Redescription and molecular characterization of Baruscapillaria spiculata (Nematoda: Capillariidae) parasitizing the Neotropic cormorant Phalacrocorax brasilianus from two Argentinian lagoons

Two species of intestinal Capillariidae were hitherto known from the Neotropic cormorant Phalacrocorax brasilianus, Baruscapillaria spiculata (Freitas, 1933), and Baruscapillaria appendiculata (Freitas, 1933). The original descriptions are very short and brief, and further reports of both species are scarce and/or confusing. This paper provides a morphological redescription and molecular characterization, based on the partial 18S rDNA gene, of B. spiculata specimens parasitizing the Neotropic cormorant in two continental lagoons from Buenos Aires province, Argentina. Both morphological and morphometrical differences between B. spiculata and B. appendiculata are highlighted on the examination of available type material. Additionally, two previous reports of B. appendiculata from Mexico and Brazil are discussed. A phylogenetic analysis conducted on specimens of B. spiculata and 46 other capillariid isolates available from the GenBank demonstrated a sister-taxon relationship between our specimens and the type species of Baruscapillaria. But, at the same time, significant genetic distances between both taxa showed an interesting variability of the genus Baruscapillaria. The probable division of this genus into multiple genera could probably be confirmed through integrative studies including more species.

Although a few morphological studies of B. appendiculata exist (Moravec et al. 2000;Monteiro 2006), there is no study including a detailed description of B. spiculata since the first brief study by Freitas 1933a. The original descriptions of both B. spiculata and B. appendiculata are short and lacking several morphological details which may have caused confusion in identifying the capillariid species parasitizing cormorants. For instance, studies by Monteiro (2006) have reported B. appendiculata from Ph. brasilianus in Brazil, but the morphological description seems to belong to the species B. spiculata.
Hence in this study, we aimed to describe morphological details of B. spiculata parasitizing Ph. brasilianus in Buenos Aires province, Argentina, in order to clarify the species status. Also, a molecular characterization of this species using the partial 18S rDNA gene was performed.

Sampling
Nine Ph. brasilianus individuals from the San Miguel del Monte Lagoon (SMML) (35°27′ 35.46″ S; 58°48′ 11.05″ W) and another 7 ones from the Chis-Chis Lagoon (CCL) (35°45′ 43.76″ S, 57°57′ 6.75″ W), both from the Buenos Aires province, Argentina, were collected as dead animals during 2014-2017. All birds were necropsied and their complete digestive tracts were kept frozen at −20°C until they could be examined. Digestive tracts were analyzed for helminths and capillariid nematodes were isolated from cloacae. Nematodes for morphological study were fixed in 5% formalin or 70% ethanol. Specimens used to amplify specific DNA fragments from both SMML and CCL were fixed in absolute ethanol.

Morphological study
Thirty-six adult capillariid nematodes from Ph. brasilianus were examined. Nematodes were cleared in lactophenol and studied with both light microscopes Leica DM2500® (Wetzlar, Germany) with a drawing attachment, and Olympus BX51® (Tokyo, Japan) with a camera QImaging® Go-3. Types of Capillaria spiculata Freitas, 1933 (CHIOC 2833 and3079) were studied with a light microscope Carl Zeiss Axiophot equipped with a Canon Power Shot S80 camera at the Coleção Helmintológica do Instituto Oswaldo Cruz, Rio de Janeiro, Brazil .
Some specimens were processed for scanning electron microscopy, dried by the critical point method, and observed using a JEOL/JSMT 6360 LV® Scanning Electron Microscope (JEOL Ltd, Tokyo, Japan). Several characters considered diagnostic for capillariid nematodes (Freitas et al. 1959;Moravec 1982) were analyzed. Mean measurements are expressed in millimeters, except otherwise indicated, usually as the range followed by the mean in parentheses. Prevalence of infection was calculated for worm specimens recovered from both sampling sites according to Bush et al. (1997). Voucher specimens were stored in 70% ethanol and deposited in the Helminthological Collection of Museo de La Plata, La Plata, Argentina.
DNA extraction, amplification, and sequencing DNA of 2 male and 10 female capillariid specimens from both sampling sites, kept in 70 % alcohol, was extracted using a Promega Wizard® Genomic DNA Purification Kit according to the manufacturer's instructions. The PCR amplification of overlapping 18S ribosomal RNA gene (18S rDNA) segments was performed using different primer combinations of forward and reverse universal eukaryotic primers as previously described by Sato et al. (2010) and Tamaru et al. (2015): (1) NSF4/18 and 18S-1192R/20, (2) NSF4/18 and NSR1438/20, (3) NSF573/19 and NSR1787/18, and (4) NSF573/19 and SSU18R. The DNA polymerase used was GoTaq® Green Master Mix following the manufacturer's instructions and PCRs in 20-25-μl reaction solution were conducted in a thermal cycler Eppendorf AG (Mastercycler® Nexus) using the following cycling protocol: 2 min at 94°C, then 40 cycles at 94°C for 30 s, 64 or 62°C for 30 s, and 72°C for 90 s, followed by a final extension at 72°C for 7 min. The PCR products were purified using a Promega Wizard® SV Gel and PCR Clean-Up System; and sequenced by Macrogen Inc. (Seoul, Korea) directly with the primers for amplification. The nucleotide sequences reported in the present study are available from the DDBJ/EMBL/GenBank databases under the accession numbers MT068208, and MT068209.

Phylogenetic analysis
Contrasting 18S rDNA sequences from 46 capillariid isolates with more than 80% identity were analyzed using the Standard Nucleotide BLAST from GenBank. The 18S rRNA sequences of capillariid nematodes obtained in this study were optimized by eye using Geneious R11 under free-trial (http://www.geneious.com) (Drummond et al. 2012), and compared with other capillariid sequences from GenBank. Alignments were assembled using the online version of MAFFT v.7 (Katoh and Standley 2016). The online Gblocks v0.91 (Castresana 2000;Talavera and Castresana 2007) was used to detect and exclude from the analyses the hypervariable regions in the 18S rRNA.
The best partitioning scheme and substitution model for the 18S rRNA was chosen under the Bayesian Information Criterion (BIC) (Schwarz 1978) using the "greedy" search strategy in Partition Finder v.1.1.1 (Lanfear et al. 2012). The appropriate nucleotide substitution model implement for the 18S rRNA matrix resulting after Gblock TVMef+I+G. The phylogenetic analysis was performed by Bayesian Inference (BI) through MrBayes v. 3.2.1 (Ronquist et al. 2012) using the compared capillariid sequences and Trichuris suis (EU790668) as outgroup. The phylogenetic trees were reconstructed using two parallel analyses of Metropolis-Coupled Markov Chain Monte Carlo (MCMC) for 10 million generations each to estimate the posterior probability (PP) distribution. Topologies were sampled every 1000 generations, once the average standard deviation of split frequencies was less than 0.01. The robustness of the clades was assessed using Bayesian PP, where PP > 0.95 was considered strongly supported. A majority consensus tree with branch lengths was reconstructed after discarding the first 25% sampled trees. The final trees were visualized in FigTree software v 1.4.2 (Rambaut 2009;Rambaut et al. 2018). The proportion (p) of absolute nucleotide sites (p-distance) (Nei and Kumar 2000) was obtained to compare the genetic distance among and between linages using Mega X, with bootstrap method (1000 replicates) and with nucleotide substitution (transition + transversions) uniform rate (Kumar et al. 2018). In addition, the evolutionary history was inferred by using the Maximum Likelihood (ML) method based on the Jukes-Cantor model (Jukes and Cantor 1969). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis involved 52 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 948 positions in the final dataset. Evolutionary analyses were conducted on MEGA X.

Results
Baruscapillaria spiculata (Freitas, 1933) Moravec 1982 =Capillaria spiculata Freitas, 1933 Morphological description General (based on 18 males and 18 females from SMML and CCL): Body long and thin. Maximum body width at esophago-intestinal junction. Males smaller than females. Anterior end narrow and rounded. Oral aperture terminal, slit-like, oriented dorsoventrally. Nerve ring barely visible situated mostly within proximal fifth of muscular esophagus. Dividing line between muscular esophagus and stichosome not always observed (Fig. 1a). Stichosome composed of single row of stichocytes subdivided into 9-11 annuli, provided with large nuclei (Fig. 1b, c). Two medium-sized glandular cells or pseudo-coelomocytes observed at esophago-intestinal junction ( Fig. 1c). Two distinct lateral bacillary bands present ( Fig. 1d). Male (measurements in Table 1): Spicule well sclerotized, with proximal end widely expanded and distal end rounded (Fig. 2a,d). Spicular sheath bearing ornamentations with 4 distinct sections with a regular pattern: proximal section with annulations fine and compact (Fig. 2a); second section with annulations wide and reticulate (Fig. 2b); third section with annulations wide and loose, sometimes oblique to the longitudinal axis giving the appearance of a spiral lining (Fig. 2c); fourth and distal section with annulations fine and compact becoming wider and looser when the spicular sheath is extruded ( Fig. 2d-f). Cloacal opening subterminal (Fig. 2e, f). Caudal end with well-developed membranous bursa composed of one dorsal and two lateroventral lobes, these latter bearing one large papilla each. Dorsal lobe shorter than lateroventral ones (Fig. 2c, d). Caudal lateral alae absent.
Female (measurements in Table 2): Vulvar appendage present in all females, arising basally as a protrusion of the anterior vulvar lip and continuing as a heart-shaped cuticular fold (Fig 1c, d). Oval thick-walled eggs with polar plugs protruding in immature eggs, less protruding in fully mature eggs. Content of mature eggs uncleaved. Posterior extremity rounded. Anus subterminal.

Taxonomic summary
Host: Phalacrocorax brasilianus (Gmelin, 1789) (type host). Remarks: the specimens studied in this work strongly resemble B. spiculata as described by Freitas (1933a). The morphology of the membranous bursa, the spicule measurements, the distance between the vulva and the esophago-intestinal junction, and the site of infection allowed us to identify our specimens as B. spiculata rather than to B. appendiculata (Tables 1 and 2). Freitas (1933a) did not describe in details the spicular sheath ornamentation of B. spiculata but he mentions a "spiral striation," in contrast to the spicular sheath of B. appendiculata, which is described as smooth (Freitas 1933b). The third section of the spicular sheath of our specimens exhibited wide and loose annulations sometimes oblique to the main axis of the spicule giving the appearance of a spiral lining. Moreover, the specimens of C. spiculata examined from the CHIOC (type male, Fig. 3a-d) showed the same pattern of ornamentation on the spicular sheath-four distinct sections with a regular pattern-present in our specimens. Based on all these observations, we identified the capillariids present in SMML and CCL as B. spiculata.

Molecular characterization and phylogenetic analysis
Only two 18S rDNA consensus sequences of 1623 and 1624 bp-isolates MT068208 and MT068209, respectively-were obtained from two specimens of Baruscapillaria spiculata from SMML. The amplicons from CCL specimens failed on sequencing. Both isolates showed 99.45 % identity among each other (1615/1624), and only nine nucleotide substitutions were observed at different positions. The pairwise genetic distance among both isolates was 0.1 % (Table 3). Consequently, these specimens can be considered belonging to the same species.
Available molecular characterizations of Baruscapillaria species only concerned the type species, Baruscapillaria obsignata (Madsen, 1945). Therefore, we conducted a phylogenetic analysis including the newly obtained sequences of B. spiculata in order to confirm the taxonomic status and generic assignment of this latter species. With this aim, we largely relied on the work of Tamaru et al. (2015), and that of Sakaguchi et al. (2020) who compared several 18S rDNA sequences of different capillariid species parasitizing mainly birds and mammals. The BLAST searching revealed that our Fig. 1 Baruscapillaria spiculata (Freitas, 1933)   isolates differed from the other sequences available in the GenBank. The closest matches were species of the family Capillariidae (80-96% identity) with a sequence length coverage greater than 80%. The top matches corresponded to sequences of Baruscapillaria obsignata (GenBank Acc. Nos. LC052336 and LC052337). The phylogenetic analysis based on 46 sequences of 18S rDNA from 20 Capillariidae species was performed by BI and ML producing nearly identical well-resolved topologies (Fig. 4). Pairwise DNA analyses revealed that the interspecific variability among different capillariid sequences expressed as pairwise genetic distance (p-distance) ranged from 0.3 to 9.0% (Table 3). Isolates MT068208 and MT068209 formed a wellsupported clade (Clade B, bootstrap 100) with both isolates of B. obsignata parasitizing chickens in Kagoshima, Japan, and captive swans in Yamaguchi, Japan, respectively (Tamaru et al. 2015).

Discussion
As stated, Baruscapillaria spiculata and B. appendiculata are the only two species of intestinal Capillariidae described from the Neotropic cormorant. Reports of both species after their original descriptions are scarce. Fedynich et al. (1997) reported B. spiculata parasitizing both cormorants Ph. brasilianus and Phalacrocorax auritus (Lesson, 1831) from Texas (USA), although their report is not accompanied by either a description or illustration. Instead, there are a few studies available for B. appendiculata (Moravec et al. 2000;Monteiro 2006;Monteiro et al. 2011). However, the morphological and morphometric descriptions described in these reports do not agree with the original description of B. appendiculata, which causes confusion in the discrimination of these two species (Tables 1 and 2). For example, in the work of Moravec et al. (2000), morphological and metrical differences with respect to the Fig. 2 Baruscapillaria spiculata (Freitas, 1933)   original description of B. appendiculata are evident which renders, in our opinion, the report of B. appendiculata from Mexico rather dubious. The specimens described by Monteiro (2006) from Brazil seem to agree with B. spiculata rather than with B. appendiculata (Tables 1 and 2). Monteiro et al. (2011) again listed B. appendiculata, without a description in a survey of helminths parasitizing the Neotropic cormorant, although it is clear that the specimens concerned were those described in 2006. Redescription of B. appendiculata is necessary to solve this problem but the types of the taxon originally described as Capillaria appendiculata Freitas, 1933, deposited in the CHIOC (Acc. Numb. 7469) are lost at present, and then unavailable for comparative purposes. In absence of the type material, any attempt to identify this species should be based on the original description by Freitas (1933b). At the same time, a significant sampling effort of the type host and type locality, as well as a thorough taxonomic work should be done in order to increase the chances of finding the species originally described as C. appendiculata (Freitas 1933b) As mentioned above, the combination Ornithocapillaria appendiculata was used previously by some authors (Moravec et al. 2000;Monteiro 2006;Monteiro et al. 2011) The genus Ornithocapillaria Baruš and Sergeeva, 1990 was erected to gather some species of avian capillariids until then included in Baruscapillaria. Baruš and Sergeeva (1990a) stated that the main feature differentiating Ornithocapillaria and Baruscapillaria was the shape of the processes on the posterior end of male body, distinctly rounded and more caudally orientated in Baruscapillaria species; conical and more laterally orientated in Ornithocapillaria species. In addition, the membra nous p seud obursa in males i s l arg er i n Ornithocapillaria than in Baruscapillaria. Other main  Table 3 Pairwise genetic distance (%) from 18S ribosomal RNA gene sequences (18S rDNA) among specimens of Baruscapillaria spiculata (Freitas, 1933) Okulewicz (1993) considered unjustified the separation between both genera and synonymized Ornithocapillaria with Baruscapillaria. This author, studying several species of capillariids from Palearctic birds, observed that the main characters argued to separate Baruscapillaria and Ornithocapillaria could be found in species attributed to either genus. She also remarked that the original definition of Ornithocapillaria included only non-specific features and even characters that may be considered intraspecific such as the shape and inclination of the lateral processes supporting the pseudobursa. Moreover, Okulewicz (1993) stated that the number, position and size of bacillary bands lack a diagnostic value at a generic level assuming that most bird capillariids possess four bacillary bands: two lateral (wide), one ventral and one dorsal (narrower). Moravec et al. (2000), based on the presence of a vulvar appendage and the morphology of the male caudal end, transferred three species parasitizing cormorants from Baruscapillaria to Ornithocapillaria, and proposed the combinations Ornithocapillaria appendiculata, Ornithocapillaria carbonis (Rudolphi, 1819), and Ornithocapillaria phalacrocoraxi (Borgarenko, 1975). However, shortly after Moravec et al. (2001) adopted the synonymy of Ornithocapillaria with Baruscapillaria proposed by Okulewicz (1993), and the combinations B. appendiculata and Baruscapillaria carbonis were readily reported, especially by some European authors Frantová 2001;Kanarek and Zaleśny 2014;Moravec and Scholz 2016).
All species shown in Fig. 4 clustered robustly according to the generic arrangement of Moravec (1982). The same was observed in the surveys of Tamaru et al. (2015), and Sakaguchi et al. (2020). In these latter works, the genus Baruscapillaria was only represented by isolates of B. obsignata since until then no sequences were available for other species of the genus. The present work brings, as expected, a sister-taxon relationship between the isolates of B. obsignata and B. spiculata.
The pairwise genetic distance between our isolates and B. obsignata ranged between 1.2 and 1.4%. These distances may be seen as relatively high considering that most distances between species belonging to the same genus were usually lower than 1% (Table 3, Fig. 4). There were as well some avian Capillaria spp. which hold greater genetic distances to each other compared with the mean distances observed between species belonging to the same genus (Table 3, Fig. 4). The same pattern was remarked by Tamaru et al. (2015) and Sakaguchi et al. (2020) for species of avian Capillaria which could not be easily discriminated on morphological grounds, although their genetic characterization of 18S rDNA clearly differentiated them from one another (Sakaguchi et al. 2020). These distances are then not completely unexpected, since Moravec (1982) remarked that further studies will apparently result in breaking up the present genus Capillaria into additional independent genera. Similarly, Okulewicz (1993) considered the genus Baruscapillaria as the most heterogeneous and speciose among the bird capillariids and concluded that it seems to require revision. Therefore, we largely agree with Sakaguchi et al. (2020) that integrative approaches are highly recommended in capillariid worms.
Indeed, thorough morphological examination is necessary and should be accompanied with other approaches including different molecular genetic analysis and assessment of the geographical distribution and host ranges of multiple species. Variability within Baruscapillaria and its probable division into more genera should also be confirmed with the inclusion of more species.