NEW FACES IN AN OLD GENUS: MUSEUM COLLECTIONS AND NEW MATERIALS REVEAL NEW DIVERSITY OF ALARIA AND AN UPDATED KEY TO SPECIES

Collection

Numerous fresh adult Alaria spp. were collected from Pr. lotor in Georgia. Mesocercariae of Alaria were also collected from 2 species of frogs in North Dakota (Table I). Live digeneans were rinsed in saline, heat-killed with hot water, and preserved in 80% ethanol. Additional dead Alaria spp. specimens were collected from frozen American mink Neogale vison (Schreber), bobcat Lynx rufus (Schreber), and fisher Pekania pennanti in Wisconsin and a frozen Pr. lotor in Louisiana. Dead digeneans were immediately transferred to 80% ethanol.

Table I.Hosts, geographical origin (country or state of the United States), GenBank accession numbers, and museum accession numbers of Alaria spp. included in the present study. Museum abbreviations: Harold W. Manter Laboratory, HWML; Museum of Southwestern Biology Division of Parasites, MSB; Smithsonian National Museum of Natural History, USNM; University of Wisconsin–Stevens Point Parasitology Collection, UWSP-P.

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Morphological study

Specimens of Alaria spp. were stained with aqueous alum carmine and mounted in Damar gum on slides following the protocol of Lutz et al. (2017). Slides were studied using an Olympus BX53 compound microscope (Olympus, Tokyo, Japan) equipped with differential interference contrast optics and a digital imaging system. Illustrations were prepared with the aid of a drawing tube mounted on the BX53 microscope. All measurements are provided in micrometers. The new type series and vouchers are deposited in the collections of the H. W. Manter Laboratory (HWML), University of Nebraska, Lincoln, Nebraska, University of Wisconsin—Stevens Point Parasitology Collection (UWSP–PARA), and the Museum of Southwestern Biology Division of Parasites (MSBP), University of New Mexico, Albuquerque, New Mexico (Table I).

Type series and vouchers of several Alaria spp. were borrowed from the HWML, Smithsonian National Museum of Natural History (USNM) in Washington, D.C. and Coleção Helmintológica do Instituto Oswaldo Cruz (CHIOC) in Rio de Janeiro, Brazil. We also examined slides of Alaria spp. from coyotes, fishers, and raccoons from the parasitology teaching collections at the University of Wisconsin Oshkosh (UWO), Winona State University (WSU), and Middle Georgia State University (MGA) and slides of Alaria spp. from raccoon in Florida and stoat Mustela erminea in Washington from the private collection of Mike Kinsella. We have also examined specimens of Alaria spp. from Robert L. and Virginia R. Rausch Helminthological Collection deposited in the MSBP (Table I). Specimens from the series of A. americana sequenced by Locke et al. (2018) were examined to confirm morphology.

Molecular study

For most specimens, genomic DNA was extracted from whole or partial worms following the protocol of Tkach and Pawlowski (1999). Fragments of 28S and COI and the entire ITS region were amplified by PCR in a total volume of 25 μl using One-Taq quick load PCR mix from New England Biolabs (Ipswich, Massachusetts) according to the manufacturer’s instructions. Annealing temperatures of 53 and 45 C were used for ribosomal and mitochondrial fragments, respectively. Primers pairs used were digL2 + 1500R (28S), ITSF+300R (ITS region and 5′ end of 28S), and DiploCox5′+DiploCox3′ (COI) (Littlewood and Olson, 2001; Tkach et al., 2003; Snyder and Tkach, 2007; Achatz et al., 2019, 2022b).

PCR products were purified using Illustra ExoProStar PCR clean-up enzymatic kit (Cytiva, Marlborough, Massachusetts), cycle-sequenced directly using BrightDye Terminator Cycle Sequencing Kit (MCLAB, South San Francisco, California), cleaned using a BigDye Sequencing Clean Up Kit (MCLAB) and run on an ABI 3130, ABI 3500 or SeqStudio genetic analyzer (Thermo Fisher Scientific, Waltham, Massachusetts). The PCR primers and internal reverse primer Dipl650R (COI) were used for sequencing (Achatz et al., 2019). Contiguous sequences were assembled using Sequencher 5.4 software (GeneCodes Corp., Ann Arbor, Michigan) and deposited in GenBank (Table I). Representative isolates previously sequenced by Achatz et al. (2022a) were also used to generate new ITS and COI sequences.

Bayesian Markov chain Monte Carlo (BMCMC) methods were used to conduct 4 phylogenetic analyses on separate alignments of 28S, ITS, and COI, along with the concatenated dataset including all gene fragments. All alignments included only Alaria spp. as the ingroup and another diplostomid, Diplostomum spathaceum (Rudolphi, 1819) as the outgroup. Sequences in each alignment were aligned using ClustalW implemented in MEGA7 and then manually rechecked using Se-Al v2.0a11 (Rambaut, 2002). Gaps in alignments were treated as missing data and no internal stop codons were found in COI. The resulting alignments were trimmed to the length of the shortest sequences, and ambiguous positions were excluded from the analyses (Kumar et al., 2016). The most appropriate models of nucleotide substitution were assessed using the program MrModeltest v2.2 (Nylander, 2004) and run in PAUP* v4.0b10 (Swofford, 2002). We used the Akaike information criterion (AIC) to select the best-fit model, as estimated by MrModeltest for each fragment separately and each codon separately for COI. MrModeltest selected the following partitioned nucleotide substitution models for our data set: 28S (HKY+I+Γ), ITS region (GTR+Γ), COI codon 1 (HKY+Γ), COI codon 2 (F81), and COI codon 3 (HKY+Γ).

Phylogenetic analyses were conducted using the Bayesian inference (BI) as implemented in MrBayes v3.2.6 software (Ronquist and Huelsenbeck, 2003). The BI analyses were performed as follows: Markov chain Monte Carlo (MCMC) chains were run for 4,000,000 generations, sampling trees and parameters every 100 generations. Log-likelihood scores were plotted and only the final 75% of trees were used to produce the consensus trees. The number of generations for each analysis was determined as sufficient because the average standard deviation of split or clade frequencies across MCMC chains stabilized below 0.01. Pairwise sequence comparisons were performed using MEGA7.

For next-generation sequencing (NGS), DNA was extracted from 1 specimen of A. ovalis using a NucleoSpin Tissue XS kit (Macherey Nagel, Allentown, Pennsylvania) following manufacturer instructions and shotgun-sequenced on an HiSeq 4000 (Illumina, San Diego, California) at Azenta Life Sciences (South Plainfield, New Jersey).

The resulting 150–base pair (bp) paired-end reads were used to assemble the mt genome of A. ovalis using the mt genome of A. americana (MH536507) as a reference, using BBmap (Bushnell, 2014) and map-to-reference tools in Geneious Prime v2020.2.2 (Biomatters Ltd., Auckland, New Zealand). The mitochondrial genome assembly was annotated using MITOS (Bernt et al., 2013) and through comparison with mt genomes of Alaria americana and other diplostomoideans (MH536507–MH536513, MT679576, KR269763, KR269764, MT259035) in a MAFFT alignment (Katoh et al., 2019). All sites with gaps were removed from the alignment and a phylogenetic tree was constructed using IQTree (Nguyen et al., 2015) based on the TIM+F+R3 model of nucleotide evolution, which was selected based on the lowest Bayesian Information Criterion (BIC) (Kalyaanamoorthy et al., 2017).

Molecular phylogenies

Ribosomal and mitochondrial loci:

The 28S alignment was 1,175 bp long. The alignment included 9 named Alaria spp. and 3 species-level lineages. The topology of the phylogenetic tree resulting from the analysis of 28S was not well supported (Fig. 2). The phylogeny consisted of 2 weakly supported clades. The first clade contained 2 pairs of species: A. marcianae + Alaria sp. 3 (91% supported) and A. americana + A. arisaemoides (78% supported). The second clade positioned a subclade of A. alata + Alaria sp. 1 (less than 70% support) sister to a polytomy of several other species (80% supported). The polytomy included (A. mustelae + Alaria sp. 4 [90% supported]), Alaria trashpandae n. sp. (A. procyonis + Alaria pseudoprocyonis n. sp. [100% supported]) and A. ovalis.

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The ITS region alignment was 1,056 bp long after trimming to the length of the shortest sequence. It included 9 Alaria spp. and 1 species-level lineage. Despite our best efforts, we were unable to amplify the ITS region of Alaria sp. 4. Compared to the 28S phylogeny, the tree resulting from the analysis of the ITS region was better resolved (Fig. 3). Alaria procyonis + A. pseudoprocyonis (less than 70% support) were positioned as a sister group to the clade of other Alaria spp. (98% supported). Subsequently, A. ovalis was positioned separately from the 99% supported clade of Alaria spp. Among the remaining species, A. trashpandae was a sister species to the 95% supported clade of A. mustelae + a subclade of other Alaria spp. (100% supported). The subclade consisted of a polytomy of A. alata + Alaria sp. 1 + a 100% supported grouping of (A. marcianae + Alaria sp. 3 [100% supported] and A. americana + A. arisaemoides [100% supported]). The phylogeny resulting from the concatenated analysis had similar topology and support as the ITS phylogeny (Suppl. Data, Fig. S1).

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The COI alignment was 444 bp long after trimming to the length of the shortest sequence. It included 9 Alaria spp. and the species-level lineages. The phylogeny based on the COI analysis consisted of a basal polytomy with 4 clades (Fig. 4). The first clade (99% supported) was split between 2 subclades: A. marcianae + Alaria sp. 3 (91% supported) and (A. americana + A. arisaemoides [100%]) + (A. alata + Alaria sp. 1 [97% supported]). The second clade (98% supported) contained Alaria sp. 4 + A. mustelae. The third clade (100% supported) only consisted of A. trashpandae. The fourth clade (91% supported) included A. ovalis as a sister species to a 99% supported clade of A. procyonis + A. pseudoprocyonis.

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Next-generation sequence analysis:

Shotgun sequencing of a specimen of A. ovalis yielded 90,966,382 150-bp reads, of which 18,362 were assembled into a 13,913-bp-long mitochondrial genome (GenBank accession PQ615272). We were unable to circularize the molecule unambiguously, but all protein-coding and other expected genes were recovered in the same order and had similar lengths to other diplostomoideans, except that tRNAs for proline and asparagine were not found (Suppl Data, Table S1). Excluding the noncoding, repeat-rich region between ND5 and COIII, the coverage of the mt assembly averaged 179.5 reads per site (range 46–340). Phylogenetic analysis of the 12,808-bp gap-free mt genome alignment yielded a mostly well-resolved tree with Alaria spp. clustered together within a strongly supported clade that contained most diplostomid genera included in the analysis (Hysteromorpha Lutz, 1931; Tylodelphys Diesing, 1850; and Diplostomum von Nordmann, 1832) other than Posthodiplostomum Dubois, 1936 (Fig. 5). The phylogenetic position of the three members of the Strigeidae Railliet, 1919 (Apharyngostrigea Ciurea, 1927; Cotylurus Szidat, 1928; Cardiocephaloides Sudarikov, 1959) and Posthodiplostomum centrarchi Hoffman, 1958 indicated nonmonophyly of the Strigeidae and Diplostomidae.

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Redescription

Based on 4 specimens (USNM 1367163; holotype and 3 paratypes on the same slide). Measurements of illustrated specimen in text; measurements of entire series given in Table II. Body consisting of indistinctly separated prosoma and opisthosoma, inversely pyriform, widest at level of holdfast organ, 1,817; prosoma funnel shaped, deeply concave, 903 × 1,256; opisthosoma conical, 914 × 793. Forebody 27% of body length. Tegument unarmed. Oral sucker subterminal, 116 × 146. Ventral sucker 134 × 121. Oral:ventral sucker width ratio 1.2. Pseudosuckers associated with large masses of unicellular glands, invaginated, or very weakly lappet-like (Fig. 7). Holdfast organ elongated, often obscuring ventral sucker, 293 × 348; holdfast organ:prosoma length ratio 0.3. Prepharynx absent. Pharynx subspherical or oval, 104 × 85. Esophagus very short. Ceca slender, extend to shortly posterior to posterior margin of testes.

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Table II.Morphometric characters of Alaria spp. with opposite testes. Ranges provided followed by mean in parentheses.

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Testes 2, opposite, rounded, entire, 402–414 × 305–317. Seminal vesicle posterior to testes; ejaculatory duct not strongly muscular. Genital pore subterminal at posterior end of opisthosoma, dorsal.

Ovary transversely oval or reniform, positioned anterior to testes, 121 × 207. Oötype and Mehlis’ gland ventral to testes, near middle of body. Laurer’s canal dorsal. Vitelline follicles small, distributed throughout most of prosoma. Lateral follicles extend anterior to level of ventral sucker and posteriorly to level of testes. Median vitellarium less broadly distributed, most at level of holdfast organ. Vitelline reservoir positioned near oötype and Mehlis’ gland, ventral to testes. Uterus ventral to gonads, extends anteriorly beyond level of ovary before turning and extending posteriorly. Uterus and ejaculatory duct join to form very short hermaphroditic duct opening at tip of small genital cone. Genital cone positioned inside genital atrium, may be everted. Uterus contains 15 eggs. Eggs 90–104 × 45–61.

Excretory pore subterminal at posterior end of opisthosoma, ventral.

Taxonomic summary

Remarks

The original written description of A. adenocephala was detailed, but the illustration of adults in ventral view was incomplete. Notably, the original illustration lacked any details of terminal reproductive structure anatomy in ventral view. Unfortunately, upon our re-examination of the holotype, we were also unable to see several features typically highly diagnostic of diplostomoideans, such as most reproductive ducts. Other features, such as the pseudosuckers, were poorly delineated, which is problematic as that is 1 of the characteristic features of the species. We opted to illustrate a different specimen on the same slide (USNM 1367163) in which the important features could be seen well. Although we examined the entire type series, we only measured USNM 1367163 (holotype and paratypes slide), as many of the other specimens were also lower quality with poorly distinguishable structures. Our measurements are limited to features that could be reliably measured. Overall, the measurements were similar to the original description (Table S2). We also examined the type series of A. procyonis of Harkema (1942). As noted by Beckerdite et al. (1971), the type series of A. procyonis contained a mixture of A. adenocephala, A. procyonis, and 1 of the new species described herein (see remarks of A. procyonis in the following).

Alaria trashpandae n. sp. Young and Achatz

(Figs. 7–9)

Description

Based on 21 specimens. Measurements of holotype in text; measurements of entire series given in Table II. Body consisting of weakly separated prosoma and opisthosoma, oval, pyriform or scoop-shaped when relaxed, almost spherical when contracted, widest at level of holdfast organ in relaxed specimens and level of testes when contracted, 908; prosoma scoop-shaped, deeply concave, 487 × 411; opisthosoma conical, 421 × 299. Forebody 24% of body length. Tegument of prosoma armed with fine spines, most on ventral concavity. Oral sucker subterminal, 58 × 69. Ventral sucker 73 × 90. Oral:ventral sucker width ratio 0.8. Pseudosuckers invaginated. Holdfast organ oval, often obscuring ventral sucker, 277 × 198; holdfast organ:prosoma length ratio 0.6. Prepharynx absent. Pharynx subspherical or oval, 51 × 36. Esophagus very short. Ceca slender, do not extend posteriorly beyond anterior margin testes.

Testes 2, opposite, rounded, entire, 191–204 × 119–140. Seminal vesicle posterior to testes, well-developed; ejaculatory duct muscular, but without distinct pouch-like portion. Genital pore subterminal at posterior end of opisthosoma, dorsal.

Ovary transversely oval or reniform, positioned anterior to testes, 77 × 113. Oötype and Mehlis’ gland ventral to testes, typically anterior to and overlapping with dextral testis. Laurer’s canal dorsal. Vitelline follicles small, numerous, primarily in posterior two-thirds of prosoma, most follicles at and posterior to level of holdfast organ. Vitelline reservoir intertesticular, ventral to testes. Uterus ventral to gonads (may be partially dorsal in contracted specimens), extends anteriorly to around level of ovary before turning and extending posteriorly. Uterus and ejaculatory duct join to form very short hermaphroditic duct opening at tip of small genital cone. Genital cone positioned inside genital atrium. Uterus does not contain eggs in holotype; up to 9 eggs in paratypes. Eggs 91–108 × 54–72.

Excretory pore subterminal at posterior end of opisthosoma, ventral.

Taxonomic summary

Remarks

This new species belongs to Alaria based on several morphological features, including the presence of pseudosuckers, ventral sucker, and muscular ejaculatory duct, as well as DNA sequences. Alaria trashpandae has opposite testes, which are only shared with A. adenocephala, A. dasyuri, A. ovalis, A. procyonis, and the other new species from Pr. lotor differentiated in the following. The ceca of A. trashpandae do not extend posteriorly beyond the anterior margin of the testes, whereas the ceca of other Alaria spp. extend posterior to the level of testes. The posterior extent of the ceca was not described for A. dasyuri. Unlike A. procyonis and A. ovalis, the ejaculatory duct A. trashpandae is much less muscular and not pouch-like.

This new species is morphologically closest to A. adenocephala and A. dasyuri, neither of which has been sequenced to date. Alaria trashpandae lacks the large masses of unicellular glands associated with the pseudosuckers characteristic of A. adenocephala. The tegument of the prosoma of A. trashpandae is armed, while in A. adenocephala it is unarmed. Alaria trashpandae is smaller (body length 720–1,089, mean 881; prosoma width 328–661, mean 511) compared to A. adenocephala (body length 1,656–1,829, mean 1,774; prosoma width 1,075–1,256, mean 1,190). The oral:ventral sucker ratio is typically smaller in A. trashpandae (0.7–1, mean 0.9) compared to A. adenocephala (1.0–1.2, mean 1.1).

In relaxed specimens, the vitellarium of A. trashpandae only reaches anteriorly to about the level of the ventral sucker, whereas the vitellarium of A. dasyuri reaches the posterior margin of the pharynx. This feature is less reliable in contracted specimens of both species. The eggs of A. trashpandae (91–108, mean 100) are somewhat smaller than those of A. dasyuri (110–130, mean 118). Alaria trashpandae is distributed in common raccoons in the southeastern United States, whereas A. dasyuri is only known from the eastern quoll in Tasmania, Australia.

Alaria trashpandae differs from its sequenced congeners (including A. procyonis and A. ovalis) by 0.3–1.4% in the partial 28S sequences, 1.7–5.1% in the ITS region sequences, 14.1–16.1% in the barcoding fragment of COI sequences and 11.3–13.6% in the entire fragment of COI sequenced (Tables S3, S4). No variation was detected among the 2 isolates sequenced for 28S and the ITS region. No variation was detected among the 4 partial 28S and 3 ITS region sequences. Up to 0.9% and 0.8% variation was detected in barcoding and entire sequenced fragment of COI, respectively, of 5 sequenced isolates.

Alaria procyonis (Harkema, 1942)

(Figs. 10, 11)

Description

Based on 18 newly collected specimens. Measurements of drawn specimens in text (Fig. 10a followed by Fig. 10b in parentheses); measurements of entire series given in Table II. Body consisting of weakly separated prosoma and opisthosoma, scoop or cylindrical shaped, widest at level of holdfast organ, 1,069 (964); prosoma deeply concave 554 × 448 (549 × 380); opisthosoma conical, 515 × 285 (415 × 306). Forebody 19% (21%) of body length. Tegument of prosoma armed (Fig. 11). Oral sucker subterminal, 55 × 74 (58 × 76). Ventral sucker 74 × 88 (63 × 88). Oral:ventral sucker width ratio 0.8 (0.9). Pseudosuckers invaginated. Holdfast organ oval, 342 × 246 (246 × 145); holdfast organ:prosoma length ratio 0.6 (0.4). Prepharynx absent. Pharynx subspherical, 50 × 43 (47 × 45). Esophagus very short. Ceca slender, extend to near posterior end of opisthosoma.

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Testes 2, opposite, rounded, entire, 263–269 × 137–166 (185–186 × 121–162). Seminal vesicle posterior to testes; ejaculatory duct muscular, with distinct pouch-like portion; pouch-like part of duct 113 × 87 (149 × 73), wall 30 (17) thick. Genital pore subterminal at posterior end of opisthosoma, dorsal.

Ovary transversely oval or reniform, positioned anterior to testes, 78 × 111 (76 × 101). Oötype and Mehlis’ gland ventral to testes, dextral or sinistral. Laurer’s canal opening dorsal. Vitelline follicles small, distributed throughout most of prosoma. Lateral follicles extend to level of ventral sucker and posteriorly to level of testes. Median vitelline follicles mostly concentrated at level of holdfast organ. Vitelline reservoir positioned near oötype and Mehlis’ gland, ventral to testes. Uterus ventral to gonads. Uterus and ejaculatory duct join to form very short hermaphroditic duct opening at tip of small genital cone. Genital cone positioned inside genital atrium. Uterus contains 16 (0) eggs. Eggs 75–114 × 54–70.

Excretory pore subterminal at posterior end of opisthosoma, ventral.

Taxonomic summary

Remarks

Beckerdite et al. (1971) re-examined the type material of A. procyonis and determined it to be a mixture of A. procyonis and A. adenocephala specimens; these authors also provided a new description of experimentally obtained A. procyonis. We re-examined the type series and agree that it represents multiple species, in some cases on the same slide. However, we also identified the presence of at least 2 A. trashpandae specimens in the paratype slide USNM 1344393 from North Carolina.

The measurements of our specimens were only compared to those described by Beckerdite et al. (1971); overall our specimens were similar in size (Table S5). We do not provide a comparison to the originally described specimens of Harkema (1942) as that material represents at least 3 species. We also opted to not remeasure the specimens of Harkema (1942) as most of them were of much worse quality than those collected in the present study.

Excluding the next new species in the following, A. procyonis differs from its sequenced congeners by 0.3–1.3% in the partial 28S sequences, 1.4–4.9% in the ITS region sequences, 10.3–15.4% in the barcoding fragment of COI sequences and 8.8–11.9% in the entire fragment of COI sequenced (Suppl. Data Tables S3, S4). No variation was detected among 2 isolates sequenced for 28S and COI.

Alaria pseudoprocyonis n. sp. Young, Tkach and Achatz

Syn. Alaria procyonis sensu Achatz et al. (2022a)

(Figs. 11, 12)

Description

Based on 6 specimens. Measurements of holotype in text; measurements of entire series given in Table II. Body consisting of weakly separated prosoma and opisthosoma, scoop-shaped, widest at level of holdfast organ, 701 long; prosoma deeply concave, 514 × 300; opisthosoma conical, 187 × 231. Forebody 25% of body length. Tegument of prosoma armed with very fine spines. Oral sucker subterminal, 59 × 58. Ventral sucker 60 × 77. Oral:ventral sucker width ratio 0.8. Pseudosuckers invaginated. Holdfast organ subspherical, 169 × 172; holdfast organ:prosoma length ratio 0.3. Prepharynx absent. Pharynx subspherical, 45 × 35. Esophagus very short. Ceca slender, extend to or shortly posterior to level of testes.

Testes 2, opposite, rounded, entire, 80–87 × 54–55. Seminal vesicle posterior to testes; ejaculatory duct muscular, with distinct pouch-like portion; pouch-like part of duct 113 × 50, wall 24 thick. Genital pore subterminal at posterior end of opisthosoma, dorsal.

Ovary transversely oval, positioned anterior to testes, 45 × 63. Oötype and Mehlis’ gland ventral to testes, dextral or sinistral. Laurer’s canal not observed. Vitelline follicles small, limited to around level of holdfast organ. Lateral follicles limited to about level of holdfast organ, not reaching anteriorly to level of ventral sucker. Median vitellarium reach anteriorly to level of ventral sucker and somewhat posterior to level of holdfast organ. Vitelline reservoir positioned near oötype and Mehlis’ gland, ventral to testes. Uterus ventral to gonads. Uterus and ejaculatory duct join to form very short hermaphroditic duct opening at tip of small genital cone. Genital cone positioned inside genital atrium. Uterus does not contain eggs in holotype; up to 3 eggs in paratype. Eggs 86–99 × 50–58.

Excretory pore subterminal at posterior end of opisthosoma, ventral.

Taxonomic summary

Remarks

This new species belongs to Alaria based on several morphological features, including the presence of pseudosuckers, ventral sucker, and muscular ejaculatory duct, as well as DNA sequences. Alaria pseudoprocyonis has opposite testes, which are only shared with A. adenocephala, A. dasyuri, A. ovalis, A. procyonis, and A. trashpandae. Unlike A. adenocephala, A. dasyuri, and A. trashpandae, this new species possesses a strongly muscular ejaculatory duct that is pouch-like in appearance.

Alaria pseudoprocyonis is morphologically and genetically closest to A. ovalis and A. procyonis. The vitellarium is noticeably different between A. pseudoprocyonis and the other 2 species. It is confined to around the level of the holdfast organ and the lateral fields do not reach anteriorly to the level of ventral sucker in A. pseudoprocyonis. In contrast, in A. ovalis and A. procyonis, the vitellarium is distributed throughout most of the prosoma and the lateral fields extend anteriorly to or beyond the level of the ventral sucker. The vitellarium is also less dense in A. pseudoprocyonis compared to the other species.

The new species can be further distinguished from A. ovalis by the body shape (scoop-shaped in A. pseudoprocyonis vs. oval in A. ovalis). The oral sucker of A. pseudoprocyonis is much smaller than the ventral sucker (oral:ventral sucker width ratio 0.6–0.8), whereas the oral sucker is similar in size or slightly larger than the ventral sucker in A. ovalis. Alaria pseudoprocyonis is also smaller (body length 609–785) and much thinner (prosoma width 244–331) compared to A. ovalis (body length 1,000–1,450; prosoma width 570–725). Eggs of A. pseudoprocyonis are somewhat smaller (86–99) than those of A. ovalis (100–115).

The tegumental spines of A. pseudoprocyonis are noticeably smaller and less dense than in A. procyonis (Fig. 11). Measurement ranges of most features (e.g., body length, sucker sizes) heavily overlap. However, the holdfast organ (length), testes (length), pouch-like part of ejaculatory duct (width), and ovary (length) of A. pseudoprocyonis are smaller than in A. procyonis (Table II).

Alaria pseudoprocyonis differs from its sequenced congeners (excluding A. procyonis) by 0.3–1.3% in the partial 28S sequences, 1.3–4.8% in the ITS region sequences, 10.1–15.2% in the barcoding fragment of COI sequences, and 9.6–12.2% in the entire fragment of COI sequenced (Tables S3, S4). Alaria pseudoprocyonis and A. procyonis have identical partial 28S sequences, but the ITS region (0.1%) and COI (4.5–4.8%) differ.

Alaria marcianae (La Rue, 1917)

(Fig. 13)

Description

Based on 30 specimens. Measurements of the entire illustrated specimen in the text; measurements of the entire series are given in Table III. Body consisting of distinctly separated prosoma and opisthosoma, elongated, 1,705 long; prosoma elongated, concave, widest at the level of holdfast organ, 1,203 × 545; opisthosoma conical, 494 × 397. Forebody 23% of body length. Tegument of prosoma armed with fine spines, decreasing in density posterior to level of holdfast organ; tegument of opisthosoma unarmed. Oral sucker subterminal, 107 × 103. Ventral sucker 103 × 95. Oral:ventral sucker width ratio 1.1. Pseudosuckers well-pronounced, extend anteriorly beyond level of oral sucker when everted. Holdfast organ elongated, 566 × 195; holdfast organ:prosoma length ratio 0.5. Prepharynx short. Pharynx oval or subspherical, strongly muscular, 136 × 104. Esophagus short. Ceca slender, extend to near posterior end of opisthosoma.

Table III.Morphometric characters of some New World Alaria spp. Ranges provided followed by mean in parentheses.

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Testes 2, tandem, in opisthosoma. Anterior testis asymmetrical, dextral, 177 × 183; posterior testis trilobed, lobes oriented posteriorly, ventral to anterior testis when overlapping, 209 × 262. Seminal vesicle posterior to testes; ejaculatory duct muscular, with distinct pouch-like portion; pouch-like part of duct 127 × 72, wall 5 thick. Genital pore subterminal at posterior end of opisthosoma, dorsal.

Ovary transversely oval or reniform, positioned anterior to testes at prosoma-opisthosoma junction, 100 × 162. Oötype and Mehlis’ gland opposite to anterior testis. Laurer’s canal dorsal. Vitelline follicles small, numerous distributed throughout prosoma between near anterior margin of holdfast organ and posterior end of prosoma. Vitelline reservoir positioned opposite to anterior testis, somewhat intertesticular, near oötype and Mehlis’ gland, ventral to testes when overlapping. Uterus ventral to gonads, extends anteriorly beyond level of ovary before turning and extending posteriorly. Uterus and ejaculatory duct join to form very short hermaphroditic duct opening at tip of small genital cone. Genital cone positioned inside genital atrium. Uterus contains 0–3 eggs. Eggs 97–127 × 57–82.

Excretory pore subterminal at posterior end of opisthosoma, ventral.

Taxonomic summary

Remarks

Alaria marcianae was originally described based on mesocercariae from checkered garter snake Thamnophis marcianus (Baird and Girard) and Mexican garter snake Thamnophis eques (Reuss) collected in western Texas (La Rue, 1917). The first illustrations of adult specimens identified as A. marcianae were not provided until Burrows and Lillis (1965) based on materials from domestic cat Felis catus L. in New Jersey (Suppl. Data, Table S6). However, no formal differential diagnosis of adults has been provided before. We provide a differential diagnosis of A. marcianae based on the described materials from the American badger Taxidea taxus (Schreber) in the following.

Alaria marcianae can be easily distinguished from A. adenocephala, A. dasyuri, A. ovalis, A. procyonis, A. pseudoprocyonis, and A. trashpandae based on testes position (tandem in A. marcianae vs. opposite in the other species). Unlike A. arisaemoides, A. mustelae, A. nasuae, and A. taxideae, the pseudosuckers of A. marcianae are distinct and horn-like as opposed to weakly developed, invaginated, or very weakly lappet-like in both other species. The ejaculatory duct of A. marcianae is much more muscular with a somewhat distinct, dilated pouch-like portion compared to much less muscular and without any pouch-like portion in A. mustelae and A. taxideae.

Alaria marcianae differs from A. alata based on testes shape (e.g., posterior testis trilobed in A. marcianae vs. posterior testis with numerous small lobes in A. alata) and structure of the ejaculatory duct (ejaculatory duct more muscular with somewhat distinct pouch-like portion in A. marcianae vs. ejaculatory duct less muscular and without any pouch-like portion in A. alata). Alaria marcianae is distributed in the Nearctic, and A. alata is limited to the Palearctic.

Alaria marcianae is morphologically most similar to A. americana (see discussion to follow). Considering the complex history of these 2 species, we only compare using measurements of the sequenced material of A. marcianae and A. americana. The most apparent difference is the structure of the posterior testis: trilobed with lobes oriented posteriorly in A. marcianae vs. bilobed with lobes oriented ventrally or laterally in A. americana. Body size, as well as most organs and structures of A. marcianae, are smaller than A. americana, except for sucker and pharynx sizes which are generally larger in A. marcianae (Table III). The pouch-like part of the ejaculatory duct of A. marcianae has thinner walls (5–14, mean 10) compared to A. americana (31–55, mean 37).

Alaria marcianae differs from its sequenced congeners (including A. americana) by 0–1.4% in the partial 28S sequences, 0–4.4% in the ITS region sequences, 6.7–15.0% in the barcoding fragment of COI sequences, and 8.7–13.6% in the entire fragment of COI sequenced (Tables S3, S4). No variation was detected among 4 isolates sequenced for 28S and 2 isolates sequenced for the ITS region. Up to 1.6% variation was detected in the barcoding region of COI.

Alaria shoopi n. sp. Young, Tkach and Achatz

Syn. Alaria marcianae sensu Fischthal and Martin (1977)

(Fig. 14)

Description

Based on 7 specimens. Measurements of holotype in text; measurements of entire series given in Table III. Body consisting of distinct prosoma and opisthosoma, oval, 2,052 long; prosoma broad, almost rectangular, deeply concave, nearly uniform in width throughout, 1,396 × 1,059; opisthosoma contracted in all specimens, conical, 656 × 774. Forebody 12% of body length. Tegument of prosoma armed with fine spines. Oral sucker subterminal, 80 × 134. Ventral sucker 83 × 120. Oral:ventral sucker width ratio 1.1. Pseudosuckers horn-like, extend anteriorly beyond level of oral sucker when everted. Holdfast organ elongated, often obscuring ventral sucker, 1,045 × 365; holdfast organ:prosoma length ratio 0.7. Prepharynx absent. Pharynx oval, strongly muscular, 125 × 95. Esophagus short. Ceca slender, extend to near posterior end of opisthosoma.

Testes 2, tandem, transversely elongated, separation of testes difficult to observe. Anterior testis asymmetrical, bilobed, mostly dextral, 223 × 542; posterior testis bilobed, 330 × 671. Seminal vesicle posterior to testes or ventral and slightly overlapping posterior testis, weakly developed; ejaculatory duct muscular with apparent pouch-like portion, lumen strongly dilated, ventral to posterior testis; pouch-like part of duct 247 × 118, wall 29 thick. Genital pore subterminal at posterior end of opisthosoma, dorsal.

Ovary transversely elongated, positioned anterior to testes, ventral when overlapping, 160 × 380. Oötype, Mehlis’ gland and Laurer’s canal not observed. Vitelline follicles numerous, distribution various, but limited to prosoma at level of holdfast organ and some follicles in anterior part of opisthosoma near anterior margin of anterior testis. Vitelline reservoir small, median, ventral to testes. Uterus ventral to gonads, extends anteriorly beyond level of ovary before turning and extending posteriorly. Uterus and ejaculatory duct join to form very short hermaphroditic duct opening at tip of small genital cone. Genital cone positioned inside genital atrium. Uterus contains 0 eggs in holotype; up to 21 in paratype. Eggs 93–122 × 56–87.

Excretory pore not observed.

Taxonomic summary

Remarks

This new species belongs to Alaria based on several morphological features, including the presence of pseudosuckers, ventral sucker, and strongly muscular ejaculatory duct (see remarks on the ejaculatory pouch in discussion). The testes of this new species are tandem, whereas the testes of A. adenocephala, A. dasyuri, A. ovalis, A. procyonis, A. pseudoprocyonis, and A. trashpandae are opposite. The pseudosuckers of A. shoopi n. sp. are distinct and strongly horn-like, whereas the pseudosuckers of A. arisaemoides, A. mustelae, A. nasuae, and A. taxideae are smaller relative to body size and less distinct. The ejaculatory duct of A. shoopi is much more muscular with a distinct pouch-like portion in comparison with the much less muscular structure that lacks any pouch-like portion in A. mustelae and A. taxideae.

Alaria shoopi is morphologically closest to A. alata, A. americana, and A. marcianae (Table III). Considering the complex history of A. marcianae and A. americana, our comparison is based on the measurements of the sequenced material only. This new species is most readily differentiated from A. americana and A. marcianae based on its transversely elongated ovary compared to the reniform and ovoid ovaries of the latter 2 species. The anterior testis of A. shoopi has a median, ventral protruding part that is absent in the other species. The posterior testis of A. shoopi is bilobed, while both testes of A. alata have numerous lobes and A. marcianae has a trilobed posterior testis. The ejaculatory duct of A. shoopi has a muscular pouch-like part that is absent in A. alata. The wall of the pouch-like part of the ejaculatory duct in A. shoopi is thicker (29–36, mean 33) than in A. marcianae (5–14, mean 10) and generally thinner than A. americana (31–55, mean 37). The suckers and holdfast organ are wider in A. shoopi (oral sucker 120–148, mean 137; ventral sucker 116–144, mean 126; holdfast organ 322–436, mean 373) than A. americana (oral sucker 50–119, mean 76; ventral sucker 82–120, mean 86; holdfast organ 137–250, mean 168). The holdfast organ in A. shoopi is longer (holdfast organ length 717–1,189, mean 1,031; holdfast organ:prosoma length ratio 0.7–0.8, mean 0.7) than in A. americana (holdfast organ length 645–895, mean 766; holdfast organ:prosoma length ratio 0.5–0.6, mean 0.5).

Alaria nasuae La Rue and Townsend,1927

(Fig. 15)

Description

Based on 1 specimen. Body consisting of somewhat weakly separated prosoma and opisthosoma, 3,267 long; prosoma oval shaped, deeply concave, widest at level of holdfast organ, 2,089 × 1,729; opisthosoma conical, 1,076 × 1,178. Forebody 9% of body length. Tegument of prosoma and opisthosoma armed. Oral sucker subterminal, 98 × 100. Ventral sucker 75 × 115. Oral:ventral sucker width ratio 0.9. Pseudosuckers very weakly lappet-like. Holdfast organ massive, oval, 1,296 × 1,025; holdfast organ:prosoma length ratio 0.6. Prepharynx absent. Pharynx oval, 131 × 89. Esophagus very short. Ceca slender; ends of ceca not observed.

Testes 2, tandem, primarily in opisthosoma, not smooth. Anterior testis subspherical, dextral, lobate, 193 × 311; posterior testis much larger than anterior testis, with 2 lobes, each lobe with numerous smaller lobes, 299 × 902. Seminal vesicle posterior to testes; ejaculatory duct muscular, duct with dilated part that appears pouch-like. Genital pore subterminal at posterior end of opisthosoma, dorsal.

Ovary oval, lobate, positioned anterior to testes, 298 × 383. Oötype and Mehlis’ gland ventral to testes, near middle of opisthosoma. Laurer’s canal not observed. Vitelline follicles numerous, small, distributed throughout most of prosoma, not extending anteriorly beyond level of holdfast organ or posteriorly beyond anterior margin of posterior testis. Vitelline reservoir positioned near oötype and Mehlis’ gland, ventral to testes. Uterus ventral to gonads, extends anteriorly beyond level of ovary before turning and extending posteriorly. Uterus and ejaculatory duct join to form very short hermaphroditic duct opening at tip of small genital cone. Genital cone positioned inside genital atrium. Uterus contains 1 egg. Eggs 103 × 65.

Excretory pore not observed.

Taxonomic summary

Remarks

Shoop et al. (1989) collected the first specimens of A. nasuae from wild-caught hosts and stray dogs in Mexico. Although the original paper stated 5 specimens were collected, only 1 specimen was deposited in the USNM. At the time, Shoop et al. (1989) only provided the body length (2,670–4,830; average 3,470) and egg size (103–111 × 55–60; average 108 × 57) of the series. Therefore, we have provided a complete description of the specimen of A. nasuae deposited by Shoop et al. (1989).

Alaria sp. 4

(Fig. 16)

Description

Based on 12 specimens. Measurements of the illustrated specimen in text; measurements of entire series given in Table IV. Body consisting of distinct prosoma and opisthosoma, 1,022 long; prosoma elongated, concave, widest at level of holdfast organ, 510 × 511; opisthosoma conical, 512 × 371 long. Forebody 32% of body length. Tegument unarmed. Oral sucker subterminal, 68 × 86. Ventral sucker 66 × 73. Oral:ventral sucker width ratio 1.2. Pseudosuckers invaginated. Holdfast organ elongated, often obscuring ventral sucker, 337 × 243; holdfast organ:prosoma length ratio 0.7. Prepharynx absent. Pharynx oval, 122 × 96. Esophagus very short. Ceca slender; ends of ceca not observed.

Table IV.Morphometric comparison of Alaria sp. 4, Alaria mustelae and its current and former synonyms. Ranges provided followed by mean in parentheses.

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Testes 2, tandem, primarily in opisthosoma. Anterior testis asymmetrical, entire, sinistral or dextral, 215 × 265; posterior testis wider than anterior testis, with 3 lobes oriented posteriorly, 190 × 312. Seminal vesicle posterior to testes. Genital pore subterminal at posterior end of opisthosoma, dorsal.

Ovary transversely oval, positioned anterior to testes, 101 × 129. Oötype and Mehlis’ gland opposite to anterior testis. Laurer’s canal not observed. Vitelline follicles small, distributed throughout most of prosoma between level of ventral sucker and near midlength of anterior testis. Vitelline reservoir positioned near oötype and Mehlis’ gland, ventral to testes. Uterus ventral to gonads, extends anteriorly beyond level of ovary before turning and extending posteriorly, not well observed. Uterus and ejaculatory duct join to form very short hermaphroditic duct opening at tip of small genital cone. Genital cone positioned inside genital atrium. Uterus contains 0 eggs; up to 8 eggs in other specimen. Eggs 96–143 × 53–66.

Excretory pore not observed.

Taxonomic summary

Remarks

Unlike other specimens of the new Alaria spp. from North America, all specimens of Alaria sp. 4 originate from frozen fishers. Better quality, fresh specimens are needed to complete the description of this species. As such, we only describe it as a species-level lineage. This new species-level lineage belongs to Alaria based on several morphological features including the presence of pseudosuckers and ventral sucker, as well as molecular data. The testes of Alaria sp. 4 are tandem, whereas the testes of A. adenocephala, A. dasyuri, A. ovalis, A. procyonis, A. pseudoprocyonis, and A. trashpandae are opposite. The pseudosuckers of Alaria sp. 4 are invaginated or very weakly lappet-like, unlike the remaining congeners except for A. mustelae and A. taxideae. The prosoma of Alaria sp. 4 is elongated, whereas the prosoma of A. taxideae is more spherical. The posterior testis of Alaria sp. 4 is trilobed; in contrast, the posterior testis of A. taxideae is bilobed and each lobe is divided into ventral and dorsal sublobes (see description of testes of A. taxideae in Dubois, 1963).

Our specimens of Alaria sp. 4 (in poor condition) and A. mustelae are morphologically indistinguishable. It is likely that a collection of fresh adults, and other life stages, will reveal differences between these species. Excluding A. mustelae, Alaria sp. 4 differs from its congeners by 0.3–1.3% in the partial 28S sequences, 9.8–15.2% in the barcoding fragment of COI sequences, and 9.6–11.6% in the entire fragment of COI sequenced (Tables S3, S4). Unfortunately, we were unable to sequence ITS region of Alaria sp. 4. The partial 28S sequences of Alaria sp. 4 and A. mustelae are identical, whereas cox1 differs by 3.1–4.5% (Tables S3, S4).

Molecular phylogenetics of Alaria

Our phylogenetic tree resulting from analysis of 28S (Fig. 2) was not well resolved. In contrast, the analysis of the ITS region produced an overall well-resolved tree with strong support of most topologies (Fig. 3). The COI analysis was characterized by the presence of a basal polytomy; however, subtrees resulting from the polytomy were well resolved and supported (Fig. 4). All phylogenies demonstrated close affinities between 3 pairs of species: A. americana + A. arisaemoides, A. marcianae + Alaria sp. 3, and A. procyonis + A. pseudoprocyonis. The 28S region used in the analysis is too conserved for reconstructing the evolutionary history of this genus. On the other hand, the high mutation rate of COI resulted in lower resolution of the basal topology. The best-resolved tree based on the ITS region strongly suggests an initial origin of Alaria spp. among procyonids and subsequent radiation to other carnivores (Fig. 3).

The mt genomes of Alaria spp. emerged as sisters in a clade containing other diplostomids (Fig. 5). The topology of the mt genome trees was similar to that obtained in recent studies (Locke et al., 2018, 2021), with Po. centrarchi (a diplostomid) and Apharyngostrigea pipientis (Faust, 1918) (a strigeid) in nonmonophyletic or unresolved arrangements with other family members.

Ejaculatory pouches vs. muscular ejaculatory ducts in Alaria

The term ejaculatory pouch has been used to describe a muscular structure that surrounds the ejaculatory duct of many diplostomoideans (Dubois, 1970; Niewiadomska, 2002; Achatz et al., 2022c). Based on our detailed examination of numerous specimens of several Alaria spp., it is clear that the delineation between a true ejaculatory pouch and muscular ejaculatory duct may be difficult to discern. In some cases, such as in A. americana, A. arisaemoides, and A. procyonis, this muscular structure is well developed and appears distinctly pouch-like. The “pouch” consists of well-developed muscles rather than a dilation of the ejaculatory duct. Whereas in other species, such as A. trashpandae, the ejaculatory duct has a distinct muscular thickening, but the muscles surrounding the ejaculatory duct are not nearly as strongly developed and usually look more similar to or slightly more muscular than typical digenean ejaculatory duct. Based on museum specimens, the ejaculatory duct of A. shoopi only appears pouch-like when the lumen of the duct is dilated, rather than a true thickening of muscles. However, the ejaculatory duct wall is still rather thick. In A. alata, A. adenocephala, A. mustelae, and Alaria sp. 4, the ejaculatory duct is muscular, but most weakly developed among congeners.

The thickness of muscles of this structure is useful for differentiating among some Alaria spp. However, the gradual differences in this structure based on the state of development and dilation create a problematic situation for terminology. We opt to use the term pouch-like portion instead of ejaculatory pouch, to reflect the complex nature of this structure. We strongly encourage future researchers to examine and describe this structure closely in their materials rather than generically refer to it as an ejaculatory pouch.

The status of Alaria americana

The taxonomic histories of A. americana and A. marcianae are complex and tightly intertwined. Alaria americana was originally described based on poor specimens from the domestic dog Canis familiaris L. in Michigan (Hall and Wigdor, 1918). La Rue and Fallis (1936) described A. canis from the same host in Canada. Later, Chandler (1954) described A. minnesotae from striped skunk Mephitis mephitis (Schreber) (type host) and Felis catus L. in Minnesota. Dubois (1963) considered A. canis and A. minnesotae junior synonyms of A. americana; later, Dubois (1970) considered A. americana to be a synonym of A. marcianae.

Other authors (Johnson, 1970; Pearson and Johnson, 1988; Locke et al., 2018) have maintained A. americana as a separate species based on differences in morphology and life histories. Pearson and Johnson (1988) noted that the anatomy of A. americana and A. marcianae differed in larvae and adults. Mesocercariae of A. marcianae lacks spines that cover the entire dorsal surface, whereas in mesocercariae of A. americana the entire dorsal surface is covered with spines. Locke et al. (2018) provided DNA sequences of A. americana and demonstrated its genetic difference from A. marcianae. Our pairwise comparisons support these as different species (28S: 0.3%; ITS region: 0.8%; COI barcoding region: 8.9–12.3%; entire sequenced fragment of COI: 8.7%; Tables S3, S4).

We examined several specimens of A. americana which originate from the same host individual as those described and sequenced by Locke et al. (2018), as well as from the teaching collection at the University of Wisconsin Oshkosh and the private collection of Mike Kinsella. These materials demonstrate clear morphological differences that separate the species from A. marcianae, such as the thickness of the pouch-like portion of the ejaculatory duct and posterior testis structure (Table S6). Unfortunately, the thickness of the pouch-like portion of the ejaculatory duct was not measured for A. canis or A. minnesotae. The structure of the posterior testis was described as bilobed in A. canis (La Rue and Fallis, 1936). We agree with Dubois (1963) and Pearson and Johnson (1988), who considered A. canis a synonym of A. americana. In the monograph by Dubois (1970), the only illustration of A. marcianae provided was, in fact, A. canis. In A. minnesotae, the testes are referred to as “usually bilobed”; the testes were not drawn in the original description of A. minnesotae (La Rue and Fallis, 1936; Chandler 1954). We maintain its current synonymy with A. americana. Locke et al. (2018) provided DNA sequences accompanied by a description of their adult A. americana. However, Achatz et al. (2022a) found only 1.9–2.6% differences between A. americana and A. arisaemoides in the barcoding fragment of COI and no differences in 28S sequences. This led Achatz et al. (2022a) to conclude that these forms were likely conspecific and represented A. arisaemoides. Despite the genetic similarity, the morphology of specimens described and illustrated by Locke et al. (2018) is quite different from A. arisaemoides (original description and photographed specimen of Achatz et al., 2022a). Alaria arisaemoides has a massive holdfast organ that occupies most of the prosoma length. In contrast, the holdfast organ of A. americana of Locke et al. (2018) is much smaller. The body of A. arisaemoides reaches up to 11 mm (about 7 mm in the photographed specimen in Achatz et al., 2022a), whereas A. americana of Locke et al. (2018) does not exceed 3 mm (Table III). The pseudosuckers of A. arisaemoides are very weakly lappet-like, small, and often difficult to distinguish (Dubois, 1970; personal observations of T.J.A. and V.V.T.). Alaria americana has well-developed pseudosuckers, similar to A. alata and A. marcianae. We reexamined the slide series of A. americana of Locke et al. (2018) deposited in the MSB (27495–27501) and newly prepared slides of specimens from the same host individual. There is no doubt that these materials represent A. americana.

Alaria marcianae and A. mustelae isolates included in the present analysis (limited to those of Achatz et al., 2022a and the present study) demonstrate up to 1.6% and 2.5% intraspecific variation in the barcoding fragment of COI, respectively, which is close to or exceeds the difference between sequences of A. arisaemoides and A. americana included in the present analysis (2.0–2.5%; Table S3).

Alaria arisaemoides has 2–3 rows of ventral sucker spines, and A. americana has 3–4 rows of ventral sucker spines. Based on differences in adult and larval morphology, we consider A. americana to represent a separate species. Comparison of other loci (e.g., more variable mitochondrial genes or whole mitochondrial genomes) may help resolve this situation.

Alaria spp. in the Neotropics

Before this study, 3 Alaria spp. were known from the Neotropics: A. alata, A. marcianae, and A. nasuae. Our re-examination of A. alata and A. marcianae from South America demonstrates that both likely represent misidentified species.

Lutz (1933) conducted a life-cycle study and obtained adult specimens that he identified as A. alata; he also suggested the name Alaria nattereri Lutz, 1933 in case these specimens proved to belong to a new species. Unfortunately, only 2 incomplete illustrations were provided without a description of adult specimens. Because Lutz (1933) did not describe the species, the name Alaria nattereri Lutz, 1933 is a nomen nudum. Dubois (1938, 1970) considered this material to be A. alata, a species that is otherwise limited to the Palearctic. No author since Lutz (1933) has suggested that this material may be a different species. We re-examined the entire slide series (CHIOC 17074–17080, 17272–17289, 17291–17307, 17385, 24517, 24518, 24524–24526) of Lutz (1933) deposited in the CHIOC. Unfortunately, all specimens were in very poor condition. No specimen was complete, and organs have degraded to varying degrees in most specimens. Nevertheless, specimens in that museum series provided evidence that these specimens lacked a muscular pouch-like part of the ejaculatory duct present in A. shoopi.

We were not able to differentiate the material of Lutz (1933) from A. alata (a Palearctic species) confidently based on morphology. Nevertheless, we did observe some differences. For instance, A. alata of Lutz (1933) has a reniform-shaped ovary, and the ovary of A. alata in the Palearctic is trilobed (Dubois, 1938). We believe that the material of Lutz (1933) represents a different species. Morphological and molecular study of freshly collected high-quality specimens is necessary to resolve this longstanding question.

Lutz (1933) wrote that his material came from “Thoas cancrivorus,” also referred to as “cachorro do matto” (wild dog) and experimentally infected kittens. The slides are labeled “Raposa” (fox in Portuguese). “Cachorro do matto” is the crab-eating fox (Cerdocyon thous). However, the CHIOC catalog indicates that all slides by Lutz (1933) are from crab-eating raccoon Procyon cancrivorus (Cuvier). Dubois (1970) stated that this material was collected from Pr. cancrivorus, but does not mention the original host stated by Lutz (1933). The type host was incorrectly reported in subsequent works, such as Dubois (1970) and Fernandes et al. (2015), as well as the CHIOC database. The type host is certainly C. thous.

Fischthal and Martin (1977) collected 9 specimens identified as A. marcianae from Pu. concolor in Paraguay. No description of that material was provided. We examined these specimens and confirmed that they belonged to a new species described herein, A. shoopi. Despite the lack of molecular data from A. shoopi, the morphological differences provide sufficient evidence of its status as an independent species. The collection of new specimens of A. shoopi will lead to improved morphological characterization and allow phylogenetic analysis of its relationships with other members of the genus.

La Rue and Townsend (1927, 1932) originally described A. nasuae based on specimens from a white-nosed coati held at the National Zoological Park in Washington, D.C. At the time, it was unclear whether this infection originated from the native coati habitat or came from prey/food items in the zoo. La Rue and Townsend (1927, 1932) did not provide illustrations, but Dubois (1963, 1970) re-examined the type series and provided the first illustration of the species. Subsequently, Shoop et al. (1989) collected A. nasuae from stray dogs in Mexico.

Considering our results, there are still only 3 nominal Alaria spp. known from the Neotropics: “A. alata,” A. nasuae, and A. shoopi. Considering the high endemicity and diversity of molluscs, amphibians, and carnivores in the Neotropics, especially in the Amazon, we hypothesize that several more Alaria spp. await description in the Neotropics. No DNA sequences are available from any Alaria spp. in the Neotropics. It will be interesting to see what changes to biogeographical patterns may be revealed by their inclusion in future molecular phylogenetic analyses.

The status of Alaria sp. 4

Alaria sp. 4 is the fourth unknown species-level genetic lineage of the genus. Based on COI comparisons, Alaria sp. 2 represents A. marcianae, while the identities of Alaria sp. 1 and 3 remain unknown. This is the second study to report any Alaria species from fishers (Pe. pennanti). Alaria sp. 4 is genetically distinct from A. mustelae (Fig. 4; Table S4). Unfortunately, the quality of our specimens of Alaria sp. 4 originating from frozen fishers was poor. Although major features could be observed, fine details of these specimens could not be seen. Our adult specimens of Alaria sp. 4 are morphologically consistent with A. mustelae and its synonyms (Table IV). The COI sequences of Alaria sp. 4 and A. mustelae differ by 3.1–4.5% (Table S4), despite some specimens of both species being collected in the same general part of Wisconsin (Table S4). Other A. mustelae isolates, including 2 from fishers in Wisconsin, vary up to 2.5%. In contrast, A. marcianae collected in California (GenBank OL439161) differed from isolates of the same species collected in the midwestern United States, including Wisconsin, by only 0.9–1.6% in COI. Interspecific differences among other closely related diplostomoideans were reported at 3.4–4.1% (see Achatz et al., 2021 and references therein). Similarly low levels of interspecific differences in COI sequences have been detected within other digenean genera, such as in Ochoterenatrema Caballero, 1943 whose members exhibit as low as 3.3% interspecific difference in COI (Tkach et al., 2024). The 28S and COI phylogenetic analyses positioned Alaria sp. 4 (from fishers) as a sister group to A. mustelae. This pattern is consistent with the relationships of definitive hosts. Gulonines (which include fishers) are the sister group to mustelines (Law et al., 2017).

In 2011, K. Bates examined 93 fishers collected in Wisconsin by licensed trappers; 45 of which were infected with Alaria specimens. Locke et al. (2018) sequenced 2 specimens (MH581269, MH581270) from these materials which are genetically consistent with A. mustelae (0.4–2.0% difference from other compared A. mustelae isolates; Table S4). We examined the morphology of all available specimens (UWSP–PARA 14322–14344) to determine if these materials represent multiple species. Unfortunately, the condition of these materials did not allow for a confident conclusion.

General remarks

Alaria robusta Verma, 1936, and Alaria michiganensis Hall and Wigdor, 1918 were previously considered members of the genus (“Species inquirenda”) by Dubois (1963, 1970). Both species appear to belong to Neodiplostomum Railliet, 1919 rather than to Alaria. Both species lack pseudosuckers, similar to Neodiplostomum spp. The vitellarium of A. michiganensis is distributed in the opisthosoma as is typical of Neodiplostomum spp. (Dubois, 1963:fig. 12). Alaria robusta was not illustrated (Verma, 1936). Until morphological and molecular data based on freshly collected quality specimens become available, the taxonomic placement of both species remains unclear; however, both species are clearly not Alaria spp., based on morphological evidence.

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Considering the number of new species and nomenclatural changes, we provide a new dichotomous key to Alaria spp. based on adult morphology. The features used to separate A. americana and A. marcianae are based on sequenced adults (Locke et al., 2018; Achatz et al., 2022a; present study).

Key to nominal Alaria spp.

• 1a.

  Testes tandem2

• 1b.

  Testes opposite10

• 2a.

  Pseudosuckers small or difficult to discern, may appear very weakly lappet-like3

• 2b.

  Pseudosuckers distinct, clearly horn-like when extended6

• 3a.

  Holdfast organ massive, occupying about three-quarters or more, of the prosoma length; body is very large4

• 3b.

  Holdfast organ not massive, occupying less than two-thirds of prosoma length; body is of regular size for the genus5

• 4a.

  Body up to 12 mm long. Prosoma elongated. Posterior testis with slight lobation. Pouch-like part of ejaculatory duct longer (more than 1,000 μm long). In the Nearctic (northern United States and Canada)Alaria arisaemoides Augustine and Uribe, 1927

• (Syn. Alaria oregonensis La Rue and Barone, 1927)

• 4b.

  Body up to 4.5 mm long. Prosoma subspherical. Posterior testis highly lobular. Pouch-like part of ejaculatory duct shorter (less than 500 μm long). In the Neotropics (Mexico)Alaria nasuae La Rue and Townsend, 1927

• 5a.

  Lateral vitelline fields extend anteriorly beyond level of ventral sucker. Posterior testis bilobed, each with ventral and dorsal sublobes. In the NearcticAlaria taxideae Swanson and Erickson, 1946

• 5b.

  Lateral vitelline fields extend anteriorly up to level of ventral sucker. Posterior testis trilobed. In the NearcticAlaria mustelae Bosma, 1931(syns. Alaria canadensis Webster and Wolfgang, 1956, Alaria dubia Chandler and Rausch, 1946; Alaria freundi Sphrehn, 1932; Alaria intermedia (Olivier and Odlaug, 1938), Alaria minuta (Chandler and Rausch, 1946))

• 6a.

  Both testes with numerous small lobes. Ejaculatory duct muscular but not pouch-like in appearance. In the PalearcticAlaria alata (Goeze, 1782)

• 6b.

  Anterior testis asymmetrical. Posterior testis with up to 3 lobes. Ejaculatory duct muscular, with pouch-like appearance (may appear strongly dilated). In the Nearctic and Neotropics7

• 7a.

  Posterior testis trilobed. Wall of pouch-like part of ejaculatory duct less than 20 μm thick. In the NearcticAlaria marcianae (La Rue, 1917)

• 7b.

  Posterior testis bilobed. Wall of pouch-like part of ejaculatory duct more than 20 μm thick8

• 8a.

  Ovary transversely elongated. Holdfast organ occupies at least 70% of prosoma length. In the Neotropics (South America)Alaria shoopi n. sp.

• 8b.

  Ovary transversely oval, sometimes bilobed. Holdfast organ occupies less than 60% of prosoma length. In the Nearctic (Canada and United States)Alaria americana Hall and Wigdor, 1918

• (Syns. Alaria canis La Rue and Fallis, 1936; Alaria minnesotae Chandler, 1954)

• 9a.

  Ejaculatory duct strongly muscular, with distinct pouch-like part10

• 9b.

  Ejaculatory duct either not muscular, or muscular without pouch-like part12

• 10a.

  Body broad (oval-shaped) when relaxed. Holdfast organ longer than 350 μm. Pouch-like part of ejaculatory duct longer than 200 μm. In the NearcticAlaria ovalis (Chandler and Rausch, 1946)

• 10b.

  Body narrower (funnel, scoop, or cylindrical shaped) when relaxed. Holdfast organ shorter than 350 μm. Pouch-like part of ejaculatory duct shorter (less than 200 μm)11

• 11a.

  Vitellarium anterior and posterior to level of the holdfast organ; its ventro-lateral fields reach anteriorly to or beyond level of ventral sucker. Tegumental spines well-defined. In the NearcticAlaria procyonis (Harkema, 1942)

• 11b.

  Vitellarium confined to around the level of the holdfast organ and the ventro-lateral fields do not reach anteriorly to level of ventral sucker. Tegumental spines present, inconspicuous. In the NearcticAlaria pseudoprocyonis n. sp.

• 12a.

  Pseudosuckers associated with large masses of unicellular glands. In NearcticAlaria adenocephala (Beckerdite, Miller and Harkema, 1971)

• 12b.

  Pseudosuckers without large masses of unicellular glands13

• 13a.

  Anterior margin of vitellarium around level of ventral sucker (in relaxed specimens). Anterior margin of lateral vitellarium somewhat more anterior to median vitellarium. Eggs smaller than 100 μm. In the NearcticAlaria trashpandae n. sp.

• 13b.

  Anterior margin of vitellarium around level of pharynx (in relaxed specimens). Eggs larger than 110 μm. In AustralasiaAlaria dasyuri (Dubois and Angel, 1972)

Other diplostomids of common raccoons

Including the present study, common raccoons (Pr. lotor) are known to host 5 Alaria spp. as well as 3 other diplostomids: Parallelorchis diglossus Harkema and Miller, 1961; Procyotrema marsupiformis Harkema and Miller, 1959; and Neodiplostomum cratera (Barker and Noll, 1915). It is noteworthy that A. marcianae sequenced by Achatz et al. (2022a) were immature and likely resulted from an accidental infection. Diplostomids of raccoons are primarily known from the southeastern United States (Texas to North Carolina) (Harkema, 1942; Harkema and Miller, 1959, 1961; Beckerdite et al., 1971; Bafundo et al., 1980; Schaffer et al., 1981). Only A. ovalis (Michigan, type locality; Mississippi and Louisiana, origin of sequenced specimens) and A. pseudoprocyonis (Minnesota, type and only known locality) are known to be distributed elsewhere (Chandler and Rausch, 1946; Achatz et al., 2022a; present study). It will be interesting to see if Pr. lotor in other parts of the United States, such as the Pacific Northwest, will harbor further diversity of Alaria. We anticipate that Pr. cancrivorus in South and Central America may also have its own species of Alaria.

Parallelorchis diglossus has only been reported from common raccoons (Harkema and Miller, 1961; Beckerdite et al., 1971; Bafundo et al., 1980; Schaffer et al., 1981). The monotypic Parallelorchis Harkema and Miller, 1961 was synonymized with Alaria by Dubois (1966), but maintained by Beckerdite et al. (1971). Achatz et al. (2022a) agreed with Beckerdite et al. (1971) based on the unique holdfast structure of Pa. diglossus (= consisting of 2 tongue-like lobes that exist as a continuation of the ventral body without a clear point of constriction). Most other features of Pa. diglossus are similar to Alaria spp. from raccoons, such as opposite testes and similar structure of pseudosuckers. However, the genital atrium of Pa. diglossus is quite deep, with well-developed muscles, and the genital cone appears to be absent, whereas Alaria spp. from raccoons have more shallow genital atriums that lack well-developed muscles (Harkema and Miller, 1961). Alaria spp. from raccoons possess a genital cone, albeit the structure may be small and difficult to observe.

Likewise, Pc. marsupiformis, the only member of its genus, is morphologically similar to Alaria spp. from raccoons (e.g., opposite testes and similar structure of pseudosuckers) and species from other hosts (e.g., extremely elongated holdfast organ, relatively large pharynx compared to suckers). Interestingly, Pc. marsupiformis was originally described from Pr. lotor but later reported from a diversity of carnivorous mammals, such as N. vison and gray fox Urocyon cinereoargenteus (Schreber) (Harkema and Miller, 1959; Dubois, 1970). This species is one of the most unusual members of the Superfamily Diplostomoidea. Unlike all other diplostomoideans that inhabit the intestines of their definitive host, Pc. marsupiformis parasitizes the pancreatic ducts of carnivorous mammals. DNA sequences from this unusual digenean are needed to determine its systematic position and relationships with Alaria spp.

Raccoon biology and the evolutionary history of Alaria

Based on the sampling in the present study, and other studies referenced in the foregoing, Alaria spp. of raccoons seem to be predominantly found in the southeastern United States. Except for A. pseudoprocyonis from Minnesota, the Alaria spp. found at more northern latitudes tend to be more frequently found, and in greater diversity, in other mammalian hosts such as mustelids and canids. This matches our expectations, given that the historical distribution of common raccoons was Middle America and the southern United States, and over time this species has spread throughout North America and even been translocated into the Palearctic (Larivière, 2004; Cunze et al., 2023).

Understanding parasite life cycles is paramount to (1) understanding their current and past distributions, (2) understanding their evolutionary history, and (3) differentiating among closely related species. Experimental life cycle work is critical to understanding mechanisms of host specificity, but it can be logistically unlikely for more than 1 species and take a considerable amount of time, particularly for helminths. There is a rich history of life cycle studies of Alaria spp. (e.g., Bosma 1931, 1934; Odlaug, 1940; Shoop and Corkum, 1981), but many such studies would not be feasible today because of animal care and safety restrictions. The persistence of parasites with complex life cycles such as digeneans, including Alaria spp., often relies on hosts belonging to different phyla and trophic levels, and their overlap in time and space (Combes, 1991). Alterations in host behavior and environmental conditions of the same host species in different habitats might prompt variations in life cycles that do not follow a universal set of rules (e.g., Combes, 1991, 1996; Bolek et al., 2016; Mateus and Poulin, 2024). In part, differences in host ecology and behavior may provide some insights helpful for discerning closely related species, especially when morphological differences are not obvious. It is important to remember that broadly distributed hosts, such as common raccoons, exhibit differences in their biology depending on location. For example, reproductive timing, litter size, body weight, and foraging behavior differ between common raccoon populations in the southeastern and northern United States (Lagoni-Hansen, 1981; Tesky, 1995; Zeveloff, 2002; Feldhamer et al., 2003; Reid, 2006). It should be expected that variation in host biology will impact their parasites, particularly their transmission. A recent study on another parasite of common raccoons, Baylisascaris procyonis (Stefanski and Zarnowski, 1951), has demonstrated that the genetic structure of the parasites closely resembles that of their hosts (Carlson et al., 2021). It would not be surprising if Alaria spp. of raccoons follow a similar trend.

The need for museum depositions

Given the unusually complex life history of Alaria spp. combined with their broad distribution and host spectrum create a high potential for evolutionary radiation. This makes the deposition of a series of specimens into museums and access to them even more important, both for morphology and molecular evaluation, to be able to assess past diversity and detect changes over time. Although the present study focused on exploring the diversity and relationships of Alaria spp., this work demonstrates the usefulness of depositing specimens in museum collections. In this study, we utilized specimens deposited in major museum collections (CHIOC, HWML, MSB, USNM), a smaller university museum collection (UWSP), and teaching collections (MGA, UWO, WSU) along with personal working collections. We believe that personal working collections need to be deposited in museums as soon as possible to enable their future use and accessibility by the research community.

Unfortunately, in most cases, these historical collections lacked specimens that could be used for DNA sequencing. The ability to conduct molecular studies is critical for species differentiation, especially when morphological evidence alone is not sufficient. For instance, our ITS region phylogeny (Fig. 3) contained a clade of Alaria spp. that predominantly parasitize canine/feline definitive hosts (A. alata, A. americana, A. arisaemoides, A. marcianae, and Alaria sp. 3). Based on the host associations of the members of the above clade, adults of Alaria sp. 1 will likely be found in a canid, or felid, definitive host. No DNA sequences are available for A. shoopi and A. nasuae; these species were only studied using museum specimens. Based on host associations, we hypothesize that these 2 species would fall into the clade of other Alaria spp. from canids/felids. Although there is no doubt about the identities of A. shoopi and A. nasuae based on morphology, our examination of museum and teaching collection specimens of A. cf. americana, A. cf. mustelae and A. cf. taxideae (Table I) revealed inconsistent patterns of morphology that may suggest that these materials represent up to 3 new species. However, the lack of suitable material to conduct genetic comparisons of these species prevents us from making definitive differentiation without clearer morphological differences.

The availability of specimens for morphological characterizations and re-examination is critical for the re-evaluation of previously published results, as shown in this work. But equally important to morphology is to deposit specimens that are preserved for genomic work that has several advantages: (1) the ability to connect life-cycle stages and multiple individuals across time and space and confirm species identifications (Fig. 1); (2) understanding intraspecific variation across geographical scales and or hosts will improve estimations of diversification, life cycle/ecosystem connections; and (3) study genetics of host–parasite interactions. It is common practice for vertebrate studies to deposit tissues that may be destructively sampled in the future to generate molecular data (e.g., Winker, 2000; Galbreath et al., 2019; Thompson et al., 2021). This has led to significant advances in the evolutionary reconstructions of several lineages of vertebrates. If even a small amount of parasite tissue had been deposited separately and available for destructive sampling, then we may have been able to include these species in our analyses and better understand the diversity and evolutionary history of Alaria. Although molecular study was not commonplace at the time of these specimen collections, some researchers routinely deposited vials of specimens in various fixatives that could be utilized in the future (Lutz et al., 2017; Galbreath et al., 2019). We strongly encourage that future studies either (1) deposit slides of specimens associated with molecular data or/and (2) deposit specimens suitable for both morphological and molecular analyses in future studies.

Concluding remarks

In total, the present study has provided descriptions of 3 new Alaria spp. (A. pseudoprocyonis, A. shoopi, and A. trashpandae), a new species-level lineage (Alaria sp. 4), A. procyonis and A. marcianae (associated with DNA sequences), and A. nasuae. We have provided the first description of a new species of Alaria from South America and found evidence that there is likely at least 1 additional Alaria sp. in South America. Although many hosts of Alaria spp. are rather frequently collected and have been extensively studied for digeneans, questions remain regarding the diversity and relationships of Alaria spp.

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