Phylogenetic Relationships of Avian Cestodes from Brine Shrimp and Congruence with Larval Morphology

Citation: Redón, S.; Quiroz, M.; Luki´c, D.; Green, A.J.; Gajardo, G. Phylogenetic Relationships of Avian Cestodes from Brine Shrimp and Congruence with Larval Morphology. Animals 2024,14, 397. https:// doi.org/10.3390/ani14030397 Academic Editor: Kenji Saitoh Received: 10 December 2023 Revised: 20 January 2024 Accepted: 22 January 2024 Published: 25 January 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). animals Article Phylogenetic Relationships of Avian Cestodes from Brine Shrimp and Congruence with Larval Morphology Stella Redón1,2,* , Mauricio Quiroz 2, Dunja Luki´c 3, Andy J. Green 3and Gonzalo Gajardo 2 1Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Av. Reina Mercedes, 41012 Seville, Spain 2Departamento de Ciencias Biológicas y Biodiversidad, Universidad de Los Lagos, Av. Fuchslocher 1305, Osorno 5290000, Chile; [email protected] (M.Q.); ggajar[email protected] (G.G.) 3Department of Conservation Biology and Global Change, Estación Biológica de Doñana, EBD-CSIC, C/Américo Vespucio 26, 41092 Seville, Spain; [email protected] (D.L.); ajgr[email protected] (A.J.G.) *Correspondence: [email protected] or [email protected] Simple Summary: Hypersaline lakes provide services like nest sites and food for migratory waterbirds. The crustacean Artemia (brine shrimp) is an intermediate host for many cestode parasites completing their life cycle in multiple waterbirds (flamingos, grebes, waders, and ducks) associated with the hypersaline lakes. The molecular identification of larval stages of six cestodes in Artemia from three countries correlated well with the morphology of advanced larval stages. This study provides the first parasite molecular data on Artemia-cestode-bird relationships to facilitate our understanding of food web dynamics and service provisioning in hypersaline lakes, which are threatened by human-caused perturbations, including climate change. Abstract: Determining molecular markers for parasites provides a useful tool for their identification, particularly for larval stages with few distinguishable diagnostic characters. Avian cestodes play a key role in the food webs and biodiversity of hypersaline wetlands, yet they remain understudied. Using naturally infected Artemia, we identified cestode larvae (cysticercoids), assessed their genetic diversity, and explored phylogenetic relationships in relation to larval morphology and waterbird final hosts. We obtained partial 18S rDNA sequences for 60 cysticercoids of the family Hymenolepidae infecting Artemia spp. from seven localities and three countries (Spain, the USA, and Chile). We present the first DNA sequences for six taxa: Confluaria podicipina,Fimbriarioides sp., Flamingolepis liguloides, Flamingolepis sp. 1, Flamingolepis sp. 2, and Hymenolepis californicus. Intraspecific sequence variation (0.00–0.19% diversity) was lower than intergroup genetic distance (0.7–14.75%). Phylogenetic analysis revealed three main clades: 1—Flamingolepis, 2—Fimbriarioides, 3—Confluaria and Hymenolepis, all of which separated from hymenolepidids from mammals and terrestrial birds. This clear separation among taxa is congruent with previous morphological identification, validating the 18S gene as a useful marker to discriminate at generic/species level. Working with intermediate hosts allows the expansion of knowledge of taxonomic and genetic diversity of cestodes in wildlife, as well as elucidation of their life cycles. Keywords: Platyhelminthes; Hymenolepididae; ribosomal 18S; Anostraca; waterbirds; molecular phylogeny; phylogeography 1. Introduction Parasites have crucial roles in ecosystem functioning and services, and are important sources of information on the ecology of their hosts [ 1 ]. However, knowledge of the diversity of endoparasites of aquatic birds, such as cestodes, remains limited [ 2 ]. Studies aimed at delimiting parasitic species are mostly based on both the morphology of adult specimens and life cycle characteristics. Larval stages have a limited number of morphological features and their identification can be problematic. However, working with adult parasites Animals 2024,14, 397. https://doi.org/10.3390/ani14030397 https://www.mdpi.com/journal/animals Animals 2024,14, 397 2 of 15 implies post-mortem examination of the final hosts, which can be restricted for waterbirds, particularly protected species such as South American flamingos, which are affected by climate change and mining impacts [3,4]. Accurate identification of larval parasites is relevant to understanding the life cycle of parasites, host–parasite interactions, disease transmission and diagnosis (reviewed by [ 5 ]), and for a more comprehensive knowledge of global biodiversity [ 6 ]. Molecular tools are increasingly used in parasitological studies and can help identify the final hosts of parasites, detect them, and characterize their communities [ 7 , 8 ]. However, there are important biases in the research on helminth parasites related to specific taxa and geographical areas [ 9 ]. In addition, parasites in wildlife have received little attention compared to taxa with medical, zoonotic, or veterinary importance [9]. Cestodes are a group of obligate helminth endoparasites with multiple-host life cycles and trophic transmission through predator–prey interactions. Adults of most species parasitize mammals, but others have fish or birds as final hosts. The life cycle includes an invertebrate as intermediate hosts in which the parasite develops to an infective larval stage ready to reach the vertebrate host, where it matures and reproduces. Although cestodes are involved in major diseases in humans and wildlife, knowledge of their biodiversity is limited, being particularly scarce for those infecting wild aquatic birds in the Neotropics [ 2 , 10 ]. Flamingos are major hosts, yet the cestode fauna of flamingos from South America is currently understudied. A unique cestode (Flamingolepis chilensis) was described from the Andean flamingo Phoenicoparrus andinus [ 11 ], but there is no previous information for the Puna flamingo Phoenicoparrus jamesi and the Chilean flamingo Phoenicopterus chilensis. Early stages of parasites (i.e., eggs and larvae), obtained from fecal samples or intermediate hosts, can be used to study parasite biodiversity, host associations and biogeography, overcoming methodological limitations of working with vertebrate final hosts. Recent morphological studies of larvae in crustacean intermediate hosts (Artemia spp.) from Chile provided evidence of additional Flamingolepis species, as well as the first reports of the progynotaeniid Gynandrotaenia stammeri from South American flamingos [10,12]. The application of molecular techniques to helminth parasites can discover cryptic parasite species [ 13 , 14 ], characterize larval cestodes [ 15 , 16 ], and reconstruct phylogenetic relationships [ 17 – 19 ]. The genes encoding the nuclear ribosomal RNA are widely employed for diagnosis of cestodes infecting mammals [ 20 – 23 ]. In the last decade, large and small subunit rRNA gene (i.e., 28S and 18S) have been used to explore the phylogeny within the cestode family Hymenolepididae Ariola, 1899 (the most diverse with 923 recognized species, [2]), with emphasis on mammalian parasites [24–29]. The brine shrimp Artemia spp. (Crustacea: Branchiopoda: Anostraca) has a key role in the life cycle and transmission of avian cestodes in hypersaline lagoons, lakes and coastal salterns [ 10 , 30 – 34 ], which are valuable habitats and breeding grounds for resident and migratory birds (e.g., in South America: [ 3 , 35 , 36 ]). It serves as an intermediate host for at least 22 cestode taxa, 15 of them hymenolepidids [ 37 ], parasitizing flamingos, grebes, waders, gulls, and ducks [ 12 ]. Three putative cestode species new to science have recently been recorded in South American Artemia populations [ 10 , 12 ]. However, all studies on Artemia parasites in any continent are based on morphological and morphometric data obtained from microscopic examination, without any genetic information. Here, we present the first study to molecularly identify avian cestodes recorded in brine shrimps. We sequenced 18S rDNA gene fragments of hymenolepidid cestodes infecting Artemia, including specimens from localities in the Nearctic, Palaearctic, and Neotropical realms. We focused on the Hymenolepididae family since it is the most speciose and prevalent in brine shrimp hosts, and includes parasites of different waterbird families. Our main objectives were firstly to compare the genetic diversity of larvae with their diversity according to morphology, to establish the extent to which a molecular phylogeny is consistent with taxonomy based on larvae morphology, and secondly, to estimate genetic variation within morphospecies recorded in different continents, and/or in different host species. We compared sequences for larvae assigned to the six morphospecies from the Animals 2024,14, 397 3 of 15 genera Confluaria (parasite of grebes), Fimbriarioides (parasites of ducks), Flamingolepis (parasite of flamingos), and Hymenolepis (parasites of Charadriiforms), including species yet to be fully described. We looked for cryptic species, i.e., extensive sequence divergence in cases where the same morphospecies has been described in different biogeographical realms. We discussed our results in relation to the movement ecology of bird species acting as final hosts. 2. Materials and Methods 2.1. Artemia Samples and Parasite Material Artemia samples were collected between 2007 and 2018 in several hypersaline wetlands from Europe and the Americas as part of different research projects. Samples from seven localities and three countries were used in the present study, including the asexual Artemia parthenogenetica from two coastal salterns in Spain (Bras del Port salterns in Alicante province and San Pedro del Pinatar salterns in Murcia province), and two sexual species: Artemia franciscana from an inland lake in USA (Great Salt Lake in Utah) and from two lagoons in Northern Chile (Tebenquiche lagoon and Barros Negros lagoon in Salar de Atacama, Region of Antofagasta) and Artemia persimilis from two lagoons in the south of Chile (Amarga lagoon and Los Cisnes lagoon in Patagonia, Region of Magallanes and Chilean Antarctica). A total of 8262 Artemia individuals were examined under the microscope for cysticercoids in previous works (see [ 10 , 12 , 33 , 38 ] for details of sampling and fixing procedures). From those, 120 infected brine shrimps (45 A. persimilis, 58 A. franciscana, and 17 A. parthenogenetica; see Table S1) were preserved in absolute or 70% ethanol at 4 ◦ C for molecular analysis. Some cysticercoids of Flamingolepis liguloides (bigger size) were isolated from the Artemia tissue and fixed in absolute ethanol individually and in pools of 2–5 cysts (Table S1). This allowed us to test if parasite isolation and/or the quantity of material increased the quality of DNA and thus the 18S amplification. Most DNA extractions were performed on an Artemia individual infected with one cysticercoid (see Table S1). Indeed, a single Artemia infected with one cestode cysticercoid was enough for positive amplification (independently of the cysticercoid size), indicating that isolation of parasites from the Artemia tissue was not essential for a successful PCR reaction. Pools of 3 Artemia individuals from Los Cisnes lagoon infected with Flamingolepis sp. 1 (the most prevalent parasite in the south of Chile) were also used in the analyzes (N = 4), but no differences in the results were obtained compared to individual samples. Parasites isolated from individuals with multiple infections (i.e., with more than one cestode species) were used for the less abundant cestode taxa. Cysticercoids were morphologically identified as Confluaria podicipina,Fimbriarioides sp., Flamingolepis liguloides,Flamingolepis sp. 1, Flamingolepis sp. 2, and Hymenolepis californicus (after [ 10 , 12 , 30 , 33 , 38 , 39 ]). Identifications were mainly based on the size of the cysts, type of rostellum, number, size and shape of rostellar hooks, and length of cercomer (see above references for more details). Three uninfected Artemia individuals from Los Cisnes lagoon collected in November 2017 were considered as negative control for the PCR reaction, allowing us to exclude non-specific Artemia genomic DNA amplification. 2.2. Genomic DNA Extraction Genomic DNA was extracted from infected Artemia individuals preserved in ethanol using phenol chloroform-isoamyl (see details in Appendix A). DNA samples were kept at 4 ◦ C (or − 20 ◦ C for a longer period). The same procedure was used for the isolated F. liguloides parasites and for non-infected individuals used as negative control. A 1.2% agarose gel was prepared for screening genomic DNA extractions with 4 µL of sample. 2.3. Primer Design, PCR Amplification and Sequencing A pair of primers for amplification and sequencing of 18S rDNA of our cestodes were designed based on ten sequences of hymenolepidids available from GenBank (for primer Animals 2024,14, 397 4 of 15 information, see details in Table S2). Sequences were aligned with MEGA 11 software [ 40 ] to identify suitable regions for primer design. We selected a gene fragment including three variable regions (V3, V4, V5) flanked by more conserved ones. The following non-degenerate primers were designed accordingly: forward HymF1 (5 ′ -GATCAATTGGAGGGCAAGT-3 ′ ) and reverse HymR1 (5 ′ -CTGTCATGACGGTGATTGA3 ′ ). We attempted to amplify a fragment with an expected size of 1462 base pairs (bp) of the 18S gene of cysticercoids of the four genera tested. A PCR amplification was performed in total volume of 10 µ L containing: 1 µ L 10 × Invitrogen PCR buffer, 1 µ L MgCl 2 (50 mM), 1 µ L dNTP (2 mM), 1 µ L primer set mix HymF1 + HymR1 (2 µ M), 0.1 µ L (0.6 U) Taq polymerase (Invitrogen, Carlsbad, CA, USA), 4.9 µ L ultrapure water, and 0.5–1 µ L DNA template. The PCR consisted of an initial denaturation step at 94 ◦C for 5 min, followed by 32 cycles (denaturation at 94 ◦C for 40 s; primer annealing at 54 ◦ C for 40 s; extension at 72 ◦ C for 50 s) and a final extension at 72 ◦ C for 10 min. PCR products were visualized by electrophoresis on 1.5% TAE agarose gel with SYBR green staining under ultraviolet transillumination and using a ladder of 1Kb (Invitrogen) to determine fragment size. DNA re-amplifications were performed using PCR products as templates in a second round of PCR. To do that, each DNA band was excised from the gel with sterile carbon steel, transferred to a 0.2 mL sterilized microtube with 50–70 µ L of ultrapure water and kept at 4 ◦ C overnight. PCR for re-amplifications was performed in a volume of 30 µ L containing: 3 µ L of 10 × PCR buffer, 3 µ L of 50 mM MgCl 2 , 3 µ L of 2 mM dNTP, 3 µ L of 2 µ M primer set mix, 0.36 µ L Taq polymerase (Invitrogen), 16.64 µ L ultrapure water, and 1 µ L amplified DNA, and selecting the same thermocycling conditions described above. PCR products (2 µ L) were loaded on 1.5% TAE agarose gel before sequencing. Amplicons were sent to Macrogen Inc. (Seoul, Republic of Korea) for purification and bi-directional sequencing with the designed Hym-F1/Hym-R1 primers. Amplicons were obtained for a total of 83 samples, but only sequences with the best quality were used for the analyzes. Sixty hymenolepidids (including specimens of the six taxa, Table 1) were successfully sequenced, which meant a success rate of 72.3%. After trimming off the two ends, the size of our sequences ranged from 1113 to 1149 nucleotides. Of the DNA samples that did not amplify (N = 43), 81.4% corresponded to samples collected more than 10 years ago. Table 1. Cestode taxa included in the genetic distance and phylogenetic analysis. Columns show the intermediate host, geographic origin, length of 18S fragment, haplotypes, sequences access number, and final host. Sequences obtained in this study are indicated in bold. Final hosts are also indicated as G, grebes; F, flamingos; D, ducks; S, shorebirds; WB, other waterbirds; B, other birds; M, mammals. Cestode Taxa Intermediate Host Geographic Origin Size (bp) Haplotype (no. Sequences) Acc. no. GenBank Final Host Confluaria podicipina Artemia franciscana Great Salt Lake, Utah, USA (GSL) 1115 1 (8) OR995634 G: Podiceps auratus *, P. nigricollis * Confluaria podicipina Artemia persimilis Amarga lagoon, Chile (AMA) 1115 2 (2) OR995636 G: Podiceps occipitalis * Confluaria podicipina Artemia parthenogenetica San Pedro del Pinatar, Murcia, Spain (SPP) 1116 3 (2) OR995638 G: Podiceps nigricollis * Confluaria podicipina Artemia parthenogenetica Bras del Port salterns, Alicante, Spain (BRP) 1115 4 (1) OR995626 G: Podiceps nigricollis * Fimbriaria fasciolaris NA Chernihiv Oblast’, Ukraine 2039 ON350769 D: Anas platyrhynchos Fimbriaria sp. NA Lake Butte des Morts, Wisconsin, USA 2137 AF286982 D: Anas platyrhynchos Fimbriaria teresae NA Chernihiv Oblast’, Ukraine 2020 OP910109 D: Anas platyrhynchos Animals 2024,14, 397 5 of 15 Table 1. Cont. Cestode Taxa Intermediate Host Geographic Origin Size (bp) Haplotype (no. Sequences) Acc. no. GenBank Final Host Fimbriarioides sp. Artemia persimilis Amarga lagoon, Chile (AMA) 1149 1 (2) OR995637 D: Anatidae Fimbriarioides sp. Artemia persimilis Los Cisnes lagoon, Chile (CIS) 1149 1(4) OR995632 D: Anatidae Flamingolepis liguloides Artemia parthenogenetica Bras del Port salterns, Alicante, Spain (BRP) 1113 1 (1) 2 (1) 3 (2) 4 (1) 5 (1) OR995627 OR995628 OR995629 OR995630 OR995631 F: Phoenicopterus roseus Flamingolepis liguloides Artemia parthenogenetica San Pedro del Pinatar salterns, Murcia, Spain (SPP) 1113 6 (1) OR995639 F: Phoenicopterus roseus * Flamingolepis sp. 1 Artemia persimilis Los Cisnes lagoon, Chile (CIS) 1117 1 (14) OR995633 F: Phoenicopterus chilensis * Flamingolepis sp. 1 Artemia franciscana Tebenquiche lagoon, Chile (TEB) 1117 1 (7) 2 (1) OR995640 OR995641 F: Phoenicopterus chilensis * Flamingolepis sp. 1 Artemia franciscana Barros negros lagoon, Chile (BNE) 1117 1 (9) OR995625 F: Phoenicopterus chilensis * Flamingolepis sp. 2 Artemia franciscana Tebenquiche lagoon, Chile (TEB) 1113 1 (1) OR995642 F: Phoenicopterus chilensis *, Phoenicoparrus spp. * Hymenolepididae gen. sp. 1 NA Ciénega de Santa Clara, Sonora, Mexico 2076 MH699819 WB: Nannopterum auritus Hymenolepididae gen. sp. 2 NA Los Chivos, Veracruz, Mexico 2016 MH699822 WB: Nycticorax nycticorax Hymenolepididae gen. sp. 3 NA Lago de Catemaco Veracruz, Mexico 2042 MH699820 WB: Nyctanassa violacea Hymenolepididae gen. sp. 4 NA Tasman Bay, New Zealand 1971 JX173961 S: Haematopus unicolor Hymenolepis californicus Artemia franciscana Great Salt Lake, Utah, USA (GSL) 1118 1 (2) OR995635 S: Larus californicus, L. dalawarensis Hymenolepis microps Lagopoecus affinis *, Goniodes lagopi * Kattfjord, Troms County, Norway 2233 KY403995 B: Lagopus lagopus Wardoides nyrocae NA NA 2186 AJ287587 WB: Cygnus olor Coronacanthus magnihamatus NA Boyana River, Bulgaria 1996 KJ710321 M: Neomys fodiens Hymenolepis diminuta NA Lab strain 2148 AF286983 M: Rattus norvegicus Hymenolepis microstoma NA Lab strain 2140 AJ287525 M: Mus sp. Dilepis undula NA Wimbourne, Dorset, UK 2091 AF286981 B: Turdus merula * Indicates putative host species. 2.4. Cestode Sequence Analysis and Genetic Variations Raw nucleotide sequences were assembled and manually edited with GENEIOUS 11.1.5 (Biomatters Ltd., Auckland, New Zealand). The 18S sequences were checked with the Basic Local Alignment Search Tool (BLAST) and the NCBI nucleotide database in general for preliminary molecular identification [ 41 ]. We also used other available hymenolepidid sequences from GenBank for our analysis (matching sequences recorded in bird hosts— altogether 9—and 3 selected from mammalian hosts, see Table 1) and the closest available taxon outside of hymenolepidids for an outgroup (i.e., Dilepis undula; [ 29 ]). Sequences Animals 2024,14, 397 6 of 15 were aligned using the MUSCLE algorithm in MEGA 11 [ 40 ]. We assessed nucleotide composition (i.e., the relative frequencies of the four nucleotides) and GC content (%GC) for each taxon with MEGA 11, and the number of polymorphic sites and haplotype diversity in newly generated sequences with DnaSP v. 6. [ 42 ]. The most likely evolutionary model was determined based on the corrected Akaike Information Criterion (AICc) in JModelTest v 2.1.10 [43,44]. We chose to use the General Time Reversible (GTR) evolutionary model with a gamma shape parameter (+G) and invariable sites (+I) for construction of Maximum Likelihood (ML) in MEGA 11 and Bayesian inference (BI) in MrBayes [ 45 – 47 ] phylogenetic trees as this was the best scoring model (second overall) in JModelTest available in both MEGA 11 and MrBayes version 3.2.7a softwares. ML analyzes were performed with the complete deletion of sites containing gaps and 1000 bootstrap replicates. For BI analyzes, we applied the Markov Chain Monte Carlo (MCMC) method for 1.2 million generations until standard deviation of split frequencies reached <0.01. The trees were sampled every 1000 generations and initial 25% of produced trees were discarded as burn-in. We used FigTree v. 1.4.4 [48] to visualize a single tree. The pairwise genetic distance (inter- and intragroup) of cestode taxa was calculated with a p-distance and Tamura Nei evolutionary model (TN93; [ 49 ]) with a discrete γ model (+G; γ = 1.00) implemented in MEGA 11, both with 1000 bootstrap replicates and complete deletion of sites containing gaps. 3. Results PCR amplification of the genomic DNA yielded partial 18S rDNA sequences for the six cestode taxa assayed, representing four cestode genera. 3.1. Genetic Diversity Comparing the sixty 18S new sequences obtained in this study (Table 1) (1085 unambiguously aligned positions), there were 227 variable sites, of which 225 were parsimoniously informative. The nucleotide composition of sequences had a mean percentage of GC of 49.31%, with slight differences among genera. Fimbriarioides sp. had the highest percentage of GC (51.52%), followed by Confluaria (50.42%) and Hymenolepis (50.0%), whereas the genus Flamingolepis exhibited the lowest GC content (between 48.43% and 48.68%). Levels of nucleotide variation detected between pairs of the hymenolepidid taxa sequenced in this study (Table 1), and nine hymenolepidid taxa found in birds, three randomly chosen species from mammals, and the outgroup D. undula available in Gen- Bank (Table 1) are presented in Table S3. In this study, the highest similarity (i.e., lowest genetic distance) was found between Flamingolepis and between Confluaria podicipina and Hymenolepis californicus, while Flamingolepis liguloides and Fimbriarioides were the most divergent (Table 2). Table 2. Estimates of evolutionary divergence between hymenolepidid taxa sequenced in this study. The number of base differences per site from averaging over all sequence pairs between groups of sequences are shown below the diagonal. Standard error estimate(s) are shown above the diagonal and were obtained by a bootstrap procedure (1000 replicates). The analysis involved 60 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There was a total of 1085 positions in the final dataset. The analysis was conducted in MEGA11 ([40]). Cestode Taxa 1 2 3 4 5 6 1Flamingolepis sp. 1 0.007 0.007 0.011 0.010 0.010 2Flamingolepis sp. 2 0.055 0.002 0.010 0.010 0.010 3Flamingolepis liguloides 0.059 0.007 0.011 0.010 0.010 4Fimbriarioides sp. 0.147 0.143 0.147 0.010 0.010 5Confluaria podicipina 0.132 0.127 0.130 0.110 0.007 6Hymenolepis californicus 0.139 0.135 0.142 0.116 0.061 Animals 2024,14, 397 7 of 15 The interspecific differences considerably exceeded intraspecific variation (Tables 2and 3). Except for Flamingolepis sp. 1 in TEB lagoon and F. liguloides in BRP, for which genetic variability was detected (two and five haplotypes, respectively; Table 1), no variable positions in the sequences for the remaining cestode taxa were observed at intra-population level. Intraspecific genetic variation was observed for C. podicipina,F. liguloides, and Flamingolepis sp. 1 (Table 3, Figure 1). There was no indication of the existence of cryptic species (Table 2). Animals 2024, 14, x FOR PEER REVIEW 8 of 16 Figure 1. Maximum likelihood (ML) phylogenetic tree based on partial 18S rDNA sequences of 18 hymenolepidid taxa. GenBank accession numbers are shown. Identical sequences recorded at different localities are indicated with asterisks (* OR995637, ** OR995625, *** OR995633). Sequences generated in this study are marked in bold. Dilepis undula (family: Dilepididae) was used as Figure 1. Maximum likelihood (ML) phylogenetic tree based on partial 18S rDNA sequences of 18 hymenolepidid taxa. GenBank accession numbers are shown. Identical sequences recorded at Animals 2024,14, 397 8 of 15 different localities are indicated with asterisks (* OR995637, ** OR995625, *** OR995633). Sequences generated in this study are marked in bold. Dilepis undula (family: Dilepididae) was used as outgroup. The three major clades revealed by the present study, including host associations and rostellum morphology, are indicated. Numbers on branches are bootstrap values. The scale bar shows the number of substitutions per site. Table 3. Estimates of mean genetic divergence over sequence pairs within hymenolepidid taxa sequenced in this study. The number of base differences per site from averaging over all sequence pairs within each group are shown. This analysis involved 60 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 1085 positions in the final dataset. The presence of n/c in the results denotes cases in which it was not possible to estimate evolutionary distances. Cestode Taxa Mean ±Standard Error Confluaria podicipina 0.0012 ±0.0006 Flamingolepis sp. 1 0.0001 ±0.0001 Flamingolepis sp. 2 n/c Flamingolepis liguloides 0.0019 ±0.0008 Fimbriarioides sp. 0.0000 ±0.0000 Hymenolepis californicus 0.0000 ±0.0000 3.2. Phylogenetic Analyzes A phylogenetic analysis was performed on a dataset of 27 unique nucleotide sequences of hymenolepidids (15 newly produced haplotypes, 9 sequences previously available in GenBank of bird hymenolepidid parasites, and 3 sequences of mammalian hymenolepidids) and one outgroup sequence (D. undula). Maximum likelihood (ML) and Bayesian inference (BI) produced trees with similar topologies, with some differences in primary branches, but for which nodal support was generally below 50% (ML tree shown; Figure 1). Both ML tree (Figure 1) and BI tree (Figure S1) placed newly generated sequences in three separate clusters (1—Flamingolepis, 2—Fimbriarioides, and 3—Confluaria podicipina and Hymenolepis californicus clades). Hymenolepidid taxa parasitizing on mammal hosts, together with one taxon found in terrestrial birds (Hymenolepis microps found in the Willow ptarmigan Lagopus lagopus [50]), belonged to a separate cluster (Figure 1). All cestode specimens clustered well within their corresponding genera, confirming previous morphological identifications (Figure 1). We found common and local haplotypes, suggesting that both Artemia taxon and geographical distance were important. Confluaria podicipina was recorded in different Artemia species (A. franciscana,A. persimilis, and A. parthenogenetica), but a unique and different haplotype was found in each locality (Table 1, Figure 1). For the Flamingolepis sp. 1 associated with South American flamingos, a common haplotype was detected for the two host species (i.e., A. franciscana and A. persimilis; Table 1), but another haplotype was also found in A. franciscana from TEB lagoon in northern Chile. In the case of Fimbriarioides sp., recorded in A. persimilis in two lagoons from southern Chile (AMA and CIS), a common haplotype was detected at both localities. 4. Discussion Accurate identification of larval cestodes (i.e., cysticercoids) is important but, in certain cases, can be difficult due to their limited morphological characters, scarce knowledge of larval morphology, and unknown life cycles. Molecular techniques can help with identification. In this study, we provided: (1) a cheap and faster procedure for parasite DNA extraction that overcomes the generally complex and time-consuming isolation of microscopic parasites from the host (an Artemia hosting a single cysticercoid allowed amplification by PCR); (2) a pair of new primers for the 18S rDNA gene for hymenolepidids; (3) novel molecular data for cestodes of aquatic birds. Animals 2024,14, 397 9 of 15 The method described had a high rate of amplification success, avoided co-amplification of the host DNA and validated the effectiveness of the primers to amplify cysticercoids of different size, from the smallest (C. podicipina 72–147 × 36–89 µ m, [ 10 ]) to the biggest (F. liguloides 560–810 × 372–597 µ m, [ 30 ]). Our findings highlight the usefulness of DNA data for the identification of parasite larvae, this being a feasible technique for routine analysis in invertebrate intermediate hosts. 4.1. Molecular Novelties and the Diversity of Avian Cestodes Our research is a significant contribution to avian cestodes’ molecular information. A total of eighteen partial 18S rDNA sequences for six taxa of four genera, that as adults parasitize different waterbirds (flamingos, grebes, gulls, and ducks), have been deposited in GenBank [ 41 ]. These represent the first molecular data for Artemia parasites. Indeed, there were previously no sequences of any gene for the genus Flamingolepis or Fimbriarioides, and only limited data for other parasite taxa with the potential to infect brine shrimp hosts. For parasites of gulls (Laridae) of the genus Wardium, several species use Artemia as an intermediate host (i.e., W. fusa,W. stellorae, and Wardium sp., in Asia, Europe and America, respectively; [ 12 ]) but only one 28S sequence of Wardium cirrosa from polychaetes is available. For the genus Hymenolepis, most available data were for mammalian parasites such H. diminuta and H. microstoma parasitizing rodents, with H. microps from Lagopus lagopus (Phasianidae) being the only molecular record for avian parasites to date. We provided the first data of H. californicus, a parasite associated with Nearctic gulls such as Larus californicus [33]. For the genus Confluaria (parasite of grebes), there was only one pre-existing 28S sequence for Confluaria pseudofurcifera infecting Podiceps cristatus [ 51 ] and we presented the first data for C. podicipina. Our study offered molecular data for cestodes from different geographical regions and continents, with some species recorded in several brine shrimp and avian hosts species. For example, C. podicipina is the most cosmopolitan taxon with records in A. parthenogenetica from Europe, A. franciscana from North America and A. persimilis from South America, which can be associated with different grebe species acting as potential final hosts in each locality (e.g., Podiceps nigricollis,P. auritus, and P. occipitalis; Table 1), existing direct evidences confirming that the life history of this hymenolepidid occurs between brine shrimp (intermediate host) and the silvery grebe P. occipitalis (final host) [ 52 ]. The flamingo parasite Flamingolepis sp. 1 was recorded in both A. franciscana and A. persimilis from Chile, while Fimbriarioides sp. was exclusively infecting A. persimilis in southern Chile. Studies like this can provide key information about host–parasite associations, geographical distribution, and life cycles of parasites. Morphology has been the standard method for cestode larvae identification, but it usually results in provisional identifications pending complementary information from adult specimens or genetic confirmation. The scarce knowledge of larval morphology and life cycles of avian cestodes are important limitations when using comparative morphology for parasite identification. Molecular research can help to clarify the taxonomy of poorly studied genera such Flamingolepis Spasskii et Spasskaya, 1954. The differences we found among the sequences of Flamingolepis spp. indicated: (1) the Chilean species (Flamingolepis sp. 1 and sp. 2) and the European (F. liguloides) were closely related to each other (clade Flamingolepis) and distantly related to the other three genera analyzed; (2) the cysticercoids of Flamingolepis sp. 1 belonged to a different species (as already suggested based on morphology, [ 10 , 12 ]) since they clustered in a separate sub-clade (see Figure 1and Figure S1). However, this was less likely for Flamingolepis sp. 2, probably due to a single analyzed specimen providing insufficient molecular data. From now on, we recommend genetic identification as the fastest method for a preliminary diagnosis, but morphological and molecular evidence should be combined for a better classification of larval specimens.