Introduction
The phylogeography and distribution of any species are shaped by abiotic and biotic factors. In parasites that infect wild animals, the transmission mode, life cycle, dispersal and type of intermediate and definitive hosts play crucial roles in the genetic structure and diversity of species (Criscione and Blouin, Reference Criscione and Blouin2004; Goulding and Cohen, Reference Goulding and Cohen2014; van der Mescht et al., Reference van der Mescht, Matthee and Matthee2015; Perrot-Minnot et al., Reference Perrot-Minnot, Špakulová, Wattier, Kotlík, Düsen, Ayogdu and Tougard2018; García-Varela et al., Reference García-Varela, López-Jiménez, González-García, Sereno-Uribe and Andrade-Gómez2023). The recent application of molecular markers has provided insight into the taxonomy, systematics, delimitation species and phylogeographic structure of parasites, including acanthocephalans (Steinauer et al., Reference Steinauer, Nickol and Ortí2007; Rosas-Valdez et al., Reference Rosas-Valdez, Morrone and García-Varela2012, Reference Rosas-Valdez, Morrone, Pinacho-Pinacho, Domínguez-Domínguez and García-Varela2020; Alcántar-Escalera et al., Reference Alcántar-Escalera, García-Varela, Vázquez-Domínguez and Pérez-Ponce2013; Goulding and Cohen, Reference Goulding and Cohen2014; Perrot-Minnot et al., Reference Perrot-Minnot, Špakulová, Wattier, Kotlík, Düsen, Ayogdu and Tougard2018; Pinacho-Pinacho et al., Reference Pinacho-Pinacho, García-Varela, Sereno-Uribe and Pérez-ponce de León2018; García-Varela et al., Reference García-Varela, Masper, Crespo and Hérnandez-Orts2021, Reference García-Varela, López-Jiménez, González-García, Sereno-Uribe and Andrade-Gómez2023; Sereno-Uribe et al., Reference Sereno-Uribe, López-Jiménez, González-García, Pinacho-Pinacho, Macip Ríos and García-Varela2022). Members of the family Polymorphidae Meyer, 1931, are a globally distributed group of acanthocephalans whose adults reside in the intestines of fish-eating birds, waterfowl and marine mammals. The encysted cystacanth (larval form) resides in the body cavities of diverse crustaceans (amphipods, decapods and euphausiids) that serve as intermediate hosts to complete their life cycle. However, teleost fishes (paratenic hosts) play a principal role in transmission because they serve as ecological bridges facilitating infection to appropriate definitive hosts (Nickol et al., Reference Nickol, Crompton and Searle1999, Reference Nickol, Heard and Smith2002; Aznar et al., Reference Aznar, Pérez Ponce and Raga2006; Kennedy, Reference Kennedy2006; García-Varela et al., Reference García-Varela, Pérez-ponce de León, Aznar and Nadler2013; Presswell et al., Reference Presswell, García-Varela and Smales2018, Reference Presswell, Bennett and Smales2020). Currently, Polymorphidae contains 16 accepted genera based on morphological, ecological and molecular characteristics (Schmidt, Reference Schmidt1973, Reference Schmidt1975; Amin, Reference Amin2013; García-Varela et al., Reference García-Varela, Pérez-ponce de León, Aznar and Nadler2013; Presswell et al., Reference Presswell, García-Varela and Smales2018, Reference Presswell, Bennett and Smales2020; Ru et al., Reference Ru, Yang, Chen, Kuzmina, Spraker and Li2022; Rothman et al., Reference Rothman, Hill-Spanik, Wagner, Kendrick, Kingsley-Smith and de Buron2025). Of the approximately 140 described species classified in Polymorphidae, 41 belong to the genus Polymorphus Luhë 1911, and 6 belong to Pseudocorynosoma Aznar, Pérez Ponce de León and Raga, 2006. Adult Polymorphus and Pseudocorynosoma are parasites of aquatic birds and amphipods distributed across the globe (Aznar et al., Reference Aznar, Pérez Ponce and Raga2006; Amin, Reference Amin2013; García-Varela et al., Reference García-Varela, Hernández-Orts and Pinacho-Pinacho2017). To date, 4 species of Polymorphus (P. trochus Van Cleave, Reference Van Cleave1945, P. minutus (Goeze, 1782), P. obtusus Van Cleave, 1918 and P. brevis Van Cleave, Reference Van Cleave1916) and 3 species of Pseudocorynosoma (Ps. constrictum Van Cleave, 1918; Ps. anatarium Van Cleave, Reference Van Cleave1945; and Ps. tepehuanesi; García-Varela et al., Reference García-Varela, Hernández-Orts and Pinacho-Pinacho2017) have been recognized in both biogeographical regions of Mexico (García-Prieto et al., Reference García-Prieto, García-Varela, Mendoza-Garfias and Pérez-ponce de León2010; Alcántar-Escalera et al., Reference Alcántar-Escalera, García-Varela, Vázquez-Domínguez and Pérez-Ponce2013; García-Varela et al., Reference García-Varela, Hernández-Orts and Pinacho-Pinacho2017).
The current records indicate that P. brevis and Ps. constrictum are sympatrically distributed in Mexico and the USA. Adults of P. brevis have been associated with at least 5 fish-eating bird species from the family Ardeidae, including black-crowned night heron (Nycticorax nycticorax L.), little blue heron (Egretta caerulea L.), snowy egret (E. thula Molina), great egret (Ardea alba L.) and American bittern (Botaurus lentiginosus Rackett). Adults of Ps. constrictum have been documented in at least 8 waterfowl species from the family Anatidae, including green-winged teal (Anas crecca Gmelin), blue-winged teal (Spatula discors L.), cinnamon teal (A. cyanoptera Vieillot), Mexican duck (A. diazi Ridgway), gadwall (A. strepera L.), northern shoveler (A. clypeata L.), lesser scaup (Aythya affinis Eyton) and redheads (Ay. americana Eyton) (García-Prieto et al., Reference García-Prieto, García-Varela, Mendoza-Garfias and Pérez-ponce de León2010; García-Varela et al., Reference García-Varela, Pinacho-Pinacho, Sereno-Uribe and Mendoza-Garfías2013a, Reference García-Varela, Hernández-Orts and Pinacho-Pinacho2017). These definitive hosts are highly abundant in various biogeographical regions of Mexico and may play important roles in the phylogeographic structure of the populations of both studied species. During several surveys of parasites infecting aquatic birds in the Neotropical and Nearctic regions of Mexico, specimens of P. brevis and Ps. constrictum were obtained from their definitive hosts and characterized via an integrative taxonomic approach. In the present study, we generated and examined sequences of cytochrome c oxidase subunit I (cox1) from the mitochondrial DNA of specimens belonging to P. brevis and Ps. constrictum with the objective of studying the phylogeographic structure of both species of parasites from 6 biogeographic provinces (Trans-Mexican Volcanic, Pacific Lowlands, Veracruzan, Californian, Sierra Madre Occidental and Sonoran) in Mexico plus the Temperate Prairies biogeographical province.
Materials and methods
Specimen collection
Aquatic birds were collected between October 2006 and December 2021 in 12 localities across 6 biogeographic provinces (Transmexican Volcanic Belt, Pacific Lowlands, Veracruzan, Californian, Sierra Madre Occidental, and Sonoran) from Mexico, plus Teperate Praires biogeographical province from USA (Table 1; Figure 1). Birds were dissected within the following 4 h, and their viscera were placed in separate Petri dishes containing a 0.75% saline solution and examined under a dissecting microscope. The acanthocephalans recovered were washed in 0.75% saline solution and placed distilled water at 4°C overnight and subsequently preserved in 70% ethanol. Birds were identified using the field guide of Howell and Webb (Reference Howell and Webb1995).
Figure 1. Map of Mexico showing the sampled sites for the birds. Localities with a circle of blue and red colour were positive for the infection with Polymorphus brevis and Pseudocorynosoma constrictum respectively; localities correspond to those in Table 1.
Table 1. Specimen information, collection sites, host, locality, geographical coordinates, GenBank accession number for specimens studied in this work. The sample number for each locality corresponds with the same number in the Figure 1. Sequences in bold were generated in the current study
Morphological analyses
Selected adult acanthocephalans were gently punctured in the trunk with a fine needle, stained with Mayer’s paracarmine, destained in 70% acid ethanol, dehydrated in a graded ethanol series, cleared in methyl salicylate and mounted in Canada balsam. Specimens were examined using a compound microscope Leica DM 1000 LED equipped with bright field (Leica, Wetzlar, Germany). The acanthocephalans were identified by conventional morphological criteria following Petrochenko (Reference Petrochenko1958). In addition, descriptions of P. brevis and Ps. constrictum were consulted as needed (Van Cleave, Reference Van Cleave1945, Reference Van Cleave1945a). For scanning electron microscopy (SEM). Four adult specimens of each species were dehydrated with an ethanol series, critical point dried, sputter coated with gold, and examined with a Hitachi Stereoscan Model S-2469 N scanning electron microscope operating at 15 kV from the Instituto de Biología, Universidad Nacional Autónoma de México (UNAM). Adult specimens were deposited in the Colección Nacional de Helmintos (CNHE), Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City, under the numbers 5720, 5778, 5881, 6270 and 6271.
DNA isolation, amplification and sequencing
A total of 21 specimens, 11 identified morphologically as P. brevis and 10 as Ps. constrictum were placed individually in tubes and digested overnight at 56°C in a solution containing 10 mM Tris–HCl (pH 7.6), 20 mM NaCl, 100 mM Na2 EDTA (pH 8.0), 1% Sarkosyl, and 0.1 mg ml−1 proteinase K. Following digestion, DNA was extracted from the supernatant using the DNAzol reagent (Molecular Research Center, Cincinnati, Ohio) according to the manufacturer’s instructions. The cytochrome c oxidase subunit 1 (cox 1) of the mitochondrial DNA was amplified using the forward primer 5′-AGTTCTAATCATAA(R)GATAT(Y)GG-3′ and reverse primer 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ (Folmer et al., Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994). PCR reactions (25 μl) consisted of 10 μl of each primer, 2.5 μl of 10 × buffer, 2 mM MgCl2, and 1 U of Taq DNA polymerase (Platinum Taq, Invitrogen Corporation, São Paulo, Brazil). PCR cycling parameters for the molecular marker consisted of denaturation at 94°C for 1 min, 35 cycles of 94°C for 1 min, 40°C for 1 min and 72°C for 1 min, followed by a post-amplification incubation at 72°C for 10 min. Sequencing reactions were performed using ABI Big Dye (Applied Biosystems, Boston, Massachusetts) terminator sequencing chemistry, and reaction products were separated and detected using an ABI 3730 capillary DNA sequencer. Contigs were assembled and base-calling differences resolved using Codoncode Aligner version 12.0 (Codoncode Corporation, Dedham, Massachusetts) and submitted to GenBank dataset (Table 1).
Alignments, population genetic structure and historical demographic
Newly obtained sequences in the current research of P. brevis were aligned with 56 other sequences of P. brevis (DQ089717, HM636467, JX442194, EF467861, KC549447 − 497) downloaded from GenBank (Table 1), forming a data set of 67 sequences with 615 characters. New sequences of Ps. constrictum were aligned with other sequences of Ps. constrictum (EU267820, KX688132, JX202526 − 545), downloaded from GenBank (Table 1), forming a data set of 32 sequences with 655 characters. Sequences of each dataset were aligned separately using the software Clustal W with default parameters implemented in MEGA version 7.0 (Kumar et al., Reference Kumar, Stecher and Tamura2016). To analyse the molecular information in the framework of population genetics, we grouped individuals of P. brevis and Ps. constrictum into populations considering the biogeographic provinces (Transmexican Volcanic Belt, Pacific Lowlands, Veracruzan, Californian, Sierra Madre Occidental). A single specimen of Ps. constrictum from Sonora (locality 2 in Table 1and Figure 1) was analysed together with seven specimens from the Temperate Praires biogeographical province in the USA (locality 1 in Table 1 and Figure 1). Intrapopulation variation was summarized using standard statistics: number of haplotypes (H), number of segregating sites (S), haplotype diversity (Hd), nucleotide diversity (Pi) and average number of nucleotide differences (K), were all calculated using the program DnaSP v. 5. 10 (Rozas et al., Reference Rozas, Sánchez-delbarrio, Messeguer and Rozas2003). To examine haplotype frequency among the populations of P. brevis and Ps. constrictum a statistical network was constructed independently, using the program PopART with the median joining algorithm (Bandelt et al., Reference Bandelt, Forster and Röhl1999). The degree of genetic differentiation among the populations was estimated using the fixation indices Fst (Hudson et al., Reference Hudson, Boos and Kaplan1992), with the program Arlequin v.3.5 (Excoffier and Lischer, Reference Excoffier and Lischer2010). To investigate the population history and demography, Tajima’s D (Tajima, Reference Tajima1989) and Fu’s Fs (Fu, Reference Fu1997) test were calculated using DnaSP v. 5. 10 (Rozas et al., Reference Rozas, Sánchez-delbarrio, Messeguer and Rozas2003). The values were considered significant when the P-values were less than 0.05.
Results
Morphological identification
The body shape, somatic spine pattern in the trunk, proboscis hook number, proboscis shape, presence or absence of spines surrounding the genital pore and type of definitive host were compared to delineate the 2 species. For example, the acanthocephalans recovered from the intestines of 5 heron species showed similar morphological characteristics to those assigned to P. brevis by Van Cleave (Reference Van Cleave1945) and Alcántar-Escalera et al. (Reference Alcántar-Escalera, García-Varela, Vázquez-Domínguez and Pérez-Ponce2013), including an elongated cylindrical trunk with a single field of somatic spines on the anterior region of the trunk, a cylindrical proboscis with a swollen region, proboscis hooks arranged in 12–13 longitudinal rows of 17–19 hooks per row, a long neck, a double-walled proboscis receptacle, and four tubular cement glands in males (Figure 2A–C). The acanthocephalans recovered from the intestines of 8 waterfowl species presented morphological characteristics that matched those assigned to Ps. constrictum by García-Varela et al. (Reference García-Varela, Pinacho-Pinacho, Sereno-Uribe and Mendoza-Garfías2013a, Reference García-Varela, Hernández-Orts and Pinacho-Pinacho2017). These included a cylindrical trunk with a single field of somatic spines on the anterior region of the trunk, with slight constriction separating the anterior and posterior regions of the trunk, a cylindrical proboscis recovering with 16 longitudinal rows of 10 hooks each, and a cone-shaped neck and genital spines surrounding the genital pore (Figure 2D–F).
Figure 2. Scanning electron photomicrographs of Polymorphus brevis from Botaurus lentiginosus from San Quintin, Baja California, Mexico (locality 3 in Figure 1 and Table 1) and Pseudocorynosoma constrictum from Anas clypeata from Almoloya, Estado de México, Mexico (locality 17 in Figure 1 and Table 1). Adult male, whole worm (A, D); male anterior region (B, E); proboscis (C, F).
Population genetic structure and demographic analysis
The mitochondrial marker was successfully amplified for 11 P. brevis individuals and 10 Ps. constrictum individuals. The complete alignment of the cox1 dataset contained 67 P. brevis individuals with a total length of 615 bp, whereas the cox1 dataset of Ps. constrictum contained 32 individuals with a total length of 655 bp. No insertions or deletions were detected in any of the sequences, and when the sequences were translated into proteins, no stop codons were found. The haplotype network built for P. brevis did not show a phylogeographic structure for the 26 mtDNA haplotypes detected. The most frequent haplotype (H = 1) was shared with the Trans-Mexican Volcanic Belt, Pacific Lowlands and Californian biogeographical provinces (Figure 3A). The identified haplotypes were separated for a few substitutions from 1, 2, 3 and 7 (see Figure 3A). The haplotype diversity was high (Hd = 0.885), and the nucleotide diversity was low (pi = 0.00404) among the populations from the 4 biogeographic provinces sampled (Trans-Mexican Volcanic Belt, Pacific Lowlands, Veracruzan, and Californian). Neutrality tests (Tajima’s D, − 1. 977 and Fu’s FS, − 19.625) were negative for all regions (see Table 2), which indicates an excess of rare alleles greater than what would be expected under neutrality, suggesting a recent population expansion of P. brevis. The Fst values were estimated to assess genetic differentiation among the populations from the 4 biogeographic provinces analysed. Despite the large geographic distances, the Fst values were low, ranging from 0.01 to 0.08 (average 0.00963) (Table 3), indicating that the populations were poorly genetically differentiated from one another.
Figure 3. Haplotype network of samples of Polymorphus brevis, built with the gene cytochrome c oxidase subunit 1 (cox1) from mitochondrial DNA (A); host haplotype network (B). Each circle represents a haplotype, with size proportional to the haplotype’s frequency in the populations. Mutational steps are symbolized by dashes. biogeographic provinces, Trans-Mexican Volcanic Belt (TVM); Pacific Lowlands (PLN); Veracruzan (VER); Californian (CAL).
Table 2. Molecular diversity indices and neutrality tests calculated for cox1 data sets among the populations of Polymorphus brevis used in this study (n = number of sequences, H = number of haplotypes, S = number of segregating sites, hd = haplotype diversity, Pi = nucleotide diversity and K = average number of nucleotide differences). TVB = Transmexican Volcanic Belt; PL = Pacific Lowlands; VER = Veracruzan; CAL = Californian
Table 3. Pairwise fst values estimated for cox1. Significance level = 0.05. TVB = Transmexican Volcanic Belt; PL = Pacific Lowlands; VER= Veracruzan; CAL= Californian
The host haplotype network revealed that some adult specimens of P. brevis recovered from 4 definitive hosts, namely, the black-crowned night heron (N. nycticorax), little blue heron (E. caerulea), snowy egret (E. thula) and American bittern (B. lentiginosus), share the same haplotype (H = 1). In addition, 17 haplotypes from adult specimens were scattered throughout the network (Figure 3B). The paratenic hosts harboured 9 haplotypes that were found in fishes belonging to the Atherinidae, Cyprinidae and Goodeidae families, suggesting that these hosts can harbour and transmit diverse haplotypes to their definitive hosts (Figure 3B).
The haplotype network built for the species Ps. constrictum did not show a phylogeographic structure among the 22 mtDNA haplotypes detected. The most frequent haplotype (H = 1) was shared with the Trans-Mexican Volcanic Belt, Sierra Madre Occidental and Temperate Prairies biogeographical provinces (Figure 4A). The haplotypes were separated for a few substitutions. However, the H11, H13 and H15 haplotypes presented seven to 15 substitutions (see Figure 4A). The haplotype diversity was high (Hd = 0.954), and the nucleotide diversity was low (pi = 0.01035) among the populations from the 3 biogeographic provinces sampled (the Trans-Mexican Volcanic Belt, Sierra Madre Occidental and Temperate Prairies). Neutrality tests (Tajima’s D, − 1.866 and Fu’s FS, − 8.844) were negative (see Table 4), suggesting an excess of rare alleles compared with what would be expected under neutrality, which is consistent with a recent population expansion of Ps. constrictum. The Fst values were estimated to assess genetic differentiation among the populations from the 3 biogeographic provinces analysed. Despite the large geographic distances, the Fst values were low, ranging from 0.03 to 0.08 (average 0.02804), which indicates that the populations were poorly genetically differentiated from one another (Table 5).
Figure 4. Haplotype network of samples of Pseudocorynosoma constrictum, built with the gene cytochrome c oxidase subunit 1 (cox1) from mitochondrial DNA (A); host haplotype network (B). Each circle represents a haplotype, with size proportional to the haplotype’s frequency in the populations. Mutational steps are symbolized by dashes. Biogeographic provinces, Trans-Mexican Volcanic Belt (TVM); Sierra Madre Occidental (smoc); Temperate Prairies (TPR).
Table 4. Molecular diversity indices and neutrality tests calculated for cox1 data sets among the populations of Pseudocorynosoma constrictum used in this study (n = number of sequences, H = number of haplotypes, S = number of segregating sites, hd = haplotype diversity, Pi = nucleotide diversity and K = average number of nucleotide differences). TVB = Transmexican Volcanic Belt; SMOc = Sierra Madre Occidental; TPR = Temperate Praires
Table 5. Pairwise fst values estimated for cox1. Significance level = 0.05. TVB = Transmexican Volcanic Belt; SMOc = Sierra Madre Occidental; TPR = Temperate Praires
The host haplotype network shows that the 8 waterfowl species sampled, namely, the green-winged teal (A. crecca), blue-winged teal (S. discors), cinnamon teal (A. cyanoptera), Mexican duck (A. diazi), gadwall (A. strepera), northern shoveler (A. clypeata), lesser acaup (Ay. affinis) and redheads (Ay. americana) are able to harbour multiple haplotypes of Ps. constrictum. In addition, the most frequent haplotype (H = 1) was shared with three definitive host species (Figure 4B). The intermediate host, the amphipod (Hyalella azteca Saussure), also harbours multiple haplotypes (H1, H19, H22), which are dispersed throughout the network (Figure 4B).
Discussion
To the best of our knowledge, P. brevis and Ps. constrictum are 2 generalist species that use aquatic birds from the families Ardeidae (herons) and Anatidae (waterfowl) as definitive hosts and amphipods as intermediate hosts and are distributed sympatrically in Mexico and the USA (Amin, Reference Amin1988; García-Prieto et al., Reference García-Prieto, García-Varela, Mendoza-Garfias and Pérez-ponce de León2010; Alcántar-Escalera et al., Reference Alcántar-Escalera, García-Varela, Vázquez-Domínguez and Pérez-Ponce2013; García-Varela et al., Reference García-Varela, Pinacho-Pinacho, Sereno-Uribe and Mendoza-Garfías2013a, Reference García-Varela, Hernández-Orts and Pinacho-Pinacho2017). The species P. brevis was described from the American bittern (B. lentiginosus) from Baltimore, Maryland (Van Cleave, Reference Van Cleave1916). Since then, P. brevis has been recorded as adults in 6 fish-eating birds from Louisiana and Florida in the USA (see Amin, Reference Amin1988), as well as in 5 fish-eating birds from the Nearctic and Neotropical regions of Mexico (Alcántar-Escalera et al., Reference Alcántar-Escalera, García-Varela, Vázquez-Domínguez and Pérez-Ponce2013). In addition, cystacanths (larval form) of P. brevis have been found in 14 fish species from six families: Atherinidae, Cyprinidae, Goodeidae, Poeciliidae, Ictaluridae, Centrarchidae and Atherinopsidae (Amin, Reference Amin1988; Alcántar-Escalera et al., Reference Alcántar-Escalera, García-Varela, Vázquez-Domínguez and Pérez-Ponce2013). Our specimens identified as P. brevis agree morphologically with those previously assigned by Van Cleave (Reference Van Cleave1945) and Alcántar-Escalera et al. (Reference Alcántar-Escalera, García-Varela, Vázquez-Domínguez and Pérez-Ponce2013), including an elongated cylindrical trunk with a single field of somatic spines on the anterior region of the trunk, a cylindrical proboscis with a swollen region, and proboscis hooks arranged on 12–13 longitudinal rows of 17–19 hooks per row (see Figure 2A–C).
The type species Ps. constrictum was described from the surf scoter bird (Melanitta perspicillata L.) from Yellowstone, Wyoming, USA (Van Cleave, Reference Van Cleave1945a), and since then, Ps. constrictum has been recorded in more than 20 waterfowl species and is considered one of the most abundant and widely distributed species of polymorphid in wetlands from the Nearctic region (Van Cleave, Reference Van Cleave1945a, Farias and Canaris, Reference Farias and Canaris1986; García-Varela et al., Reference García-Varela, Pinacho-Pinacho, Sereno-Uribe and Mendoza-Garfías2013a, Reference García-Varela, Hernández-Orts and Pinacho-Pinacho2017). Our specimens identified as Ps. constrictum agree morphologically with those previously described by Van Cleave, (Reference Van Cleave1945a) and García-Varela et al. (Reference García-Varela, Pinacho-Pinacho, Sereno-Uribe and Mendoza-Garfías2013a) because it has somatic spines covering the majority of the anterior part of the trunk, with slight constriction separating the anterior and posterior regions of the trunk, and it has a cylindrical proboscis with a slightly swollen region covered with 16 longitudinal rows of 10 hooks each (see Figure 2D–F).
The intraspecific genetic divergence estimated in the present study among the 67 isolates of P. brevis and the 32 isolates of Ps. constrictum analysed ranged from 0.00% to 1.8% (average 0.5%) and from 0.00% to 3.9% (average 1.0%), respectively. These values of intraspecific genetic divergence are similar to those previously reported for isolates of polymorphid species such as Southwellina hispida (Van Cleave, 1925) Witenberg, 1932, a parasite of herons, gulls, cormorants, pelicans and hawks, which presented divergence values ranging from 0.00% to 1.5%; Hexaglandula corynosoma (Travassos, 1915), a specialist species that has been recorded as an adult only in the intestine of the yellow-crowned night-heron (Nyctanassa violacea L.), ranging from 0.00% to 2.6% (García-Varela et al., Reference García-Varela, López-Jiménez, González-García, Sereno-Uribe and Andrade-Gómez2023); and Andracantha sigma (Presswell, García-Varela et al., Reference García-Varela, Hernández-Orts and Pinacho-Pinacho2017), a parasite of seabirds and the Otago shag (Leucocarbo chalconotus Gray), spotted shag (Phalacrocorax punctatus Sparrman) and Otago little blue penguin, (Eudyptula novaehollandiae Forster) from New Zealand, which ranges from 0.00% to 0.32% (Presswell et al., Reference Presswell, García-Varela and Smales2018).
The haplotype network genealogy generated in this study was based on cox1 sequences from P. brevis and Ps. constrictum and did not show a clear phylogeographic structure. As a result, the haplotypes could not be grouped according to their biogeographical provinces, despite the presence of geographical barriers such as mountains (Sierra Madre Occidental and Oriental), lowlands, drylands, the Balsas depression and the central Trans-Mexican Volcanic Belt (Barrier et al., Reference Barrier, Velasquillo, Chavez and Gaulon1998; Ferrari et al., Reference Ferrari, Orozco‐Esquivel, Manea and Manea2012; Morrone et al., Reference Morrone, Escalante and Rodríguez-Tapia2017). Interestingly, the lack of population genetic structure detected in the current study agrees with previous phylogeographic studies with other polymorphid species that have a broad distribution. For example, Profilicollis altmani (Perry, 1942), associated with multiple species of definitive hosts (gulls, ducks, sanderlings, and common tern); Profilicollis novaezelandensis (Brockerhoff and Smales, Reference Brockerhoff and Smales2002), which has been found as adults in gulls (Larus spp.); and Southwellina hispida, associated with piscivorous birds throughout the world (see Brockerhoff and Smales, Reference Brockerhoff and Smales2002; Goulding and Cohen, Reference Goulding and Cohen2014; García-Varela et al., Reference García-Varela, López-Jiménez, González-García, Sereno-Uribe and Andrade-Gómez2023).
Despite the considerable geographic distances, the Fst values estimated among the populations of P. brevis and Ps. constrictum were very low (Tables 3 and 5), indicating limited genetic differentiation. This pattern can be explained by the migration patterns of the definitive hosts herons and waterfowl, which can facilitate gene flow across regions. In addition, the estimated values of Fu´s Fs and Tajima´s D were negative, indicating that the populations of both acanthocephalan species may have undergone recent population expansion. P. brevis and Ps. constrictum are considered generalist species because they parasitize a broad spectrum of aquatic birds, mainly those of the families Ardeidae (herons) and Anatidae (waterfowl), which inhabit freshwater systems, such as ponds, lakes, rivers and wetlands in the Nearctic region, extending from central Mexico to the northern USA. It is well known that amphipods serve as intermediate hosts to P. brevis and Ps. constrictum (Amin, Reference Amin1988; García-Varela et al., Reference García-Varela, Pinacho-Pinacho, Sereno-Uribe and Mendoza-Garfías2013a; Alcántar-Escalera et al., Reference Alcántar-Escalera, García-Varela, Vázquez-Domínguez and Pérez-Ponce2013). However, the main difference in the life cycles of the 2 species is the participation of the paratenic hosts: Freshwater fishes in the families Atherinidae, Cyprinidae, Goodeidae, Poeciliidae, Ictaluridae and Centrarchidae, which harbour the cystacanth (larval form), are key to the transmission of P. brevis to appropriate definitive hosts, whereas in the life cycle of Ps. constrictum, the infected amphipod (H. aztecae) exhibits notable behavioural and phenotypic shifts, including a bright orange spot in the haemocoel and altered photic behaviour, which increases predation by ducks (Bethel and Holmes, Reference Bethel and Holmes1973; Benesh et al., Reference Benesh, Duclos and Nickol2005; Duclos et al., Reference Duclos, Danner and Nickol2006).
Finally, the results of the present study allowed us to understand the genetic diversity and population genetic structure of two acanthocephalan species that parasitize a broad range of aquatic birds, mainly those of the families Ardeidae (herons) and Anatidae (waterfowl), which are distributed from central Mexico to the northern USA in the Nearctic region. Presumably, the biology and migration patterns of the definitive hosts, along with the involvement of paratenic hosts, may have played a key role in shaping the distribution of the haplotypes and the population genetic structure of the 2 acanthocephalan species studied.
Acknowledgements
We thank Berenit Mendoza for her help with the use of the SEM unit and Laura Márquez and Nelly López Ortiz from LaNabio for their help during the sequencing of the DNA fragments.
Author contributions
ALSU, MTGG, ALJ, MPOO and MGV conceived and designed the study. ALSU, MTGG, ALJ, YAP, and MPOO conducted data gathering. ALJ and MTGG performed phylogeographic analyses. ALSU, MTGG, ALJ, YAP, MPOO and MGV wrote and edited the article.
Financial support
This research was supported by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT-UNAM) IN204425 to MGV. ALJ thanks the Dirección General de Asuntos de Personal Académico (DGAPA-UNAM) Mexico for the Postdoctoral Fellowship granted.
Competing interests
The authors declare there are no conflicts of interest.
Ethical standards
The sampling in this work complies with the current laws and animal ethics regulations of Mexico. Specimens were collected under the Cartilla Nacional de Colector Científico (FAUT 0202) issued by the Secretaría del Medio Ambiente y Recursos Naturales (SEMARNAT), to M.G.V.
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