST v1.8.4 (Drummond and Rambaut 2007; Drummond et al. 2012). We used the fossil age of the Gnathostomata (468.4–420.7 Ma) (Benton et al. 2015) and the ML best tree to obtain an ultrametric starting tree using treePL (Smith and O’Meara 2012) (smoothing parameters obtained by cross validation), which was then imported into BEAUTi (Drummond and Rambaut 2007) as a starting tree to fix the tree topology. The BEAST analyses employed 14 time priors (uniform distribution) from the literature (see Supplementary Table S5 available on Dryad). We executed six independent MCMC runs on the cluster from the CIPRES Science Gateway (Miller et al. 2010) for different models (variations included Yule, birth-death process, strict clock, uncorrelated relaxed clock with lognormal distribution, and uncorrelated relaxed clock with exponential distribution); each run with 300 million generations and sampled every 1000 generations. This was followed by marginal likelihood estimation using path sampling (PS)/stepping-stone (SS) sampling (300 steps by 1,000,000 iterations) (Baele et al. 2012, 2013). Then, we compared their logarithmic marginal likelihoods for six models (see Supplementary Table S6 available on Dryad) and used Treeannotator v1.8.4 (Drummond and Rambaut 2007) to obtain the maximum clade credibility time tree from 29 001 resulting post burn-in trees (ESS >200) of the best model (Yule process and uncorrelated relaxed clock with lognormal distribution, Supplementary Table S6 available on Dryad). Testing Parasite–Host Associations and Dating Ascaridoid Phylogeny We compiled a set of parasite–host association (PHA) links from the literature and our own observations (see Supplementary Table S3 available on Dryad), and tested whether the PHA links are independent or phylogenetically related, using the eight-gene ML phylogeny of the Ascaridoidea and the ML phylogeny of the hosts. We executed both global and individual tests using the ParaFit method (Legendre and Desdevises 2002) in the APE package (Paradis et al. 2004) in R 3.3.1 (Core R Team 2016), where we ran 100,000 random permutation and considered the PHA links with the permuted $$P$$-values of both ParaFitLink 1 and ParaFitLink 2 $$\leqslant $$0.05 as significant. We recognized four pairs of significant monophyletic parasite-to-host specialist (P2H) clades (Fig. 2) in which both the host clade (H-clade) and the parasite clade (P-clade) are monophyletic, and the P-clade taxa are present only in that H-clade. The event-based cophylogeny of the four pairs of P2H clades enable us to calibrate the P-clades based on the time information from the associated H-clades. On the basis of the other host-parasite comparisons for time calibrations (Ricklefs and Outlaw 2010; Bensch et al. 2013; Shah et al. 2010), we considered the time from the appearance of the ancestor lineage of the H-clade (in the vertebrate tree) to the divergence of the most recent common ancestor (MRCA) of the H-clade as the potential switching time range of the ancestor lineage of the P-clade (in the ascaridoid tree). Therefore, we calibrated the ancestor lineage (on the stem) of the four P-clades (calibrations for the Raphidascarididae and Ascarididae are equivalent) using two strategies: (i) setting the upper limit of the P-clade stem by the upper limit of the (oldest) fossil of the parent node of the associated H-clade, and setting the lower limit of the P-clade stem using the lower limit of the fossil of the MRCA of the H-clade; (ii) using the limits of the 95% highest posterior density interval (HPD95) instead of the host fossil limits. See details in Supplementary Table S5 available on Dryad. Figure 2. View largeDownload slide Time tree of the Ascaridoidea (left) and their hosts (right), the parasite–host association links, maximum likelihood reconstruction of the ancestral host type of the Ascaridoidea and the habitat of ascaridoid terminal hosts. Bars on nodes show the 95% highest posterior density interval estimated from the resulting BEAST MCMC trees. Of the seven letter-labeled P-clades, four (A–D) are the significant monophyletic P2H clades and have the same color as the associated monophyletic H-clades on the host tree; these are connected by solid lines in the same colors. The calibration of molecular clock analyses is based on these P2H clades (see Supplementary Fig. S1 and Table S5 available on Dryad). Clades E and F are specialized P-clades, but their host clades are paraphyletic; clade G is not supported by the Bayesian inference. The grey and dashed lines are phylogenetically independent links. Pie charts on the nodes of the ascaridoid tree show the scaled marginal likelihood of each host type estimated by the maximum likelihood method using the equal-rates model (stably invariant results are not shown). Colored diamonds at the tips of the parasite tree indicate the host type of the relevant taxa. Similarly, the pie charts and the diamonds on the host tree show the estimated ancestral habitats (by the maximum likelihood method using the symmetrical model) and the present states, respectively. The “sun” marks the MRCA of the Ascaridoidea; the “moon” marks the MRCA of the Anisakinae (ANI); the “star” marks the MRCA of the “Ascaridoidea minus Heterocheilidae” clade. Starting from these nodes, the five critical “land-to-water” shifts in the early history of the Ascaridoidea that we propose are: “sun” to R1, “star” to “moon” to R2 and to R3, and “star” to R4 and R5. All these shifts and evolution took place during the Triassic to the Early Cretaceous, when the global sea level rose. Taxon abbreviations: RAP $$=$$ Raphidascarididae; ASC $$=$$ Ascarididae; TOX $$=$$ Toxocaridae; ACA $$=$$ Acanthocheilidae; ANI $$=$$ Anisakidae; HETC $$=$$ Heterocheilidae; OGHETK $$=$$ Heterakoidea (outgroup); OGCOS $$=$$ Cosmocercoidea (outgroup); MAMM $$=$$ Mammalia (Theria); AVES $$=$$ Aves (modern birds); REPT $$=$$ Reptilia (Sauria excluding birds); ACTI $$=$$ Actinopterygii (Teleostei); CHON $$=$$ Chondrichthyes (Elasmobranchii). Period abbreviations: S $$=$$ Silurian; D $$=$$ Devonian; C $$=$$ Carboniferous; P $$=$$ Permian; T $$=$$ Triassic; J $$=$$ Jurassic; K $$=$$ Cretaceous; Pg $$=$$ Palaeogene; N $$=$$ Neogene (and Quaternary). The time trees (under best model) of both the Ascaridoidea and hosts are available in the Dryad. Figure 2. View largeDownload slide Time tree of the Ascaridoidea (left) and their hosts (right), the parasite–host association links, maximum likelihood reconstruction of the ancestral host type of the Ascaridoidea and the habitat of ascaridoid terminal hosts. Bars on nodes show the 95% highest posterior density interval estimated from the resulting BEAST MCMC trees. Of the seven letter-labeled P-clades, four (A–D) are the significant monophyletic P2H clades and have the same color as the associated monophyletic H-clades on the host tree; these are connected by solid lines in the same colors. The calibration of molecular clock analyses is based on these P2H clades (see Supplementary Fig. S1 and Table S5 available on Dryad). Clades E and F are specialized P-clades, but their host clades are paraphyletic; clade G is not supported by the Bayesian inference. The grey and dashed lines are phylogenetically independent links. Pie charts on the nodes of the ascaridoid tree show the scaled marginal likelihood of each host type estimated by the maximum likelihood method using the equal-rates model (stably invariant results are not shown). Colored diamonds at the tips of the parasite tree indicate the host type of the relevant taxa. Similarly, the pie charts and the diamonds on the host tree show the estimated ancestral habitats (by the maximum likelihood method using the symmetrical model) and the present states, respectively. The “sun” marks the MRCA of the Ascaridoidea; the “moon” marks the MRCA of the Anisakinae (ANI); the “star” marks the MRCA of the “Ascaridoidea minus Heterocheilidae” clade. Starting from these nodes, the five critical “land-to-water” shifts in the early history of the Ascaridoidea that we propose are: “sun” to R1, “star” to “moon” to R2 and to R3, and “star” to R4 and R5. All these shifts and evolution took place during the Triassic to the Early Cretaceous, when the global sea level rose. Taxon abbreviations: RAP $$=$$ Raphidascarididae; ASC $$=$$ Ascarididae; TOX $$=$$ Toxocaridae; ACA $$=$$ Acanthocheilidae; ANI $$=$$ Anisakidae; HETC $$=$$ Heterocheilidae; OGHETK $$=$$ Heterakoidea (outgroup); OGCOS $$=$$ Cosmocercoidea (outgroup); MAMM $$=$$ Mammalia (Theria); AVES $$=$$ Aves (modern birds); REPT $$=$$ Reptilia (Sauria excluding birds); ACTI $$=$$ Actinopterygii (Teleostei); CHON $$=$$ Chondrichthyes (Elasmobranchii). Period abbreviations: S $$=$$ Silurian; D $$=$$ Devonian; C $$=$$ Carboniferous; P $$=$$ Permian; T $$=$$ Triassic; J $$=$$ Jurassic; K $$=$$ Cretaceous; Pg $$=$$ Palaeogene; N $$=$$ Neogene (and Quaternary). The time trees (under best model) of both the Ascaridoidea and hosts are available in the Dryad. The starting tree, prepared for use in BEAST, was from the eight-gene ML tree of the Ascaridoidea (with outgroups) and transformed by treePL with a single calibration on the root (fixed age $$=$$ 541 Ma, see Blaxter (2009)). The BEAST analyses of the Ascaridoidea employed the eight-gene matrix (see Supplementary Table S2 available on Dryad) and were performed in the same way as dating the vertebrate tree, but with 200 million generations of the standard BEAST MCMC run and a total of 200 million iterations for PS/SS sampling. We obtained the final time trees from the resulting post burn-in trees under the best model (Birth-death and uncorrelated relaxed clock with lognormal distribution using the calibrating strategy 2, Supplementary Table S6 available on Dryad) with first 5% dropped (ESS >200). Reconstruction of the Ancestral Habitat of the Hosts and the Ancestral Host Type of the Ascaridoids We separated the habitats of the hosts of the sampled ascaridoid species into three types: terrestrial, semiaquatic, and aquatic (including both fresh and sea water). According to the hosts and host habitats, we then distinguished the sampled ascaridoid representatives into six types of parasites, living in: (i) terrestrial tetrapods, (ii) semiaquatic tetrapods, (iii) aquatic tetrapods, (iv) elasmobranch fishes, (v) teleost fishes, and (vi) multiple types of hosts (more than two types). Some genera, with a single species sampled here but known to have various types of host, are assigned by the host type of the majority of their species rather than that of the sampled species. To infer the possible ancestral host type of ascaridoid clades, we mapped the known host types as discrete traits onto the ascaridoid time tree (calibrated by strategy 2) and used the ML method (Pagel 1994) to estimate the scaled likelihood of each state (host type of parasite or habitat of host) on each node. The state with the largest marginal likelihood was considered as the most probable ancestral state on that node. We fitted our data with three modes: equal-rate, symmetrical, and all-rate-different. We selected the equal-rate model as the best-fitting model for ascaridoid host types and the symmetrical model for host habitats using AIC and Akaike weight (see Supplementary Table S7 available on Dryad). Then, we defined the estimated switching time range (EST) as the period when the parasite ancestral lineage and the host ancestral lineage possibly co-occurred. Accordingly, we defined the maximum limit of the EST as the minimum value of the stem age of a parasite clade and that of its primary host clade, and defined the minimum limit as the maximum value of the crown ages of these two clades (Table 1 and Supplementary Fig. S1 available on Dryad). Considering that the Bayesian MCMC dating method (BEAST) provides interval estimates of the cladal ages, we expanded the EST into the joint 95% highest posterior density interval (jHPD95) of EST by using the minimum value between the maximum limits of the stem ages of the parasite HPD95 and the host HPD95 as the maximum limit of the jHPD95 and using the maximum value between the minimum limits of the stem ages of the parasite HPD95 and the host HPD95 as the minimum limit of the jHPD95 (Table 1 and Supplementary Fig. S1 available on Dryad). Table 1. Divergence time (in Ma) of the Ascaridoidea and their hosts estimated using strategy 2, the estimated switching time range (EST), and the time windows (TW in Myr, size of EST) and expanded time window [ETW in Myr, size of joint 95% HPD (jPHD95)] of the host switching Ascaridoid Clade Stem age (HPD95$$^{\mathrm{a}})$$ Crown age (HPD95) Host Clade Stem age (HPD95) Crown age (HPD95) EST$$^{\mathrm{b}}$$ (jHPD95$$^{\mathrm{c}})$$ TW$$^{\mathrm{d}}$$ ETW$$^{\mathrm{e}}$$ Ascaridoidea 360.47 325.27 Amniota 390.43 323.04 360.47–325.27 35.20 106.94 (456.30–280.00) (406.90–258.94) (424.94–358.70) (331.30–318.00) (424.94–318.00) Raphidascarididae 247.31 140.33 Teleostei 390.43 194.67 247.31–194.67 52.64 131.73 (292.59–209.49) (177.55–109.91) (424.94–358.70) (233.63–160.86) (292.59–160.86) Ascarididae 200.75 100.39 Boreoeutheria (terrestrial) 121.85 109.29 121.85–109.29 12.56 42.76 (247.28–153.95) (136.64–72.30) (137.82–106.17) (124.88–95.06) (137.82–95.06) Baylisascaris 71.79 55.92 Arctoidea (excluding Pinnipedia) 59.89 54.90 59.89–55.92 3.97 12.75 (96.68–52.32) (77.03–39.77) (63.00–56.12) (59.03–50.25) (63.00–50.25) Toxocaridae 200.75 144.91 ### ### ### ### ### ### (247.28–153.95) (197.13–93.65) Toxocara 144.91 61.59 Laurasiatheria 109.29 91.19 109.29–91.19 18.10 43.58 (197.13–93.65) (92.48–34.45) (124.88–95.06) (102.41–81.30) (124.88–81.30) Porrocaecum 144.91 78.23 Neoaves 79.63 67.56 79.63–78.23 1.40 28.80 (197.13–93.65) (141.92–28.37) (86.8–70.12) (76.80–58.00) (86.8–58.00) Acanthocheilidae 228.94 177.21 Elasmobranchii 427.89 343.02 ### ### ### (267.97–196.06) (195.10–171.60) (447.77–420.70) (360.27–337.50) Mawsonascaris 177.21 8.36 Rhinobatidae$$+$$Myliobatidae 192.87 178.24 (195.10–171.60) ### 23.50 (195.10–171.60) (15.06–3.36) (220.02–176.64) (200.05–171.60) Anisakidae 256.62 214.90 ### ### ### ### ### ### (304.88–216.23) (262.60–171.64) Anisakinae 214.90 133.96 ### ### ### ### ### ### (262.60–171.64) (180.56–93.54) Anisakis 63.14 56.45 Cetacea 64.18 48.65 63.14–56.45 6.69 17.76 (66.00–56.39) (63.40–48.24) (66.00–59.45) (54.73–42.38) (66.00–48.24) Pseudoterranova 50.77 13.32 Pinnipedia 51.64 40.55 50.77–40.55 10.22 21.85 (56.30–39.52) (27.66–4.16) (56.30–46.58) (46.99–34.45) (56.30–34.45) Contracaecinae 214.90 135.26 ### ### ### ### ### ### (262.60–171.64) (181.29–96.65) Contracaecum (part) 104.20 87.79 Neognathae (fish eating) 230.43 79.63 104.20–87.79 16.41 70.30 (140.42–73.13) (120.56–57.77) (263.29–190.84) (86.8–70.12) (140.42–70.12) Heterocheilidae 325.27 153.27 Amniota 390.43 323.04 325.27–323.04 2.23 88.90 (406.90–258.94) (238.07–84.02) (424.94–358.70) (331.30–318.00) (406.90–318.00) Ascaridoid Clade Stem age (HPD95$$^{\mathrm{a}})$$ Crown age (HPD95) Host Clade Stem age (HPD95) Crown age (HPD95) EST$$^{\mathrm{b}}$$ (jHPD95$$^{\mathrm{c}})$$ TW$$^{\mathrm{d}}$$ ETW$$^{\mathrm{e}}$$ Ascaridoidea 360.47 325.27 Amniota 390.43 323.04 360.47–325.27 35.20 106.94 (456.30–280.00) (406.90–258.94) (424.94–358.70) (331.30–318.00) (424.94–318.00) Raphidascarididae 247.31 140.33 Teleostei 390.43 194.67 247.31–194.67 52.64 131.73 (292.59–209.49) (177.55–109.91) (424.94–358.70) (233.63–160.86) (292.59–160.86) Ascarididae 200.75 100.39 Boreoeutheria (terrestrial) 121.85 109.29 121.85–109.29 12.56 42.76 (247.28–153.95) (136.64–72.30) (137.82–106.17) (124.88–95.06) (137.82–95.06) Baylisascaris 71.79 55.92 Arctoidea (excluding Pinnipedia) 59.89 54.90 59.89–55.92 3.97 12.75 (96.68–52.32) (77.03–39.77) (63.00–56.12) (59.03–50.25) (63.00–50.25) Toxocaridae 200.75 144.91 ### ### ### ### ### ### (247.28–153.95) (197.13–93.65) Toxocara 144.91 61.59 Laurasiatheria 109.29 91.19 109.29–91.19 18.10 43.58 (197.13–93.65) (92.48–34.45) (124.88–95.06) (102.41–81.30) (124.88–81.30) Porrocaecum 144.91 78.23 Neoaves 79.63 67.56 79.63–78.23 1.40 28.80 (197.13–93.65) (141.92–28.37) (86.8–70.12) (76.80–58.00) (86.8–58.00) Acanthocheilidae 228.94 177.21 Elasmobranchii 427.89 343.02 ### ### ### (267.97–196.06) (195.10–171.60) (447.77–420.70) (360.27–337.50) Mawsonascaris 177.21 8.36 Rhinobatidae$$+$$Myliobatidae 192.87 178.24 (195.10–171.60) ### 23.50 (195.10–171.60) (15.06–3.36) (220.02–176.64) (200.05–171.60) Anisakidae 256.62 214.90 ### ### ### ### ### ### (304.88–216.23) (262.60–171.64) Anisakinae 214.90 133.96 ### ### ### ### ### ### (262.60–171.64) (180.56–93.54) Anisakis 63.14 56.45 Cetacea 64.18 48.65 63.14–56.45 6.69 17.76 (66.00–56.39) (63.40–48.24) (66.00–59.45) (54.73–42.38) (66.00–48.24) Pseudoterranova 50.77 13.32 Pinnipedia 51.64 40.55 50.77–40.55 10.22 21.85 (56.30–39.52) (27.66–4.16) (56.30–46.58) (46.99–34.45) (56.30–34.45) Contracaecinae 214.90 135.26 ### ### ### ### ### ### (262.60–171.64) (181.29–96.65) Contracaecum (part) 104.20 87.79 Neognathae (fish eating) 230.43 79.63 104.20–87.79 16.41 70.30 (140.42–73.13) (120.56–57.77) (263.29–190.84) (86.8–70.12) (140.42–70.12) Heterocheilidae 325.27 153.27 Amniota 390.43 323.04 325.27–323.04 2.23 88.90 (406.90–258.94) (238.07–84.02) (424.94–358.70) (331.30–318.00) (406.90–318.00) Bold taxon names are those whose parasite–host links are all significantly supported by ParaFit tests. The results of strategy 1 are provided in Supplementary Table S9 available on Dryad. $$^{\mathrm{a}}$$HPD95: 95% highest posterior density interval. $$^{\mathrm{b}}$$EST: the estimated switching time range. $$^{\mathrm{c}}$$jHPD95: the joint 95% highest posterior density interval of EST, where the upper limit is defined as the minor value between the upper limits of the stem ages of the parasite HPD and the host HPD, and the lower limit is defined as the major value between the lower limits of the stem ages of the parasite HPD and the host HPD (shown in Supplementary Fig. S1, available on Dryad). $$^{\mathrm{d}}$$TW: the time windows (range size of the EST) in which the parasitism is estimated to have been established. $$^{\mathrm{e}}$$ETW: the expanded time windows, i.e. the range size of the jHPD95. $$^{\mathrm{f}}$$###: No monophyletic host clade or reasonable result here. Table 1. Divergence time (in Ma) of the Ascaridoidea and their hosts estimated using strategy 2, the estimated switching time range (EST), and the time windows (TW in Myr, size of EST) and expanded time window [ETW in Myr, size of joint 95% HPD (jPHD95)] of the host switching Ascaridoid Clade Stem age (HPD95$$^{\mathrm{a}})$$ Crown age (HPD95) Host Clade Stem age (HPD95) Crown age (HPD95) EST$$^{\mathrm{b}}$$ (jHPD95$$^{\mathrm{c}})$$ TW$$^{\mathrm{d}}$$ ETW$$^{\mathrm{e}}$$ Ascaridoidea 360.47 325.27 Amniota 390.43 323.04 360.47–325.27 35.20 106.94 (456.30–280.00) (406.90–258.94) (424.94–358.70) (331.30–318.00) (424.94–318.00) Raphidascarididae 247.31 140.33 Teleostei 390.43 194.67 247.31–194.67 52.64 131.73 (292.59–209.49) (177.55–109.91) (424.94–358.70) (233.63–160.86) (292.59–160.86) Ascarididae 200.75 100.39 Boreoeutheria (terrestrial) 121.85 109.29 121.85–109.29 12.56 42.76 (247.28–153.95) (136.64–72.30) (137.82–106.17) (124.88–95.06) (137.82–95.06) Baylisascaris 71.79 55.92 Arctoidea (excluding Pinnipedia) 59.89 54.90 59.89–55.92 3.97 12.75 (96.68–52.32) (77.03–39.77) (63.00–56.12) (59.03–50.25) (63.00–50.25) Toxocaridae 200.75 144.91 ### ### ### ### ### ### (247.28–153.95) (197.13–93.65) Toxocara 144.91 61.59 Laurasiatheria 109.29 91.19 109.29–91.19 18.10 43.58 (197.13–93.65) (92.48–34.45) (124.88–95.06) (102.41–81.30) (124.88–81.30) Porrocaecum 144.91 78.23 Neoaves 79.63 67.56 79.63–78.23 1.40 28.80 (197.13–93.65) (141.92–28.37) (86.8–70.12) (76.80–58.00) (86.8–58.00) Acanthocheilidae 228.94 177.21 Elasmobranchii 427.89 343.02 ### ### ### (267.97–196.06) (195.10–171.60) (447.77–420.70) (360.27–337.50) Mawsonascaris 177.21 8.36 Rhinobatidae$$+$$Myliobatidae 192.87 178.24 (195.10–171.60) ### 23.50 (195.10–171.60) (15.06–3.36) (220.02–176.64) (200.05–171.60) Anisakidae 256.62 214.90 ### ### ### ### ### ### (304.88–216.23) (262.60–171.64) Anisakinae 214.90 133.96 ### ### ### ### ### ### (262.60–171.64) (180.56–93.54) Anisakis 63.14 56.45 Cetacea 64.18 48.65 63.14–56.45 6.69 17.76 (66.00–56.39) (63.40–48.24) (66.00–59.45) (54.73–42.38) (66.00–48.24) Pseudoterranova 50.77 13.32 Pinnipedia 51.64 40.55 50.77–40.55 10.22 21.85 (56.30–39.52) (27.66–4.16) (56.30–46.58) (46.99–34.45) (56.30–34.45) Contracaecinae 214.90 135.26 ### ### ### ### ### ### (262.60–171.64) (181.29–96.65) Contracaecum (part) 104.20 87.79 Neognathae (fish eating) 230.43 79.63 104.20–87.79 16.41 70.30 (140.42–73.13) (120.56–57.77) (263.29–190.84) (86.8–70.12) (140.42–70.12) Heterocheilidae 325.27 153.27 Amniota 390.43 323.04 325.27–323.04 2.23 88.90 (406.90–258.94) (238.07–84.02) (424.94–358.70) (331.30–318.00) (406.90–318.00) Ascaridoid Clade Stem age (HPD95$$^{\mathrm{a}})$$ Crown age (HPD95) Host Clade Stem age (HPD95) Crown age (HPD95) EST$$^{\mathrm{b}}$$ (jHPD95$$^{\mathrm{c}})$$ TW$$^{\mathrm{d}}$$ ETW$$^{\mathrm{e}}$$ Ascaridoidea 360.47 325.27 Amniota 390.43 323.04 360.47–325.27 35.20 106.94 (456.30–280.00) (406.90–258.94) (424.94–358.70) (331.30–318.00) (424.94–318.00) Raphidascarididae 247.31 140.33 Teleostei 390.43 194.67 247.31–194.67 52.64 131.73 (292.59–209.49) (177.55–109.91) (424.94–358.70) (233.63–160.86) (292.59–160.86) Ascarididae 200.75 100.39 Boreoeutheria (terrestrial) 121.85 109.29 121.85–109.29 12.56 42.76 (247.28–153.95) (136.64–72.30) (137.82–106.17) (124.88–95.06) (137.82–95.06) Baylisascaris 71.79 55.92 Arctoidea (excluding Pinnipedia) 59.89 54.90 59.89–55.92 3.97 12.75 (96.68–52.32) (77.03–39.77) (63.00–56.12) (59.03–50.25) (63.00–50.25) Toxocaridae 200.75 144.91 ### ### ### ### ### ### (247.28–153.95) (197.13–93.65) Toxocara 144.91 61.59 Laurasiatheria 109.29 91.19 109.29–91.19 18.10 43.58 (197.13–93.65) (92.48–34.45) (124.88–95.06) (102.41–81.30) (124.88–81.30) Porrocaecum 144.91 78.23 Neoaves 79.63 67.56 79.63–78.23 1.40 28.80 (197.13–93.65) (141.92–28.37) (86.8–70.12) (76.80–58.00) (86.8–58.00) Acanthocheilidae 228.94 177.21 Elasmobranchii 427.89 343.02 ### ### ### (267.97–196.06) (195.10–171.60) (447.77–420.70) (360.27–337.50) Mawsonascaris 177.21 8.36 Rhinobatidae$$+$$Myliobatidae 192.87 178.24 (195.10–171.60) ### 23.50 (195.10–171.60) (15.06–3.36) (220.02–176.64) (200.05–171.60) Anisakidae 256.62 214.90 ### ### ### ### ### ### (304.88–216.23) (262.60–171.64) Anisakinae 214.90 133.96 ### ### ### ### ### ### (262.60–171.64) (180.56–93.54) Anisakis 63.14 56.45 Cetacea 64.18 48.65 63.14–56.45 6.69 17.76 (66.00–56.39) (63.40–48.24) (66.00–59.45) (54.73–42.38) (66.00–48.24) Pseudoterranova 50.77 13.32 Pinnipedia 51.64 40.55 50.77–40.55 10.22 21.85 (56.30–39.52) (27.66–4.16) (56.30–46.58) (46.99–34.45) (56.30–34.45) Contracaecinae 214.90 135.26 ### ### ### ### ### ### (262.60–171.64) (181.29–96.65) Contracaecum (part) 104.20 87.79 Neognathae (fish eating) 230.43 79.63 104.20–87.79 16.41 70.30 (140.42–73.13) (120.56–57.77) (263.29–190.84) (86.8–70.12) (140.42–70.12) Heterocheilidae 325.27 153.27 Amniota 390.43 323.04 325.27–323.04 2.23 88.90 (406.90–258.94) (238.07–84.02) (424.94–358.70) (331.30–318.00) (406.90–318.00) Bold taxon names are those whose parasite–host links are all significantly supported by ParaFit tests. The results of strategy 1 are provided in Supplementary Table S9 available on Dryad. $$^{\mathrm{a}}$$HPD95: 95% highest posterior density interval. $$^{\mathrm{b}}$$EST: the estimated switching time range. $$^{\mathrm{c}}$$jHPD95: the joint 95% highest posterior density interval of EST, where the upper limit is defined as the minor value between the upper limits of the stem ages of the parasite HPD and the host HPD, and the lower limit is defined as the major value between the lower limits of the stem ages of the parasite HPD and the host HPD (shown in Supplementary Fig. S1, available on Dryad). $$^{\mathrm{d}}$$TW: the time windows (range size of the EST) in which the parasitism is estimated to have been established. $$^{\mathrm{e}}$$ETW: the expanded time windows, i.e. the range size of the jHPD95. $$^{\mathrm{f}}$$###: No monophyletic host clade or reasonable result here. Results and Discussion Phylogenies Our phylogenetic analyses using eight different genes indicated that the representatives of the Ascaridoidea were divided into six distinct clades (families): Heterocheilidae (including Ortleppascaris, Dujardinascaris, Krefftascaris, and Heterocheilus), Anisa- kidae (including Anisakis, Pseudoterranova, Pulch- rascaris, Terranova, Contracaecum, and Phocascaris), Toxocaridae (including Toxocara and Porrocaecum), Acanthocheilidae (including Acanthocheilus, Pseuda- nisakis and Mawsonascaris), Ascarididae (including Baylisascaris, Ascaris, Parascaris, and Toxascaris), and Raphidascarididae (including Hysterothylacium, Raphidascaroides, Goezia, Maricostula, Iheringascaris, Raphidascaris, and Ichthyascaris). All of these groups are monophyletic, with strong support from both ML (BS $$=$$ 99–100) and Bayesian inference (BPP $$=$$ 1), except for the Toxocaridae (BPP $$=$$ 0.98, BS $$=$$ 58) (Fig. 1). These analyses further confirm the Heterocheilidae as the sister clade to the remaining Ascaridoidea, consistent with previous phylogenetic studies based on fewer genes and taxa (Nadler 1992; Nadler and Hudspeth 1998, 2000). Figure 1. View largeDownload slide Phylogenetic relationships among ascaridoid nematodes inferred from maximum likelihood and Bayesian analyses of the DNA sequences of five nuclear and three mitochondrial genes: ITS-1, ITS-2, 5.8S, 28S, 18S, cox1, cox2, and 12S representing two loci. Ascaridia galli (Heterakoidea), Cruzia americana (Cosmocercoidea), and Falcaustra sinensis (Cosmocercoidea) were used as outgroups. Branch lengths are proportional to the substitutions per site. The paired squares filled by colors on the branches indicate the support of the downstream nodes inferred by maximum likelihood bootstraps (left) and Bayesian posterior probabilities (right). Support values of key nodes are mapped near the nodes. Both maximum likelihood and Bayesian trees are available in the Dryad. Figure 1. View largeDownload slide Phylogenetic relationships among ascaridoid nematodes inferred from maximum likelihood and Bayesian analyses of the DNA sequences of five nuclear and three mitochondrial genes: ITS-1, ITS-2, 5.8S, 28S, 18S, cox1, cox2, and 12S representing two loci. Ascaridia galli (Heterakoidea), Cruzia americana (Cosmocercoidea), and Falcaustra sinensis (Cosmocercoidea) were used as outgroups. Branch lengths are proportional to the substitutions per site. The paired squares filled by colors on the branches indicate the support of the downstream nodes inferred by maximum likelihood bootstraps (left) and Bayesian posterior probabilities (right). Support values of key nodes are mapped near the nodes. Both maximum likelihood and Bayesian trees are available in the Dryad. Our present study represents the first attempt to resolve the systematic position of the Acanthocheilidae using phylogenetic analyses based on molecular data. The results support the validity of the Acanthocheilidae as a distinct family outside the Anisakidae, which is in agreement with some previous hypotheses based on morphological characters (Osche 1958; Hartwich 1974; Gibson 1973, 1983), but these results strongly reject these proposals to remove Pseudanisakis from the Acanthocheilidae (Sprent 1983; Sprent 1990; Petter et al. 1991) as these two genera Pseudanisakis and Acanthocheilus are sister groups with strong support in both ML inference and Bayesian inference (BS $$=$$ 100, BPP $$=$$ 1). Our phylogenetic analyses also confirmed Mawsonascaris as a member of the Acanthocheilidae with a sister relationship to Acanthocheilus$$+$$Pseudanisakis based on strong support from both ML inference and Bayesian inference (BS $$=$$ 100, BPP $$=$$ 1). Parasite–Host Associations We tested co-phylogenetic associations between ascaridoid species and their vertebrate hosts. The results supported a very significant global co-evolution between the Ascaridoidea and their hosts (global ParaFit Statistics $$=$$ 529.5912, $$P$$$$<$$0.001). One hundred and twenty-one of 151 PHA are significant (see Supplementary Table S8 available on Dryad). Within these significant PHAs, four clades represent uniformly monophyletic P2H clades (Fig. 2), in which both the host clade (H-clade) and the parasite clade (P-clade) are monophyletic, and the P-clade taxa are restricted to the H-clade. The H-clade can thereby be considered as the “primary hosts” of the P-clade. For example, members of the Raphidascarididae are specialized parasites of teleost fishes. There are three clades with H clades that are not strictly monophyletic because of some ecological constraints: the Ascarididae are specialized parasites of terrestrial Boreoeutheria (absent from pinnipeds and whales); species of Baylisascaris (nested within Ascarididae) are specialized in terrestrial Arctoidea (absent from pinnipeds); the derivative four Contracaecum spp. (clade G, Fig. 2) parasitize only fish-eating birds (as adult nematodes), but their monophyly was not supported by Bayesian inference (Fig. 1). These clades were not used in the calibration. Although acanthocheilids are obligate parasites of elasmobranchs, not all of their PHA links are supported here as cophylogenetic relationships. In our results, only Mawsonascaris coevolving with the Rhinobatidae $$+$$ Myliobatidae is significant. Within the Anisakidae, Anisakis and Pseudoterranova have a completely significant co-evolution with their hosts (whales and pinnipeds, respectively), that agrees well with a previous study (Mattiucci and Nascetti 2008). Other anisakids have a confusing mixture of hosts and, therefore, exhibit less cophylogenetic significance. Similarly, the Toxocaridae and Heterocheilidae lack significant PHA links. Divergence Time, Ancestral Hosts, and Switches By calibrating the four uniformly monophyletic P2H clades using the fossil/estimated time information for the relevant associated host clades (see Supplementary Table S5 available on Dryad), we estimated the time period for ascaridoid divergence. Then, we reconstructed both the host types for ascaridoids and the habitats of hosts for all of the internal nodes of the time-calibrated trees. Our results (Fig. 2 and Table 1, strategy 2 shown; strategy 1 results are provided in Supplementary Fig. S1 and Table S9 available on Dryad) showed that the ancestor of the Ascaridoidea is estimated to have appeared 360.47 Ma (95% HPD: 456.30–280.00 Ma) and the MRCA of the extant ascaridoid species occurred 325.27 (406.90–258.94) Ma (Fig. 2 and Table 1). This result is prior to, but not discordant with, an existing hypothesis of a pre-Permian origin (Osche 1963), although the latter was proposed based on “parasitophyletic rules” and considered the elasmobranch-parasitic Acanthocheilidae as the sister group to the other ascaridoids, which has been previously questioned (Sprent 1983) and is rejected herein. The host of the ancestral ascaridoid lineage was estimated, with a marginal likelihood (0.4950), as being a “terrestrial tetrapod.” In the host tree, the lineages of the amniotes, teleosts and elasmobranchs occurred within this range, but among these, only an ancestral amniote lineage is plausible, as its habitat was estimated to be “terrestrial” (marginal likelihood: 0.8918). So, we hypothesize that the ascaridoid ancestor was derived from its parent lineage by switching into a terrestrial amniote host approximately 360.47–325.27 Ma (joint 95% HPD: 424.94–318.00 Ma), which indicates that there was at least 35.20 Myr (or at most 106.94 Myr) for the fixation of this parasitism (Fig. 2 and Table 1). Moreover, the median crown age of the ascaridoid lineage is slightly older (2.23 Myr) than the amniote lineage, which suggests that the basal split of the ascaridoid lineage may predate the host divergence. If so, one might predict that the Heterocheilidae and its sister lineage would appear in both daughter host clades (the reptile-clade and the mammal-clade) of the amniote lineage (Fig. 3). In fact, heterocheilids parasitize a limited group of mammals (manatees and dugongs), whereas most heterocheilids parasitize reptiles. Members of the other five families have radiated into every major clade of amniotes. Figure 3. View largeDownload slide Scheme of the main host switches of high-level ascaridoids inferred from both cophylogenetic relationships plus the literature. Path color is used to distinguish the families. The terminal dashed lines mean that the parasitic relationships have no cophylogenetic significance. Dot-dashed lines indicate “missing paths”, where parasitism was established in the ancestor but is absent from its descendants. Loops in the paths locate possible parasitism on the host phylogeny. The placements of the three free loops (not linked to the host tree) are unresolved by our results. Dotted lines display the possible path of the Angusticaecinae and Sulcascaris (absent from our analysis). The three discontinuous bifurcations (may be multifurcate), marked with a “sun”, “star”, and “moon”, represent the three starting nodes for the “land-to-water” host shifts (as in Fig. 2). The time axis (unscaled) indicates some key points in the sea-level changes which took place during the evolution of the ascaridoids. Host profiles on the internal nodes depict the ancestors, and illustrations at the base represent not only the species but also phylogenetically related taxa. Figure 3. View largeDownload slide Scheme of the main host switches of high-level ascaridoids inferred from both cophylogenetic relationships plus the literature. Path color is used to distinguish the families. The terminal dashed lines mean that the parasitic relationships have no cophylogenetic significance. Dot-dashed lines indicate “missing paths”, where parasitism was established in the ancestor but is absent from its descendants. Loops in the paths locate possible parasitism on the host phylogeny. The placements of the three free loops (not linked to the host tree) are unresolved by our results. Dotted lines display the possible path of the Angusticaecinae and Sulcascaris (absent from our analysis). The three discontinuous bifurcations (may be multifurcate), marked with a “sun”, “star”, and “moon”, represent the three starting nodes for the “land-to-water” host shifts (as in Fig. 2). The time axis (unscaled) indicates some key points in the sea-level changes which took place during the evolution of the ascaridoids. Host profiles on the internal nodes depict the ancestors, and illustrations at the base represent not only the species but also phylogenetically related taxa. The crown age of the Heterocheilidae was estimated to be 153.27 (238.07–84.02) Ma, and its possible host MRCA was estimated to be a “semiaquatic tetrapod” (marginal likelihood: 0.8928). The dates estimated based on ML topology suggested that the divergence of the heterocheilids does not follow the evolution of hosts but is associated with the break-up of Pangaea. The Alligator-parasitic species separated about 153.27 (238.07–84.02) Ma, coinciding with the separation of Laurasia from Gondwana (since 175 Ma), and the divergence of Heterocheilus (parasitizing Trichechus in Africa and South America) and Krefftascaris (parasitizing Chelodina in Australia and New Guinea) took place 114.02 (191.97–47.78) Ma. At the same time, Gondwana was dividing into multiple continents (Africa, South America, India, Antarctica, and Australia). Extant heterocheilid species, mainly parasitic in the species of Crocodilia (not merely Alligator), Sirenia and Chelodina, all live in regions derived from the coastal areas of the Palaeo-Tethys Ocean (most of the hosts are Gondwanan in origin, except for Alligator). We consider that the stem heterocheilids likely inhabited both semiaquatic diapsids and Mammaliaformes distributed across Gondwana and, perhaps, along the shores of the Palaeo-Tethys Ocean, where they may have transferred hosts via predation or food webs (Fig. 3). The estimated switching time (range) of the heterocheilid lineage parasitizing such semiaquatic amniotes was 325.27–323.04 (406.90–318.00) Ma. The oldest known ascaridoid fossil (240 Ma) is most likely to have been a heterocheilid, judging by both superficial morphological traits and its herbivorous/carnivorous Gondwanan mammal-like host (Silva et al. 2014). The “Ascaridoidea minus Heterocheilidae” clade is estimated to have diverged into six major clades during the Triassic. Members of the Raphidascarididae are specialized parasites of teleosts, although some Goezia species also parasitize crocodiles. The estimated switching time of the Raphidascarididae to teleosts was 247.31–194.67 (292.59–160.86) Ma, which suggests that it took 52.64 (at most 131.73) Myr to fix this parasitism. However, the Raphidascarididae did not diversify with the radiation of teleosts until the beginning of the Cretaceous (about 140.33 Ma). Acanthocheilids only parasitize elasmobranchs, but both their stem and crown ages are far later than the age of elasmobranchs, which indicates that acanthocheilids parasitized elasmobranchs secondarily. Therefore, the hypothesis of an aquatic or marine origin for the Ascaridoidea (Osche 1958) is not supported by our results. The Ascarididae parasitize terrestrial boreoeuthe- rians, which they may have switched to this group during the period 121.85–109.29 (137.82–95.06) Ma, and almost immediately diversified with their hosts, but did not parasitize aquatic hosts. The Angusticaecinae is a derivative group within the Ascarididae. Its members mainly parasitize frogs, snakes, lizards, chameleons, and armadillos. As they are missing from our analysis, we tentatively assume that they are sister to other ascaridid (Ascarididae) subgroups, colonized lizards and snakes (Squamata) and then transferred to tailless amphibians through predation or food webs. The crown age of the Toxocaridae is estimated to be 144.91 (197.13–93.65) Ma, and the ancestral host type is considered to be “terrestrial tetrapods” (marginal likelihood: 0.9747, Fig. 2). This family used to be considered as a subfamily within the Ascarididae, but its members have much more diverse infection patterns than ascaridids (Sprent 1983). Owing to the temporal limitations, the stem lineage of the Toxocaridae would have been unable to colonize the stem of the amniotes, but had opportunities to switch to therians and modern birds. We, therefore speculate that the common ancestor of ascaridids and toxocarids first parasitized ancient therians based on the latter’s alternative infection patterns, e.g. species of Porrocaecum, which opportunistically infect mammals, have transferred to birds via their intermediate hosts (e.g., earthworms) (Fig. 3). The Ascarididae and the Toxocaridae are the only extant ascaridoids that still parasitize terrestrial tetrapods. The Anisakidae is one of the most species-rich families in the Ascaridoidea. Our estimates show that it split into two major clades (Anisakinae and Contracaecinae) 214.90 (262.60–171.64) Ma. Members of the Anisakinae parasitize diverse vertebrates: elasmobranchs (Pulchrascaris and Terranova), pinnipeds (Pseudoterranova), cetaceans (Anisakis and Terranova), sea turtles (Sulcascaris), and crocodiles and snakes (Terranova). Our estimates of the ancestral hosts of anisakines had low likelihoods, precluding additional speculation. Members of the Contracaecinae parasitize fish-eating birds (supported by significant PHA tests) and pinnipeds (no significant support). The stem lineage of this subfamily appeared 214.90 (262.60–171.64) Ma, but diverged 135.26 (181.29–96.65) Ma, during a period when only birds and their stem groups (bird-line archosaurs) were possible hosts (pinnipeds had not yet appeared). The ancestral host type is estimated to be “semiaquatic tetrapods” (marginal likelihood: 0.9939). Nevertheless, we cannot draw further conclusions on the primary host of the Contracaecinae, because the ML and Bayesian trees conflict regarding relationships. Our results show that the MRCA of the Anisakidae was likely to parasitize a “semiaquatic tetrapod” (marginal likelihood: 0. 5675) 214.90 (259.34–169.76) Ma. The stem age of the Anisakidae, 254.03 (302.72–215.34) Ma, just postdates the crown age of diapsids (265.62 (285.12–255.9) Ma), which is roughly concordant with the previous divergence time analyses using the Cytochrome C and globin amino acid sequences (i.e., the Anisakidae and Ascarididae diverged about 150–250 Ma) (Vanfleteren et al. 1994). Land-Water Shifts with Sea-Level Changes Our dating results indicated that ascaridoids were derived from other “ascaridomorph” nematodes in the Early Carboniferous (Mississippian) and were parasites of terrestrial amniotes, which agrees with some earlier hypotheses of a terrestrial origin of ascaridoid nematodes (Chitwood 1950; Dougherty 1951); it is also reasonable because our results suggest that the origin of ascaridoids occurred at a time when the sea-level dropped, falling to present levels or lower (Haq et al. 1987; Haq and Schutter 2008) (Fig. 2). This time was at the onset of the Permo-Carboniferous Glaciation and the Carboniferous Rainforest Collapse, causing a downturn in amphibian diversity and the adaptive radiation of reptiles (Sahney et al. 2010), which may be the reason why the crown ascaridoids are rare in amphibians, but rich in reptiles. During the entire Permian (a period of low sea-level), we found no diversification of crown groups of either ascaridoids or their hosts. But shortly after the recovery from the Permian-Triassic extinction, when the sea-level began to rise, the “Ascaridoidea minus Heterocheilidae” clade began an abrupt diversification into five distinct subclades. We also recognize five fundamental switches of host types in ascaridoid evolution, all of which likely occurred at the time of a rise in sea-level during the Triassic to the Early Cretaceous periods and all from terrestrial to aquatic or semiaquatic environments (Fig. 2). However, this pattern cannot be deduced from changes in host habitat or from host evolution alone. Our results suggest that early host switches of ascaridoids appear to be correlated with global climate patterns, particularly sea-level change. Moreover, our schematic model (Fig. 3) shows that “lateral host switches” (yielding increased host range in parasite groups and diverse parasitological patterns in host groups) occurred very recently, possibly in the Cenozoic, consistent with more host switching rather than cophylogenetic patterns. It is worth noting that it should have been possible for terrestrial ascaridids and terrestrial/semiaquatic toxocarids to colonize marine mammals, such as pinnipeds or cetaceans (Fig. 3), as predicted by parasite-host cophylogeny. Perhaps the ancestors of pinnipeds and cetaceans lost their ascaridids and toxocarids during their transition to aquatic life as new food-web associations resulted in colonization by other ascaridoids, although our current knowledge offers no evidence for these missing associations. Conclusion Our molecular phylogenetic analyses divide this superfamily into six main monophyletic clades, i.e. the Heterocheilidae, Acanthocheilidae, Anisakidae, Ascarididae, Toxocaridae, and Raphidascarididae, among which the Heterocheilidae, rather than the Acanthocheilidae, appears to be the sister group to the remaining ascaridoid lineages. We further calibrated the phylogeny using an approach that involves time priors from fossils of the co-evolving hosts, rather than the rare and unreliable ascaridoid egg fossils, and date the common ancestor of the Ascaridoidea back to the Early Carboniferous (approximately 360.47–325.27 Ma). We then estimated the host types of the ancestral ascaridoid and its major clades, and reconstructed the habitat type of the relevant host clades. Accordingly, we established a host-switching model for the high-level groups. The dates and ancestral host types indicated by our results both suggest that members of the Ascaridoidea primarily inhabited terrestrial tetrapods and subsequently extended their range to elasmobranchs and teleosts. We also determined that the fundamental terrestrial-to-aquatic switches of these nematodes coincided with rises in sea-level during the Triassic to the Early Cretaceous. Supplementary Material Data available from the Dryad Digital Repository: http://doi.org/10.5061/dryad.28g16. Acknowledgements The authors wish to thank Dr Guy Baele for suggestions on the use of marginal likelihood estimation using PS/SS sampling. We are also grateful to Professor Hideo Hasegawa (Oita University, Japan) for providing useful literature. Funding This study was supported by the National Natural Science Foundation of China [NSFC-31572231, NSFC-31750002]; Natural Science Foundation of Hebei Province [NSFH-C2016205088]; and the Youth Top Talent Support Program of Hebei Province to L.L. 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Relationships among some ascaridoid nematodes based on ribosomal DNA sequence data. Parasitol. Res. 86 : 738 – 744 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Systematic Biology Oxford University Press

Molecular Phylogeny and Dating Reveal a Terrestrial Origin in the Early Carboniferous for Ascaridoid Nematodes

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Abstract

Abstract Ascaridoids are among the commonest groups of zooparasitic nematodes (roundworms) and occur in the alimentary canal of all major vertebrate groups, including humans. They have an extremely high diversity and are of major socio-economic importance. However, their evolutionary history remains poorly known. Herein, we performed a comprehensive phylogenetic analysis of the Ascaridoidea. Our results divided the Ascaridoidea into six monophyletic major clades, i.e., the Heterocheilidae, Acanthocheilidae, Anisakidae, Ascarididae, Toxocaridae, and Raphidascarididae, among which the Heterocheilidae, rather than the Acanthocheilidae, represents the sister clade to the remaining ascaridoids. The phylogeny was calibrated using an approach that involves time priors from fossils of the co-evolving hosts, and dates the common ancestor of the Ascaridoidea back to the Early Carboniferous (approximately 360.47–325.27 Ma). The divergence dates and ancestral host types indicated by our study suggest that members of the Ascaridoidea first parasitized terrestrial tetrapods, and subsequently, extended their host range to elasmobranchs and teleosts. We also propose that the fundamental terrestrial-aquatic switches of these nematodes were affected by changes in sea-level during the Triassic to the Early Cretaceous. Host switching, molecular dating, parasite–host association, phylogeny, sea-level change, terrestrial origin The Ascaridoidea is a superfamily of parasitic nematodes (roundworms) containing more than 800 described species that parasitize all major lineages of vertebrates (Hartwich 1974; Hodda 2011), and as adults usually inhabit the alimentary canal. Ascaridoids are of veterinary, medical and economic importance, causing disease in domestic animals, wildlife, and humans (e.g., ascariasis, toxocariasis, baylisascariasis, and anisakiasis) (Mozgovoi 1953; Anderson 2000; Despommier 2003; Hochberg and Hamer 2010; Bauer 2013). Current classifications of the Ascaridoidea have been proposed based mainly on morphological characters, including the form of the excretory system (Hartwich 1954, 1957, 1974; Chabaud 1965; Gibson 1983), the structure of the esophago-intestinal junction (Baylis 1920; Mozgovoi 1953; Yamaguti 1961), the labial structure (Osche 1958) and the number and arrangement of caudal papillae (Fagerholm 1991). The different weighting of these morphological characters by individual investigators has led to conflicting classifications of this superfamily. The origin and the early evolutionary history of the Ascaridoidea have long been uncertain and contentious. Some authors, through strict application of “parasitophyletic rules” [e.g., Szidat’s Rule (hosts resembling the ancestral state are more likely to harbor parasites resembling the ancestral state); Fahrenholz’s Rule (host and parasite phylogenies tend to mirror one another) (MacIntosh and Frias 2017)], hypothesized that ascaridoids originated in an aquatic (marine) environment and that members of the Acanthocheilidae, parasites of elasmobranch fishes, must represent the earliest subgroup (Mozgovoi 1953; Osche 1958, 1963; Sprent 1962; Gibson 1983). Others have disagreed with this hypothesis and speculated that ascaridoids originally parasitized early terrestrial tetrapods with a secondary radiation to aquatic hosts (Chitwood and Chitwood 1950; Dougherty 1951; Chabaud 1955; Sprent 1983; Anderson 1984). These varying hypotheses result from different interpretations of observations of life history patterns and/or morphological traits. To understand the early evolution of the Ascaridoidea, a reliable and representative phylogeny is essential. Recently, some authors have made efforts to establish the phylogenetic relationships of certain taxa and to solve particular problems using nuclear rDNA sequence data and mitochondrial genes (Nadler 1992; Nadler and Hudspeth 1998, 2000; Nadler et al. 2000, 2005; Zhu et al. 2000; Mattiucci and Nascetti 2006, 2008; Mattiucci et al. 2008; Li et al. 2012, 2017; Mohandas et al. 2014; Liu et al. 2015, 2016). However, due to the scarcity and inaccessibility of suitable material for some taxa (e.g., the Acanthocheilidae, Heterocheilidae, and Raphidascarididae), these molecular phylogenetic studies have included only a very small representation of ascaridoid diversity. Hence, our knowledge of the phylogeny of the Ascaridoidea is still far from comprehensive. Estimating the age of the ascaridoid clades has also been difficult. By scaling divergence events to geological time, one can determine the timing of key innovations during the history of the group. Unfortunately, fossils of ascaridoids are extremely rare due to their soft body (Silva et al. 2014). To date, only three species represented by fossilised eggs (all tentatively assigned to the Ascarididae: i.e. † Ascarites) have been found in the coprolites of cynodonts or dinosaurs (Poinar and Boucot 2006; Poinar 2011; Silva et al. 2014), but the systematic placements of these eggs are questionable (superficially they resemble the Ascarididae, Toxocaridae and Heterocheilidae). This hinders efforts to use calibration information from ascaridoid evolutionary history along with molecular sequence data to estimate divergence times using standard molecular clock approaches. Two studies have made such attempts by estimation using eukaryotes (Douzery et al. 2004) or based on molecular evolutionary rates of other groups of organisms (Vanfleteren et al. 1994). Their results are largely in conflict. Douzery et al. (2004) estimated the divergence of the orders Spirurida and Ascaridida at $$\sim$$150 Ma, whereas Vanfleteren et al. (1994) estimated the divergence of the Anisakidae and Ascaridae to be 250–150 Ma. In this study, we performed phylogenetic analyses of ascaridoid nematodes based on five nuclear and three mitochondrial genes. These molecular phylogenetic analyses included the most comprehensive taxon sampling of the Ascaridoidea to date, yielding a robust evolutionary hypothesis for the superfamily. The phylogeny was calibrated using an approach that involves time priors from hosts rather than ascaridoid calibrations per se. We then estimated the host types of the ancestor of the ascaridoids and some of its major clades with the aim of establishing an original host-switching model for the group. Materials and Methods Taxon Sampling The in-group samples include 65 ascaridoid species belonging to 26 genera, including all of the major families (Acanthocheilidae, Anisakidae, Ascarididae, Heterocheilidae, Raphidascarididae, Toxocaridae) according to the generally accepted classifications of the Ascaridoidea (Hartwich 1974; Fagerholm 1991) (see Supplementary Table S1 available on Dryad at http://doi.org/10.5061/dryad.28g16). Representatives of the family Crossophoridae (containing only the genera Crossophorus Ehrenberg 1828 and Dartevellenia Ezzat 1954, parasites of hyracoids) were unavailable for inclusion in the analysis. Many previous phylogenetic studies based on various loci strongly support the Heterakoidea and Cosmocercoidea as being groups closely related to the Ascaridoidea, but not part of this clade (Smythe et al. 2006; Holterman et al. 2006; Nadler et al. 2007; Ross et al. 2010; Cernotíková et al. 2011; Kim et al. 2014; Liu et al. 2016; Choudhury and Nadler 2016). Consequently, three species of the Heterakoidea (Ascaridia galli) and Cosmocercoidea (Cruzia americana and Falcaustra sinensis) were used as outgroups to root the trees (see Supplementary Table S1 available on Dryad). DNA Extraction, Amplification, and Sequencing To study the evolutionary history of the Ascaridoidea, we constructed a database, including nuclear sequences [partial sequences of small ribosomal subunit (18S), large ribosomal subunit (28S), internal transcribed spacer 1 (ITS-1), 5.8S and internal transcribed spacer 2 (ITS-2)] plus mitochondrial data [partial cytochrome $$c$$ oxidase subunit 1 (cox1), cytochrome $$c$$ oxidase subunit 2 (cox2), and 12S small subunit ribosomal RNA gene] from 68 taxa, including 123 new sequences (see Supplementary Table S2 available on Dryad). Sequence data were also obtained from GenBank. Genomic DNA from an individual nematode was extracted using a Column Genomic DNA Isolation Kit (Shanghai Sangon, China) according to the manufacturer’s instructions and then stored at $$-$$20℃ until the molecular analysis. For amplifying these target sequences, the following published primers were used: the near-complete 18S rDNA by the primers 18SF and 18SR (Floyd et al. 2005), the partial ITS region by the primers A and B (D’Amelio et al. 2000), the partial 28S rDNA by the primers 28SF and 28SR (Nadler and Hudspeth 1998), the partial cox1 by the primers CO1F and CO1R (Lazarova et al. 2006), the partial cox2 by the primers CO2F and CO2R (Nadler and Hudspeth 2000), and the partial 12S by the primers 12SF and 12SR (Li et al. 2012). All polymerase chain reactions (PCRs) were performed in 50 $$\mu$$L of PCR reaction buffer with 10 mM Tris HCl at pH 8.4, 50 mM KCl, 3.0 mM MgCl$$_{{2}}$$, 250 $$\mu $$M of each dNTP, 50 pmol of each primer, and 1.5 U of Taq polymerase (Takara) in a thermocycler (2720, Applied Biosystems) under the following conditions: 94° C, 5 min (initial denaturation), followed by 30 cycles of 94° C, 30 s (denaturation), 55° C, 30 s (annealing), 72° C, 70 s (extension), and a final extension of 72° C for 7 min. PCR products were checked on GoldView-stained 1.5% agarose gel and purified by the Column PCR Product Purification Kit (Shanghai Sangon, China). Sequencing for each sample was completed for both strands using a Dideoxy Terminator Cycle Sequencing Kit (v.2, Applied Biosystems, CA, USA) and an automated sequencer (ABI-PRISM 377). All the DNA sequences obtained herein (18S, ITS-1, 5.8S, ITS-2, 28S, 12S, cox1, and cox2) are deposited in the GenBank database (http://www.ncbi.nlm. nih.gov) (Table S1). Sequence Alignment Molecular sequences of each gene (nucleotides for non-coding genes and amino acids for coding genes) were initially aligned using the E-INS-i algorithm in the online version of MAFFT v7.3 (Katoh et al. 2002; Katoh and Standley 2013) with adjustment to the direction of the first sequence, gappy regions retained and other alignment parameters left as default. The nucleotides of coding genes were aligned to the alignments of amino acids. The program MEGA v7.0.14 (Kumar et al. 2016) was used to refine the primary single-gene alignment manually. Alignment positions with unique or few ($$\leqslant $$3) indels were deleted; the ends were trimmed; the third position of coding genes was removed if indicated by the DAMBE mutational saturation test (Xia et al. 2003). Multiple genes were concatenated into one merged matrix using Mesquite v3.04 (Maddison and Maddison 2016). Data Partitions, Model Selection, and Phylogenetic Analyses We used PartitionFinder v2.0.0 (Lanfear et al. 2017) to select both a data-partitioning scheme and a best-fitting substitution model for each partition using the greedy algorithm (Lanfear et al. 2012) with the Bayesian information criterion (Schwarz 1978). The partitioning schemes and the optimal model selected for each combination of partition and tree inference program are detailed in Supplementary Table S2 available on Dryad. We performed the maximum likelihood (ML) inference using RAxML v8.2.11 (Stamatakis 2014). The best-scoring tree was found with the rapid hill-climbing algorithm (Stamatakis et al. 2007) and bootstrap support (BS) was estimated using the rapid bootstrap option (Stamatakis et al. 2008) and 1000 replicates (GTR$$+$$Gamma model, partitioned for the multigene matrices). The bipartition values for ML BS were mapped to the best-scoring tree(s). ML BS values $$\geqslant $$80 were considered to constitute strong nodal support, whereas BS values $$\geqslant $$50 and $$<$$80 were considered to constitute moderate nodal support. We performed a Bayesian inference analysis for each matrix using MrBayes v3.2.6 (Huelsenbeck and Ronquist 2001; Ronquist et al. 2012) executed on the CIPRES Science Gateway cluster (Miller et al. 2010) using partitions and models defined by PartitionFinder (see above). We made two simultaneous runs of Markov Chain Monte Carlo (MCMC) sampling, each with four chains, 20 million generations, and sampled every 1000 generations. The standard deviation of split frequencies converged at 0.003818. We used the program Tracer v1.6 (Rambaut and Drummond 2013) to diagnose convergence, determine an appropriate burn-in and verify the effective sample size (ESS) of every parameter >200 for the combined MCMC samples of each matrix. Accordingly, the first 25% of the samples of each run were dropped when generating the 50% consensus trees and estimating nodal Bayesian posterior probabilities (BPP). BPP $$\geqslant $$0.98 were considered to constitute strong nodal support, whereas BPP values $$\geqslant $$0.95 and $$<$$0.98 were considered to constitute moderate nodal support. The resulting trees were rooted with the outgroups. Phylogeny of Hosts To build the host phylogeny, we sampled a total of 89 genera of vertebrates (without outgroups), which include all the genus-level hosts of the Ascaridoidea known to us in this study (see Supplementary Table S3 available on Dryad). It was necessary to estimate such a phylogeny because a tree with these particular vertebrate genera was not available in the published literature. We used six genes, including the nuclear recombination activating gene 1 (RAG1, intron excluded) and mitochondrial cytochrome b (Cyt b), cox1, cox2, 12S, and 16S rDNA. All these sequences were mined from the GenBank database (see Supplementary Table S4 available on Dryad). We used RAxML v8.2.11 (Stamatakis 2014) to build the ML phylogenetic tree of the hosts in the same way as described for the parasites, and with a mixed matrix concatenated from protein sequences (RAG1, Cyt b, cox1, cox2) and nucleotide sequences (12S, 16S) (Table S2). The divergence times of the hosts were estimated using BEAST v1.8.4 (Drummond and Rambaut 2007; Drummond et al. 2012). We used the fossil age of the Gnathostomata (468.4–420.7 Ma) (Benton et al. 2015) and the ML best tree to obtain an ultrametric starting tree using treePL (Smith and O’Meara 2012) (smoothing parameters obtained by cross validation), which was then imported into BEAUTi (Drummond and Rambaut 2007) as a starting tree to fix the tree topology. The BEAST analyses employed 14 time priors (uniform distribution) from the literature (see Supplementary Table S5 available on Dryad). We executed six independent MCMC runs on the cluster from the CIPRES Science Gateway (Miller et al. 2010) for different models (variations included Yule, birth-death process, strict clock, uncorrelated relaxed clock with lognormal distribution, and uncorrelated relaxed clock with exponential distribution); each run with 300 million generations and sampled every 1000 generations. This was followed by marginal likelihood estimation using path sampling (PS)/stepping-stone (SS) sampling (300 steps by 1,000,000 iterations) (Baele et al. 2012, 2013). Then, we compared their logarithmic marginal likelihoods for six models (see Supplementary Table S6 available on Dryad) and used Treeannotator v1.8.4 (Drummond and Rambaut 2007) to obtain the maximum clade credibility time tree from 29 001 resulting post burn-in trees (ESS >200) of the best model (Yule process and uncorrelated relaxed clock with lognormal distribution, Supplementary Table S6 available on Dryad). Testing Parasite–Host Associations and Dating Ascaridoid Phylogeny We compiled a set of parasite–host association (PHA) links from the literature and our own observations (see Supplementary Table S3 available on Dryad), and tested whether the PHA links are independent or phylogenetically related, using the eight-gene ML phylogeny of the Ascaridoidea and the ML phylogeny of the hosts. We executed both global and individual tests using the ParaFit method (Legendre and Desdevises 2002) in the APE package (Paradis et al. 2004) in R 3.3.1 (Core R Team 2016), where we ran 100,000 random permutation and considered the PHA links with the permuted $$P$$-values of both ParaFitLink 1 and ParaFitLink 2 $$\leqslant $$0.05 as significant. We recognized four pairs of significant monophyletic parasite-to-host specialist (P2H) clades (Fig. 2) in which both the host clade (H-clade) and the parasite clade (P-clade) are monophyletic, and the P-clade taxa are present only in that H-clade. The event-based cophylogeny of the four pairs of P2H clades enable us to calibrate the P-clades based on the time information from the associated H-clades. On the basis of the other host-parasite comparisons for time calibrations (Ricklefs and Outlaw 2010; Bensch et al. 2013; Shah et al. 2010), we considered the time from the appearance of the ancestor lineage of the H-clade (in the vertebrate tree) to the divergence of the most recent common ancestor (MRCA) of the H-clade as the potential switching time range of the ancestor lineage of the P-clade (in the ascaridoid tree). Therefore, we calibrated the ancestor lineage (on the stem) of the four P-clades (calibrations for the Raphidascarididae and Ascarididae are equivalent) using two strategies: (i) setting the upper limit of the P-clade stem by the upper limit of the (oldest) fossil of the parent node of the associated H-clade, and setting the lower limit of the P-clade stem using the lower limit of the fossil of the MRCA of the H-clade; (ii) using the limits of the 95% highest posterior density interval (HPD95) instead of the host fossil limits. See details in Supplementary Table S5 available on Dryad. Figure 2. View largeDownload slide Time tree of the Ascaridoidea (left) and their hosts (right), the parasite–host association links, maximum likelihood reconstruction of the ancestral host type of the Ascaridoidea and the habitat of ascaridoid terminal hosts. Bars on nodes show the 95% highest posterior density interval estimated from the resulting BEAST MCMC trees. Of the seven letter-labeled P-clades, four (A–D) are the significant monophyletic P2H clades and have the same color as the associated monophyletic H-clades on the host tree; these are connected by solid lines in the same colors. The calibration of molecular clock analyses is based on these P2H clades (see Supplementary Fig. S1 and Table S5 available on Dryad). Clades E and F are specialized P-clades, but their host clades are paraphyletic; clade G is not supported by the Bayesian inference. The grey and dashed lines are phylogenetically independent links. Pie charts on the nodes of the ascaridoid tree show the scaled marginal likelihood of each host type estimated by the maximum likelihood method using the equal-rates model (stably invariant results are not shown). Colored diamonds at the tips of the parasite tree indicate the host type of the relevant taxa. Similarly, the pie charts and the diamonds on the host tree show the estimated ancestral habitats (by the maximum likelihood method using the symmetrical model) and the present states, respectively. The “sun” marks the MRCA of the Ascaridoidea; the “moon” marks the MRCA of the Anisakinae (ANI); the “star” marks the MRCA of the “Ascaridoidea minus Heterocheilidae” clade. Starting from these nodes, the five critical “land-to-water” shifts in the early history of the Ascaridoidea that we propose are: “sun” to R1, “star” to “moon” to R2 and to R3, and “star” to R4 and R5. All these shifts and evolution took place during the Triassic to the Early Cretaceous, when the global sea level rose. Taxon abbreviations: RAP $$=$$ Raphidascarididae; ASC $$=$$ Ascarididae; TOX $$=$$ Toxocaridae; ACA $$=$$ Acanthocheilidae; ANI $$=$$ Anisakidae; HETC $$=$$ Heterocheilidae; OGHETK $$=$$ Heterakoidea (outgroup); OGCOS $$=$$ Cosmocercoidea (outgroup); MAMM $$=$$ Mammalia (Theria); AVES $$=$$ Aves (modern birds); REPT $$=$$ Reptilia (Sauria excluding birds); ACTI $$=$$ Actinopterygii (Teleostei); CHON $$=$$ Chondrichthyes (Elasmobranchii). Period abbreviations: S $$=$$ Silurian; D $$=$$ Devonian; C $$=$$ Carboniferous; P $$=$$ Permian; T $$=$$ Triassic; J $$=$$ Jurassic; K $$=$$ Cretaceous; Pg $$=$$ Palaeogene; N $$=$$ Neogene (and Quaternary). The time trees (under best model) of both the Ascaridoidea and hosts are available in the Dryad. Figure 2. View largeDownload slide Time tree of the Ascaridoidea (left) and their hosts (right), the parasite–host association links, maximum likelihood reconstruction of the ancestral host type of the Ascaridoidea and the habitat of ascaridoid terminal hosts. Bars on nodes show the 95% highest posterior density interval estimated from the resulting BEAST MCMC trees. Of the seven letter-labeled P-clades, four (A–D) are the significant monophyletic P2H clades and have the same color as the associated monophyletic H-clades on the host tree; these are connected by solid lines in the same colors. The calibration of molecular clock analyses is based on these P2H clades (see Supplementary Fig. S1 and Table S5 available on Dryad). Clades E and F are specialized P-clades, but their host clades are paraphyletic; clade G is not supported by the Bayesian inference. The grey and dashed lines are phylogenetically independent links. Pie charts on the nodes of the ascaridoid tree show the scaled marginal likelihood of each host type estimated by the maximum likelihood method using the equal-rates model (stably invariant results are not shown). Colored diamonds at the tips of the parasite tree indicate the host type of the relevant taxa. Similarly, the pie charts and the diamonds on the host tree show the estimated ancestral habitats (by the maximum likelihood method using the symmetrical model) and the present states, respectively. The “sun” marks the MRCA of the Ascaridoidea; the “moon” marks the MRCA of the Anisakinae (ANI); the “star” marks the MRCA of the “Ascaridoidea minus Heterocheilidae” clade. Starting from these nodes, the five critical “land-to-water” shifts in the early history of the Ascaridoidea that we propose are: “sun” to R1, “star” to “moon” to R2 and to R3, and “star” to R4 and R5. All these shifts and evolution took place during the Triassic to the Early Cretaceous, when the global sea level rose. Taxon abbreviations: RAP $$=$$ Raphidascarididae; ASC $$=$$ Ascarididae; TOX $$=$$ Toxocaridae; ACA $$=$$ Acanthocheilidae; ANI $$=$$ Anisakidae; HETC $$=$$ Heterocheilidae; OGHETK $$=$$ Heterakoidea (outgroup); OGCOS $$=$$ Cosmocercoidea (outgroup); MAMM $$=$$ Mammalia (Theria); AVES $$=$$ Aves (modern birds); REPT $$=$$ Reptilia (Sauria excluding birds); ACTI $$=$$ Actinopterygii (Teleostei); CHON $$=$$ Chondrichthyes (Elasmobranchii). Period abbreviations: S $$=$$ Silurian; D $$=$$ Devonian; C $$=$$ Carboniferous; P $$=$$ Permian; T $$=$$ Triassic; J $$=$$ Jurassic; K $$=$$ Cretaceous; Pg $$=$$ Palaeogene; N $$=$$ Neogene (and Quaternary). The time trees (under best model) of both the Ascaridoidea and hosts are available in the Dryad. The starting tree, prepared for use in BEAST, was from the eight-gene ML tree of the Ascaridoidea (with outgroups) and transformed by treePL with a single calibration on the root (fixed age $$=$$ 541 Ma, see Blaxter (2009)). The BEAST analyses of the Ascaridoidea employed the eight-gene matrix (see Supplementary Table S2 available on Dryad) and were performed in the same way as dating the vertebrate tree, but with 200 million generations of the standard BEAST MCMC run and a total of 200 million iterations for PS/SS sampling. We obtained the final time trees from the resulting post burn-in trees under the best model (Birth-death and uncorrelated relaxed clock with lognormal distribution using the calibrating strategy 2, Supplementary Table S6 available on Dryad) with first 5% dropped (ESS >200). Reconstruction of the Ancestral Habitat of the Hosts and the Ancestral Host Type of the Ascaridoids We separated the habitats of the hosts of the sampled ascaridoid species into three types: terrestrial, semiaquatic, and aquatic (including both fresh and sea water). According to the hosts and host habitats, we then distinguished the sampled ascaridoid representatives into six types of parasites, living in: (i) terrestrial tetrapods, (ii) semiaquatic tetrapods, (iii) aquatic tetrapods, (iv) elasmobranch fishes, (v) teleost fishes, and (vi) multiple types of hosts (more than two types). Some genera, with a single species sampled here but known to have various types of host, are assigned by the host type of the majority of their species rather than that of the sampled species. To infer the possible ancestral host type of ascaridoid clades, we mapped the known host types as discrete traits onto the ascaridoid time tree (calibrated by strategy 2) and used the ML method (Pagel 1994) to estimate the scaled likelihood of each state (host type of parasite or habitat of host) on each node. The state with the largest marginal likelihood was considered as the most probable ancestral state on that node. We fitted our data with three modes: equal-rate, symmetrical, and all-rate-different. We selected the equal-rate model as the best-fitting model for ascaridoid host types and the symmetrical model for host habitats using AIC and Akaike weight (see Supplementary Table S7 available on Dryad). Then, we defined the estimated switching time range (EST) as the period when the parasite ancestral lineage and the host ancestral lineage possibly co-occurred. Accordingly, we defined the maximum limit of the EST as the minimum value of the stem age of a parasite clade and that of its primary host clade, and defined the minimum limit as the maximum value of the crown ages of these two clades (Table 1 and Supplementary Fig. S1 available on Dryad). Considering that the Bayesian MCMC dating method (BEAST) provides interval estimates of the cladal ages, we expanded the EST into the joint 95% highest posterior density interval (jHPD95) of EST by using the minimum value between the maximum limits of the stem ages of the parasite HPD95 and the host HPD95 as the maximum limit of the jHPD95 and using the maximum value between the minimum limits of the stem ages of the parasite HPD95 and the host HPD95 as the minimum limit of the jHPD95 (Table 1 and Supplementary Fig. S1 available on Dryad). Table 1. Divergence time (in Ma) of the Ascaridoidea and their hosts estimated using strategy 2, the estimated switching time range (EST), and the time windows (TW in Myr, size of EST) and expanded time window [ETW in Myr, size of joint 95% HPD (jPHD95)] of the host switching Ascaridoid Clade Stem age (HPD95$$^{\mathrm{a}})$$ Crown age (HPD95) Host Clade Stem age (HPD95) Crown age (HPD95) EST$$^{\mathrm{b}}$$ (jHPD95$$^{\mathrm{c}})$$ TW$$^{\mathrm{d}}$$ ETW$$^{\mathrm{e}}$$ Ascaridoidea 360.47 325.27 Amniota 390.43 323.04 360.47–325.27 35.20 106.94 (456.30–280.00) (406.90–258.94) (424.94–358.70) (331.30–318.00) (424.94–318.00) Raphidascarididae 247.31 140.33 Teleostei 390.43 194.67 247.31–194.67 52.64 131.73 (292.59–209.49) (177.55–109.91) (424.94–358.70) (233.63–160.86) (292.59–160.86) Ascarididae 200.75 100.39 Boreoeutheria (terrestrial) 121.85 109.29 121.85–109.29 12.56 42.76 (247.28–153.95) (136.64–72.30) (137.82–106.17) (124.88–95.06) (137.82–95.06) Baylisascaris 71.79 55.92 Arctoidea (excluding Pinnipedia) 59.89 54.90 59.89–55.92 3.97 12.75 (96.68–52.32) (77.03–39.77) (63.00–56.12) (59.03–50.25) (63.00–50.25) Toxocaridae 200.75 144.91 ### ### ### ### ### ### (247.28–153.95) (197.13–93.65) Toxocara 144.91 61.59 Laurasiatheria 109.29 91.19 109.29–91.19 18.10 43.58 (197.13–93.65) (92.48–34.45) (124.88–95.06) (102.41–81.30) (124.88–81.30) Porrocaecum 144.91 78.23 Neoaves 79.63 67.56 79.63–78.23 1.40 28.80 (197.13–93.65) (141.92–28.37) (86.8–70.12) (76.80–58.00) (86.8–58.00) Acanthocheilidae 228.94 177.21 Elasmobranchii 427.89 343.02 ### ### ### (267.97–196.06) (195.10–171.60) (447.77–420.70) (360.27–337.50) Mawsonascaris 177.21 8.36 Rhinobatidae$$+$$Myliobatidae 192.87 178.24 (195.10–171.60) ### 23.50 (195.10–171.60) (15.06–3.36) (220.02–176.64) (200.05–171.60) Anisakidae 256.62 214.90 ### ### ### ### ### ### (304.88–216.23) (262.60–171.64) Anisakinae 214.90 133.96 ### ### ### ### ### ### (262.60–171.64) (180.56–93.54) Anisakis 63.14 56.45 Cetacea 64.18 48.65 63.14–56.45 6.69 17.76 (66.00–56.39) (63.40–48.24) (66.00–59.45) (54.73–42.38) (66.00–48.24) Pseudoterranova 50.77 13.32 Pinnipedia 51.64 40.55 50.77–40.55 10.22 21.85 (56.30–39.52) (27.66–4.16) (56.30–46.58) (46.99–34.45) (56.30–34.45) Contracaecinae 214.90 135.26 ### ### ### ### ### ### (262.60–171.64) (181.29–96.65) Contracaecum (part) 104.20 87.79 Neognathae (fish eating) 230.43 79.63 104.20–87.79 16.41 70.30 (140.42–73.13) (120.56–57.77) (263.29–190.84) (86.8–70.12) (140.42–70.12) Heterocheilidae 325.27 153.27 Amniota 390.43 323.04 325.27–323.04 2.23 88.90 (406.90–258.94) (238.07–84.02) (424.94–358.70) (331.30–318.00) (406.90–318.00) Ascaridoid Clade Stem age (HPD95$$^{\mathrm{a}})$$ Crown age (HPD95) Host Clade Stem age (HPD95) Crown age (HPD95) EST$$^{\mathrm{b}}$$ (jHPD95$$^{\mathrm{c}})$$ TW$$^{\mathrm{d}}$$ ETW$$^{\mathrm{e}}$$ Ascaridoidea 360.47 325.27 Amniota 390.43 323.04 360.47–325.27 35.20 106.94 (456.30–280.00) (406.90–258.94) (424.94–358.70) (331.30–318.00) (424.94–318.00) Raphidascarididae 247.31 140.33 Teleostei 390.43 194.67 247.31–194.67 52.64 131.73 (292.59–209.49) (177.55–109.91) (424.94–358.70) (233.63–160.86) (292.59–160.86) Ascarididae 200.75 100.39 Boreoeutheria (terrestrial) 121.85 109.29 121.85–109.29 12.56 42.76 (247.28–153.95) (136.64–72.30) (137.82–106.17) (124.88–95.06) (137.82–95.06) Baylisascaris 71.79 55.92 Arctoidea (excluding Pinnipedia) 59.89 54.90 59.89–55.92 3.97 12.75 (96.68–52.32) (77.03–39.77) (63.00–56.12) (59.03–50.25) (63.00–50.25) Toxocaridae 200.75 144.91 ### ### ### ### ### ### (247.28–153.95) (197.13–93.65) Toxocara 144.91 61.59 Laurasiatheria 109.29 91.19 109.29–91.19 18.10 43.58 (197.13–93.65) (92.48–34.45) (124.88–95.06) (102.41–81.30) (124.88–81.30) Porrocaecum 144.91 78.23 Neoaves 79.63 67.56 79.63–78.23 1.40 28.80 (197.13–93.65) (141.92–28.37) (86.8–70.12) (76.80–58.00) (86.8–58.00) Acanthocheilidae 228.94 177.21 Elasmobranchii 427.89 343.02 ### ### ### (267.97–196.06) (195.10–171.60) (447.77–420.70) (360.27–337.50) Mawsonascaris 177.21 8.36 Rhinobatidae$$+$$Myliobatidae 192.87 178.24 (195.10–171.60) ### 23.50 (195.10–171.60) (15.06–3.36) (220.02–176.64) (200.05–171.60) Anisakidae 256.62 214.90 ### ### ### ### ### ### (304.88–216.23) (262.60–171.64) Anisakinae 214.90 133.96 ### ### ### ### ### ### (262.60–171.64) (180.56–93.54) Anisakis 63.14 56.45 Cetacea 64.18 48.65 63.14–56.45 6.69 17.76 (66.00–56.39) (63.40–48.24) (66.00–59.45) (54.73–42.38) (66.00–48.24) Pseudoterranova 50.77 13.32 Pinnipedia 51.64 40.55 50.77–40.55 10.22 21.85 (56.30–39.52) (27.66–4.16) (56.30–46.58) (46.99–34.45) (56.30–34.45) Contracaecinae 214.90 135.26 ### ### ### ### ### ### (262.60–171.64) (181.29–96.65) Contracaecum (part) 104.20 87.79 Neognathae (fish eating) 230.43 79.63 104.20–87.79 16.41 70.30 (140.42–73.13) (120.56–57.77) (263.29–190.84) (86.8–70.12) (140.42–70.12) Heterocheilidae 325.27 153.27 Amniota 390.43 323.04 325.27–323.04 2.23 88.90 (406.90–258.94) (238.07–84.02) (424.94–358.70) (331.30–318.00) (406.90–318.00) Bold taxon names are those whose parasite–host links are all significantly supported by ParaFit tests. The results of strategy 1 are provided in Supplementary Table S9 available on Dryad. $$^{\mathrm{a}}$$HPD95: 95% highest posterior density interval. $$^{\mathrm{b}}$$EST: the estimated switching time range. $$^{\mathrm{c}}$$jHPD95: the joint 95% highest posterior density interval of EST, where the upper limit is defined as the minor value between the upper limits of the stem ages of the parasite HPD and the host HPD, and the lower limit is defined as the major value between the lower limits of the stem ages of the parasite HPD and the host HPD (shown in Supplementary Fig. S1, available on Dryad). $$^{\mathrm{d}}$$TW: the time windows (range size of the EST) in which the parasitism is estimated to have been established. $$^{\mathrm{e}}$$ETW: the expanded time windows, i.e. the range size of the jHPD95. $$^{\mathrm{f}}$$###: No monophyletic host clade or reasonable result here. Table 1. Divergence time (in Ma) of the Ascaridoidea and their hosts estimated using strategy 2, the estimated switching time range (EST), and the time windows (TW in Myr, size of EST) and expanded time window [ETW in Myr, size of joint 95% HPD (jPHD95)] of the host switching Ascaridoid Clade Stem age (HPD95$$^{\mathrm{a}})$$ Crown age (HPD95) Host Clade Stem age (HPD95) Crown age (HPD95) EST$$^{\mathrm{b}}$$ (jHPD95$$^{\mathrm{c}})$$ TW$$^{\mathrm{d}}$$ ETW$$^{\mathrm{e}}$$ Ascaridoidea 360.47 325.27 Amniota 390.43 323.04 360.47–325.27 35.20 106.94 (456.30–280.00) (406.90–258.94) (424.94–358.70) (331.30–318.00) (424.94–318.00) Raphidascarididae 247.31 140.33 Teleostei 390.43 194.67 247.31–194.67 52.64 131.73 (292.59–209.49) (177.55–109.91) (424.94–358.70) (233.63–160.86) (292.59–160.86) Ascarididae 200.75 100.39 Boreoeutheria (terrestrial) 121.85 109.29 121.85–109.29 12.56 42.76 (247.28–153.95) (136.64–72.30) (137.82–106.17) (124.88–95.06) (137.82–95.06) Baylisascaris 71.79 55.92 Arctoidea (excluding Pinnipedia) 59.89 54.90 59.89–55.92 3.97 12.75 (96.68–52.32) (77.03–39.77) (63.00–56.12) (59.03–50.25) (63.00–50.25) Toxocaridae 200.75 144.91 ### ### ### ### ### ### (247.28–153.95) (197.13–93.65) Toxocara 144.91 61.59 Laurasiatheria 109.29 91.19 109.29–91.19 18.10 43.58 (197.13–93.65) (92.48–34.45) (124.88–95.06) (102.41–81.30) (124.88–81.30) Porrocaecum 144.91 78.23 Neoaves 79.63 67.56 79.63–78.23 1.40 28.80 (197.13–93.65) (141.92–28.37) (86.8–70.12) (76.80–58.00) (86.8–58.00) Acanthocheilidae 228.94 177.21 Elasmobranchii 427.89 343.02 ### ### ### (267.97–196.06) (195.10–171.60) (447.77–420.70) (360.27–337.50) Mawsonascaris 177.21 8.36 Rhinobatidae$$+$$Myliobatidae 192.87 178.24 (195.10–171.60) ### 23.50 (195.10–171.60) (15.06–3.36) (220.02–176.64) (200.05–171.60) Anisakidae 256.62 214.90 ### ### ### ### ### ### (304.88–216.23) (262.60–171.64) Anisakinae 214.90 133.96 ### ### ### ### ### ### (262.60–171.64) (180.56–93.54) Anisakis 63.14 56.45 Cetacea 64.18 48.65 63.14–56.45 6.69 17.76 (66.00–56.39) (63.40–48.24) (66.00–59.45) (54.73–42.38) (66.00–48.24) Pseudoterranova 50.77 13.32 Pinnipedia 51.64 40.55 50.77–40.55 10.22 21.85 (56.30–39.52) (27.66–4.16) (56.30–46.58) (46.99–34.45) (56.30–34.45) Contracaecinae 214.90 135.26 ### ### ### ### ### ### (262.60–171.64) (181.29–96.65) Contracaecum (part) 104.20 87.79 Neognathae (fish eating) 230.43 79.63 104.20–87.79 16.41 70.30 (140.42–73.13) (120.56–57.77) (263.29–190.84) (86.8–70.12) (140.42–70.12) Heterocheilidae 325.27 153.27 Amniota 390.43 323.04 325.27–323.04 2.23 88.90 (406.90–258.94) (238.07–84.02) (424.94–358.70) (331.30–318.00) (406.90–318.00) Ascaridoid Clade Stem age (HPD95$$^{\mathrm{a}})$$ Crown age (HPD95) Host Clade Stem age (HPD95) Crown age (HPD95) EST$$^{\mathrm{b}}$$ (jHPD95$$^{\mathrm{c}})$$ TW$$^{\mathrm{d}}$$ ETW$$^{\mathrm{e}}$$ Ascaridoidea 360.47 325.27 Amniota 390.43 323.04 360.47–325.27 35.20 106.94 (456.30–280.00) (406.90–258.94) (424.94–358.70) (331.30–318.00) (424.94–318.00) Raphidascarididae 247.31 140.33 Teleostei 390.43 194.67 247.31–194.67 52.64 131.73 (292.59–209.49) (177.55–109.91) (424.94–358.70) (233.63–160.86) (292.59–160.86) Ascarididae 200.75 100.39 Boreoeutheria (terrestrial) 121.85 109.29 121.85–109.29 12.56 42.76 (247.28–153.95) (136.64–72.30) (137.82–106.17) (124.88–95.06) (137.82–95.06) Baylisascaris 71.79 55.92 Arctoidea (excluding Pinnipedia) 59.89 54.90 59.89–55.92 3.97 12.75 (96.68–52.32) (77.03–39.77) (63.00–56.12) (59.03–50.25) (63.00–50.25) Toxocaridae 200.75 144.91 ### ### ### ### ### ### (247.28–153.95) (197.13–93.65) Toxocara 144.91 61.59 Laurasiatheria 109.29 91.19 109.29–91.19 18.10 43.58 (197.13–93.65) (92.48–34.45) (124.88–95.06) (102.41–81.30) (124.88–81.30) Porrocaecum 144.91 78.23 Neoaves 79.63 67.56 79.63–78.23 1.40 28.80 (197.13–93.65) (141.92–28.37) (86.8–70.12) (76.80–58.00) (86.8–58.00) Acanthocheilidae 228.94 177.21 Elasmobranchii 427.89 343.02 ### ### ### (267.97–196.06) (195.10–171.60) (447.77–420.70) (360.27–337.50) Mawsonascaris 177.21 8.36 Rhinobatidae$$+$$Myliobatidae 192.87 178.24 (195.10–171.60) ### 23.50 (195.10–171.60) (15.06–3.36) (220.02–176.64) (200.05–171.60) Anisakidae 256.62 214.90 ### ### ### ### ### ### (304.88–216.23) (262.60–171.64) Anisakinae 214.90 133.96 ### ### ### ### ### ### (262.60–171.64) (180.56–93.54) Anisakis 63.14 56.45 Cetacea 64.18 48.65 63.14–56.45 6.69 17.76 (66.00–56.39) (63.40–48.24) (66.00–59.45) (54.73–42.38) (66.00–48.24) Pseudoterranova 50.77 13.32 Pinnipedia 51.64 40.55 50.77–40.55 10.22 21.85 (56.30–39.52) (27.66–4.16) (56.30–46.58) (46.99–34.45) (56.30–34.45) Contracaecinae 214.90 135.26 ### ### ### ### ### ### (262.60–171.64) (181.29–96.65) Contracaecum (part) 104.20 87.79 Neognathae (fish eating) 230.43 79.63 104.20–87.79 16.41 70.30 (140.42–73.13) (120.56–57.77) (263.29–190.84) (86.8–70.12) (140.42–70.12) Heterocheilidae 325.27 153.27 Amniota 390.43 323.04 325.27–323.04 2.23 88.90 (406.90–258.94) (238.07–84.02) (424.94–358.70) (331.30–318.00) (406.90–318.00) Bold taxon names are those whose parasite–host links are all significantly supported by ParaFit tests. The results of strategy 1 are provided in Supplementary Table S9 available on Dryad. $$^{\mathrm{a}}$$HPD95: 95% highest posterior density interval. $$^{\mathrm{b}}$$EST: the estimated switching time range. $$^{\mathrm{c}}$$jHPD95: the joint 95% highest posterior density interval of EST, where the upper limit is defined as the minor value between the upper limits of the stem ages of the parasite HPD and the host HPD, and the lower limit is defined as the major value between the lower limits of the stem ages of the parasite HPD and the host HPD (shown in Supplementary Fig. S1, available on Dryad). $$^{\mathrm{d}}$$TW: the time windows (range size of the EST) in which the parasitism is estimated to have been established. $$^{\mathrm{e}}$$ETW: the expanded time windows, i.e. the range size of the jHPD95. $$^{\mathrm{f}}$$###: No monophyletic host clade or reasonable result here. Results and Discussion Phylogenies Our phylogenetic analyses using eight different genes indicated that the representatives of the Ascaridoidea were divided into six distinct clades (families): Heterocheilidae (including Ortleppascaris, Dujardinascaris, Krefftascaris, and Heterocheilus), Anisa- kidae (including Anisakis, Pseudoterranova, Pulch- rascaris, Terranova, Contracaecum, and Phocascaris), Toxocaridae (including Toxocara and Porrocaecum), Acanthocheilidae (including Acanthocheilus, Pseuda- nisakis and Mawsonascaris), Ascarididae (including Baylisascaris, Ascaris, Parascaris, and Toxascaris), and Raphidascarididae (including Hysterothylacium, Raphidascaroides, Goezia, Maricostula, Iheringascaris, Raphidascaris, and Ichthyascaris). All of these groups are monophyletic, with strong support from both ML (BS $$=$$ 99–100) and Bayesian inference (BPP $$=$$ 1), except for the Toxocaridae (BPP $$=$$ 0.98, BS $$=$$ 58) (Fig. 1). These analyses further confirm the Heterocheilidae as the sister clade to the remaining Ascaridoidea, consistent with previous phylogenetic studies based on fewer genes and taxa (Nadler 1992; Nadler and Hudspeth 1998, 2000). Figure 1. View largeDownload slide Phylogenetic relationships among ascaridoid nematodes inferred from maximum likelihood and Bayesian analyses of the DNA sequences of five nuclear and three mitochondrial genes: ITS-1, ITS-2, 5.8S, 28S, 18S, cox1, cox2, and 12S representing two loci. Ascaridia galli (Heterakoidea), Cruzia americana (Cosmocercoidea), and Falcaustra sinensis (Cosmocercoidea) were used as outgroups. Branch lengths are proportional to the substitutions per site. The paired squares filled by colors on the branches indicate the support of the downstream nodes inferred by maximum likelihood bootstraps (left) and Bayesian posterior probabilities (right). Support values of key nodes are mapped near the nodes. Both maximum likelihood and Bayesian trees are available in the Dryad. Figure 1. View largeDownload slide Phylogenetic relationships among ascaridoid nematodes inferred from maximum likelihood and Bayesian analyses of the DNA sequences of five nuclear and three mitochondrial genes: ITS-1, ITS-2, 5.8S, 28S, 18S, cox1, cox2, and 12S representing two loci. Ascaridia galli (Heterakoidea), Cruzia americana (Cosmocercoidea), and Falcaustra sinensis (Cosmocercoidea) were used as outgroups. Branch lengths are proportional to the substitutions per site. The paired squares filled by colors on the branches indicate the support of the downstream nodes inferred by maximum likelihood bootstraps (left) and Bayesian posterior probabilities (right). Support values of key nodes are mapped near the nodes. Both maximum likelihood and Bayesian trees are available in the Dryad. Our present study represents the first attempt to resolve the systematic position of the Acanthocheilidae using phylogenetic analyses based on molecular data. The results support the validity of the Acanthocheilidae as a distinct family outside the Anisakidae, which is in agreement with some previous hypotheses based on morphological characters (Osche 1958; Hartwich 1974; Gibson 1973, 1983), but these results strongly reject these proposals to remove Pseudanisakis from the Acanthocheilidae (Sprent 1983; Sprent 1990; Petter et al. 1991) as these two genera Pseudanisakis and Acanthocheilus are sister groups with strong support in both ML inference and Bayesian inference (BS $$=$$ 100, BPP $$=$$ 1). Our phylogenetic analyses also confirmed Mawsonascaris as a member of the Acanthocheilidae with a sister relationship to Acanthocheilus$$+$$Pseudanisakis based on strong support from both ML inference and Bayesian inference (BS $$=$$ 100, BPP $$=$$ 1). Parasite–Host Associations We tested co-phylogenetic associations between ascaridoid species and their vertebrate hosts. The results supported a very significant global co-evolution between the Ascaridoidea and their hosts (global ParaFit Statistics $$=$$ 529.5912, $$P$$$$<$$0.001). One hundred and twenty-one of 151 PHA are significant (see Supplementary Table S8 available on Dryad). Within these significant PHAs, four clades represent uniformly monophyletic P2H clades (Fig. 2), in which both the host clade (H-clade) and the parasite clade (P-clade) are monophyletic, and the P-clade taxa are restricted to the H-clade. The H-clade can thereby be considered as the “primary hosts” of the P-clade. For example, members of the Raphidascarididae are specialized parasites of teleost fishes. There are three clades with H clades that are not strictly monophyletic because of some ecological constraints: the Ascarididae are specialized parasites of terrestrial Boreoeutheria (absent from pinnipeds and whales); species of Baylisascaris (nested within Ascarididae) are specialized in terrestrial Arctoidea (absent from pinnipeds); the derivative four Contracaecum spp. (clade G, Fig. 2) parasitize only fish-eating birds (as adult nematodes), but their monophyly was not supported by Bayesian inference (Fig. 1). These clades were not used in the calibration. Although acanthocheilids are obligate parasites of elasmobranchs, not all of their PHA links are supported here as cophylogenetic relationships. In our results, only Mawsonascaris coevolving with the Rhinobatidae $$+$$ Myliobatidae is significant. Within the Anisakidae, Anisakis and Pseudoterranova have a completely significant co-evolution with their hosts (whales and pinnipeds, respectively), that agrees well with a previous study (Mattiucci and Nascetti 2008). Other anisakids have a confusing mixture of hosts and, therefore, exhibit less cophylogenetic significance. Similarly, the Toxocaridae and Heterocheilidae lack significant PHA links. Divergence Time, Ancestral Hosts, and Switches By calibrating the four uniformly monophyletic P2H clades using the fossil/estimated time information for the relevant associated host clades (see Supplementary Table S5 available on Dryad), we estimated the time period for ascaridoid divergence. Then, we reconstructed both the host types for ascaridoids and the habitats of hosts for all of the internal nodes of the time-calibrated trees. Our results (Fig. 2 and Table 1, strategy 2 shown; strategy 1 results are provided in Supplementary Fig. S1 and Table S9 available on Dryad) showed that the ancestor of the Ascaridoidea is estimated to have appeared 360.47 Ma (95% HPD: 456.30–280.00 Ma) and the MRCA of the extant ascaridoid species occurred 325.27 (406.90–258.94) Ma (Fig. 2 and Table 1). This result is prior to, but not discordant with, an existing hypothesis of a pre-Permian origin (Osche 1963), although the latter was proposed based on “parasitophyletic rules” and considered the elasmobranch-parasitic Acanthocheilidae as the sister group to the other ascaridoids, which has been previously questioned (Sprent 1983) and is rejected herein. The host of the ancestral ascaridoid lineage was estimated, with a marginal likelihood (0.4950), as being a “terrestrial tetrapod.” In the host tree, the lineages of the amniotes, teleosts and elasmobranchs occurred within this range, but among these, only an ancestral amniote lineage is plausible, as its habitat was estimated to be “terrestrial” (marginal likelihood: 0.8918). So, we hypothesize that the ascaridoid ancestor was derived from its parent lineage by switching into a terrestrial amniote host approximately 360.47–325.27 Ma (joint 95% HPD: 424.94–318.00 Ma), which indicates that there was at least 35.20 Myr (or at most 106.94 Myr) for the fixation of this parasitism (Fig. 2 and Table 1). Moreover, the median crown age of the ascaridoid lineage is slightly older (2.23 Myr) than the amniote lineage, which suggests that the basal split of the ascaridoid lineage may predate the host divergence. If so, one might predict that the Heterocheilidae and its sister lineage would appear in both daughter host clades (the reptile-clade and the mammal-clade) of the amniote lineage (Fig. 3). In fact, heterocheilids parasitize a limited group of mammals (manatees and dugongs), whereas most heterocheilids parasitize reptiles. Members of the other five families have radiated into every major clade of amniotes. Figure 3. View largeDownload slide Scheme of the main host switches of high-level ascaridoids inferred from both cophylogenetic relationships plus the literature. Path color is used to distinguish the families. The terminal dashed lines mean that the parasitic relationships have no cophylogenetic significance. Dot-dashed lines indicate “missing paths”, where parasitism was established in the ancestor but is absent from its descendants. Loops in the paths locate possible parasitism on the host phylogeny. The placements of the three free loops (not linked to the host tree) are unresolved by our results. Dotted lines display the possible path of the Angusticaecinae and Sulcascaris (absent from our analysis). The three discontinuous bifurcations (may be multifurcate), marked with a “sun”, “star”, and “moon”, represent the three starting nodes for the “land-to-water” host shifts (as in Fig. 2). The time axis (unscaled) indicates some key points in the sea-level changes which took place during the evolution of the ascaridoids. Host profiles on the internal nodes depict the ancestors, and illustrations at the base represent not only the species but also phylogenetically related taxa. Figure 3. View largeDownload slide Scheme of the main host switches of high-level ascaridoids inferred from both cophylogenetic relationships plus the literature. Path color is used to distinguish the families. The terminal dashed lines mean that the parasitic relationships have no cophylogenetic significance. Dot-dashed lines indicate “missing paths”, where parasitism was established in the ancestor but is absent from its descendants. Loops in the paths locate possible parasitism on the host phylogeny. The placements of the three free loops (not linked to the host tree) are unresolved by our results. Dotted lines display the possible path of the Angusticaecinae and Sulcascaris (absent from our analysis). The three discontinuous bifurcations (may be multifurcate), marked with a “sun”, “star”, and “moon”, represent the three starting nodes for the “land-to-water” host shifts (as in Fig. 2). The time axis (unscaled) indicates some key points in the sea-level changes which took place during the evolution of the ascaridoids. Host profiles on the internal nodes depict the ancestors, and illustrations at the base represent not only the species but also phylogenetically related taxa. The crown age of the Heterocheilidae was estimated to be 153.27 (238.07–84.02) Ma, and its possible host MRCA was estimated to be a “semiaquatic tetrapod” (marginal likelihood: 0.8928). The dates estimated based on ML topology suggested that the divergence of the heterocheilids does not follow the evolution of hosts but is associated with the break-up of Pangaea. The Alligator-parasitic species separated about 153.27 (238.07–84.02) Ma, coinciding with the separation of Laurasia from Gondwana (since 175 Ma), and the divergence of Heterocheilus (parasitizing Trichechus in Africa and South America) and Krefftascaris (parasitizing Chelodina in Australia and New Guinea) took place 114.02 (191.97–47.78) Ma. At the same time, Gondwana was dividing into multiple continents (Africa, South America, India, Antarctica, and Australia). Extant heterocheilid species, mainly parasitic in the species of Crocodilia (not merely Alligator), Sirenia and Chelodina, all live in regions derived from the coastal areas of the Palaeo-Tethys Ocean (most of the hosts are Gondwanan in origin, except for Alligator). We consider that the stem heterocheilids likely inhabited both semiaquatic diapsids and Mammaliaformes distributed across Gondwana and, perhaps, along the shores of the Palaeo-Tethys Ocean, where they may have transferred hosts via predation or food webs (Fig. 3). The estimated switching time (range) of the heterocheilid lineage parasitizing such semiaquatic amniotes was 325.27–323.04 (406.90–318.00) Ma. The oldest known ascaridoid fossil (240 Ma) is most likely to have been a heterocheilid, judging by both superficial morphological traits and its herbivorous/carnivorous Gondwanan mammal-like host (Silva et al. 2014). The “Ascaridoidea minus Heterocheilidae” clade is estimated to have diverged into six major clades during the Triassic. Members of the Raphidascarididae are specialized parasites of teleosts, although some Goezia species also parasitize crocodiles. The estimated switching time of the Raphidascarididae to teleosts was 247.31–194.67 (292.59–160.86) Ma, which suggests that it took 52.64 (at most 131.73) Myr to fix this parasitism. However, the Raphidascarididae did not diversify with the radiation of teleosts until the beginning of the Cretaceous (about 140.33 Ma). Acanthocheilids only parasitize elasmobranchs, but both their stem and crown ages are far later than the age of elasmobranchs, which indicates that acanthocheilids parasitized elasmobranchs secondarily. Therefore, the hypothesis of an aquatic or marine origin for the Ascaridoidea (Osche 1958) is not supported by our results. The Ascarididae parasitize terrestrial boreoeuthe- rians, which they may have switched to this group during the period 121.85–109.29 (137.82–95.06) Ma, and almost immediately diversified with their hosts, but did not parasitize aquatic hosts. The Angusticaecinae is a derivative group within the Ascarididae. Its members mainly parasitize frogs, snakes, lizards, chameleons, and armadillos. As they are missing from our analysis, we tentatively assume that they are sister to other ascaridid (Ascarididae) subgroups, colonized lizards and snakes (Squamata) and then transferred to tailless amphibians through predation or food webs. The crown age of the Toxocaridae is estimated to be 144.91 (197.13–93.65) Ma, and the ancestral host type is considered to be “terrestrial tetrapods” (marginal likelihood: 0.9747, Fig. 2). This family used to be considered as a subfamily within the Ascarididae, but its members have much more diverse infection patterns than ascaridids (Sprent 1983). Owing to the temporal limitations, the stem lineage of the Toxocaridae would have been unable to colonize the stem of the amniotes, but had opportunities to switch to therians and modern birds. We, therefore speculate that the common ancestor of ascaridids and toxocarids first parasitized ancient therians based on the latter’s alternative infection patterns, e.g. species of Porrocaecum, which opportunistically infect mammals, have transferred to birds via their intermediate hosts (e.g., earthworms) (Fig. 3). The Ascarididae and the Toxocaridae are the only extant ascaridoids that still parasitize terrestrial tetrapods. The Anisakidae is one of the most species-rich families in the Ascaridoidea. Our estimates show that it split into two major clades (Anisakinae and Contracaecinae) 214.90 (262.60–171.64) Ma. Members of the Anisakinae parasitize diverse vertebrates: elasmobranchs (Pulchrascaris and Terranova), pinnipeds (Pseudoterranova), cetaceans (Anisakis and Terranova), sea turtles (Sulcascaris), and crocodiles and snakes (Terranova). Our estimates of the ancestral hosts of anisakines had low likelihoods, precluding additional speculation. Members of the Contracaecinae parasitize fish-eating birds (supported by significant PHA tests) and pinnipeds (no significant support). The stem lineage of this subfamily appeared 214.90 (262.60–171.64) Ma, but diverged 135.26 (181.29–96.65) Ma, during a period when only birds and their stem groups (bird-line archosaurs) were possible hosts (pinnipeds had not yet appeared). The ancestral host type is estimated to be “semiaquatic tetrapods” (marginal likelihood: 0.9939). Nevertheless, we cannot draw further conclusions on the primary host of the Contracaecinae, because the ML and Bayesian trees conflict regarding relationships. Our results show that the MRCA of the Anisakidae was likely to parasitize a “semiaquatic tetrapod” (marginal likelihood: 0. 5675) 214.90 (259.34–169.76) Ma. The stem age of the Anisakidae, 254.03 (302.72–215.34) Ma, just postdates the crown age of diapsids (265.62 (285.12–255.9) Ma), which is roughly concordant with the previous divergence time analyses using the Cytochrome C and globin amino acid sequences (i.e., the Anisakidae and Ascarididae diverged about 150–250 Ma) (Vanfleteren et al. 1994). Land-Water Shifts with Sea-Level Changes Our dating results indicated that ascaridoids were derived from other “ascaridomorph” nematodes in the Early Carboniferous (Mississippian) and were parasites of terrestrial amniotes, which agrees with some earlier hypotheses of a terrestrial origin of ascaridoid nematodes (Chitwood 1950; Dougherty 1951); it is also reasonable because our results suggest that the origin of ascaridoids occurred at a time when the sea-level dropped, falling to present levels or lower (Haq et al. 1987; Haq and Schutter 2008) (Fig. 2). This time was at the onset of the Permo-Carboniferous Glaciation and the Carboniferous Rainforest Collapse, causing a downturn in amphibian diversity and the adaptive radiation of reptiles (Sahney et al. 2010), which may be the reason why the crown ascaridoids are rare in amphibians, but rich in reptiles. During the entire Permian (a period of low sea-level), we found no diversification of crown groups of either ascaridoids or their hosts. But shortly after the recovery from the Permian-Triassic extinction, when the sea-level began to rise, the “Ascaridoidea minus Heterocheilidae” clade began an abrupt diversification into five distinct subclades. We also recognize five fundamental switches of host types in ascaridoid evolution, all of which likely occurred at the time of a rise in sea-level during the Triassic to the Early Cretaceous periods and all from terrestrial to aquatic or semiaquatic environments (Fig. 2). However, this pattern cannot be deduced from changes in host habitat or from host evolution alone. Our results suggest that early host switches of ascaridoids appear to be correlated with global climate patterns, particularly sea-level change. Moreover, our schematic model (Fig. 3) shows that “lateral host switches” (yielding increased host range in parasite groups and diverse parasitological patterns in host groups) occurred very recently, possibly in the Cenozoic, consistent with more host switching rather than cophylogenetic patterns. It is worth noting that it should have been possible for terrestrial ascaridids and terrestrial/semiaquatic toxocarids to colonize marine mammals, such as pinnipeds or cetaceans (Fig. 3), as predicted by parasite-host cophylogeny. Perhaps the ancestors of pinnipeds and cetaceans lost their ascaridids and toxocarids during their transition to aquatic life as new food-web associations resulted in colonization by other ascaridoids, although our current knowledge offers no evidence for these missing associations. Conclusion Our molecular phylogenetic analyses divide this superfamily into six main monophyletic clades, i.e. the Heterocheilidae, Acanthocheilidae, Anisakidae, Ascarididae, Toxocaridae, and Raphidascarididae, among which the Heterocheilidae, rather than the Acanthocheilidae, appears to be the sister group to the remaining ascaridoid lineages. We further calibrated the phylogeny using an approach that involves time priors from fossils of the co-evolving hosts, rather than the rare and unreliable ascaridoid egg fossils, and date the common ancestor of the Ascaridoidea back to the Early Carboniferous (approximately 360.47–325.27 Ma). We then estimated the host types of the ancestral ascaridoid and its major clades, and reconstructed the habitat type of the relevant host clades. Accordingly, we established a host-switching model for the high-level groups. The dates and ancestral host types indicated by our results both suggest that members of the Ascaridoidea primarily inhabited terrestrial tetrapods and subsequently extended their range to elasmobranchs and teleosts. We also determined that the fundamental terrestrial-to-aquatic switches of these nematodes coincided with rises in sea-level during the Triassic to the Early Cretaceous. Supplementary Material Data available from the Dryad Digital Repository: http://doi.org/10.5061/dryad.28g16. Acknowledgements The authors wish to thank Dr Guy Baele for suggestions on the use of marginal likelihood estimation using PS/SS sampling. We are also grateful to Professor Hideo Hasegawa (Oita University, Japan) for providing useful literature. Funding This study was supported by the National Natural Science Foundation of China [NSFC-31572231, NSFC-31750002]; Natural Science Foundation of Hebei Province [NSFH-C2016205088]; and the Youth Top Talent Support Program of Hebei Province to L.L. 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Systematic BiologyOxford University Press

Published: Sep 1, 2018

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