Nuclear and mitochondrial phylogenomics of the Diplostomoidea and Diplostomida (Digenea, Platyhelminthes)1

Higher systematics within the Digenea, Carus 1863 have been relatively stable since a phylogenetic analysis of partial nuclear ribosomal markers (rDNA) led to the erection of the Diplostomida Olson, Cribb, Tkach, Bray, and Littlewood, 2003. However, recent mitochondrial (mt) genome phylogenies suggest this order might be paraphyletic. These analyses show members of two diplostomidan superfamilies are more closely related to the Plagiorchiida La Rue, 1957 than to other members of the Diplostomida. In one of the groups implicated, the Diplostomoidea Poirier, 1886, a recent phylogeny based on mt DNA also indicates the superfamily as a whole is non-monophyletic. To determine if these results were robust to additional taxon sampling, we analyzed mt genomes from seven diplostomoids in three families. To choose between phylogenetic alternatives based on mt genomes and the prior rDNA-based topology, we also analyzed hundreds of ultra-conserved elements (UCEs) assembled from shotgun sequencing. The Diplostomida was paraphyletic in the mt genome phylogeny, but supported in the UCE phylogeny. We speculate this mitonuclear discordance is related to ancient, rapid radiation in the Digenea. Both UCEs and mt genomes support the monophyly of the Diplostomoidea and show congruent relationships within it. The Cyathocotylidae Mühling, 1898 are early diverging descendants of a paraphyletic clade of Diplostomidae Poirier, 1886, in which were nested members of the Strigeidae Railliet, 1919; the results support prior suggestions that the Crassiphialinae Sudarikov, 1960 will rise to the family level. Morphological traits of diplostomoid metacercariae appear to be more useful for differentiating higher taxa than those of adults. We describe a new species of Cotylurus Szidat, 1928, resurrect a species of Hysteromorpha Lutz, 1931, and find support for a species of Alaria Schrank, 1788 of contested validity. Complete rDNA operons are provided as a resource for future studies.

superfamily as a whole is non-monophyletic. To determine if these results were robust to 23 additional taxon sampling, we analyzed mt genomes from seven diplostomoids in three families. 24 To choose between phylogenetic alternatives based on mt genomes and the prior rDNA-based 25 topology, we also analyzed hundreds of ultra-conserved elements (UCEs) assembled from 26 shotgun sequencing. The Diplostomida was paraphyletic in the mt genome phylogeny, but 27 supported in the UCE phylogeny. We speculate this mitonuclear discordance is related to 28 ancient, rapid radiation in the Digenea. Both UCEs and mt genomes support the monophyly of 29 the Diplostomoidea and show congruent relationships within it. The Cyathocotylidae Mühling, and were chosen to represent major clades in Blasco-Costa and Locke (2017). DNA was 126 extracted from individual worms, or subsamples, using Qiagen's DNEasy blood and tissue kit 127 (GmbH, Germany), following the manufacturer's protocol with two 200-μL elutions. Of these 25 128 worms, we selected seven (Table 1)       The mitochondrial genomes were 13,665 -15,107 bp in length (supplementary Table 1). The 12 199 coding regions occurred in the same order as in digeneans other than S. haematobium, S. 200 spindale, and S. mansoni. In C. prussica, two pairs of tRNA genes were reversed in order 201 compared with other diplostomoids. One of these reversals, the occurrence of trnN prior to trnP, 202 is also a feature of Clinostomum complanatum (KM923964). The order of three tRNA genes 203 occurring between nad6 and nad5 in C. medioconiger differed from that seen in other 204 diplostomoids, and these genes were positioned adjacent to a 774-bp non-coding span which, 205 among diplostomoids and Clinostomum, was unique to C. medioconiger. 206 Phylogenetic analysis of nucleotide or translated amino acid sequences from the 12 concatenated 207 coding regions of the mitochondrial genomes did not support the order Diplostomida (Fig. 2).

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The diplostomoids and Clinostomum emerged as early diverging descendants from common 209 ancestors of the Plagiorchiida, rather than grouping with Schistosoma and Trichobilharzia.  Relationships among diplostomoids were similar to those recovered by analysis of mt genomes, 217 differing slightly within a clade of Diplostominae Alaria + Hysteromorpha + Tylodelphys + 218 Diplostomum (Figs. 2, 3). Within the diplostomoids, Cyathocotyle prussica was basal to a clade 219 12 in which the Diplostomidae were paraphyletic. The diplostomid Posthodiplostomum centrarchi 220 was basal to a clade of both strigeids and other diplostomids. The two strigeids were sister taxa.

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The non-molecular characters of metacercariae showed higher correspondence with the UCE 222 topology than did the characters of adults (Fig 3, supplementary Fig. 1 Table 1) were subjected to BLAST searches, which supported 241 identifications in all cases, as described further below.     Table 2): Two of nine specimens were up to 75 greater in total length; in 4/9 specimens, we 282 observed the hindbody to be widest at the anterior testis (e.g., Fig 5), rather than at the level of

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Copulatory bursa 1010-1919 long. Hindbody length 3.6-5.6 times that of copulatory bursa 312 (means, standard deviations, n structures measured in Supplementary Table 3).     divergence between C. strigeoides and JX977781, we believe all these data originate from C. 386 strigeoides, but JX977781 is identified as C. gallinulinae. The material we examined was 387 distinguished from C. gallinulinae by a large pharynx (5% of total length, and over half the size 388 of the oral sucker, versus ≤2% of total length, and less than half the size of the oral sucker in C.     3.4.6 Hysteromorpha triloba (Rudolphi, 1819) (Fig. 8, Supplementary Fig. 3   Sequences of CO1 from metacercariae of H. triloba from Italy differed by 6.9-9.7 (mean 8.7)% 487 from material collected in North and Central America (Fig. 4). Among the Italian isolates, CO1  Table 6). We consider these 500 European and American isolates of Hysteromorpha to be separate species, even if meristic or 501 morphological differences were not discerned between American and European adults of     Table 7), although he did not comment on the tegument; a pseudosucker on one   (Figs. 1, 2). There were about one quarter as many variable sites in the alternatives, yielded unequivocal support for the order (Fig. 3). 560 Discordance between nuclear and mt phylogenies is not uncommon, and although it is

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Within the Diplostomoidea, however, the mt and UCE phylogenies are congruent, which 574 suggests they reflect evolutionary relationships among the species studied. Figures 2 and 3 also 575 share elements that recur in prior work. In most molecular phylogenies, the superfamily is

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The initial aim of this study was a phylogenomic evaluation of higher relationships 629 among a small number of worms. To this end, we selected seven specimens identified to genus 630 that were promising for shotgun sequencing. After closer examination of vouchers, we recorded 631 a new species of Cotylurus, resurrected a species of Hysteromorpha, and found support for a 632 species of Alaria of contested validity, and these findings were supported with molecular data.