Ancylostoma ailuropodae n. sp. (Nematoda: Ancylostomatidae), a new hookworm parasite isolated from wild giant pandas in Southwest China

Background Hookworms belonging to the genus Ancylostoma (Dubini, 1843) cause ancylostomiasis, a disease of considerable concern in humans and domestic and wild animals. Molecular and epidemiological data support evidence for the zoonotic potential among species of Ancylostoma where transmission to humans is facilitated by rapid urbanization and increased human-wildlife interactions. It is important to assess and describe these potential zoonotic parasite species in wildlife, especially in hosts that have physiological similarities to humans and share their habitat. Moreover, defining species diversity within parasite groups that can circulate among free-ranging host species and humans also provides a pathway to understanding the distribution of infection and disease. In this study, we describe a previously unrecognized species of hookworm in the genus Ancylostoma in the giant panda, including criteria for morphological and molecular characterization. Methods The hookworm specimens were obtained from a wild giant panda that died in the Fengtongzai Natural Reserve in Sichuan Province of China in November 2013. They were microscopically examined and then genetically analyzed by sequencing the nuclear internal transcribed spacer (ITS, ITS1-5.8S-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes in two representative specimens (one female and one male, FTZ1 and FTZ2, respectively). Results Ancylostoma ailuropodae n. sp. is proposed for these hookworms. Morphologically the hookworm specimens differ from other congeneric species primarily based on the structure of the buccal capsule in males and females, characterized by 2 pairs of ventrolateral and 2 pairs of dorsolateral teeth; males differ in the structure and shape of the copulatory bursa, where the dorsal ray possesses 2 digitations. Pairwise nuclear and mitochondrial DNA comparisons, genetic distance analysis, and phylogenetic data strongly indicate that A. ailuropodae from giant pandas is a separate species which shared a most recent common ancestor with A. ceylanicum Looss, 1911 in the genus Ancylostoma (family Ancylostomatidae). Conclusion Ancylostoma ailuropodae n. sp. is the fourth species of hookworm described from the Ursidae and the fifteenth species assigned to the genus Ancylostoma. A sister-species association with A. ceylanicum and phylogenetic distinctiveness from the monophyletic Uncinaria Frölich, 1789 among ursids and other carnivorans indicate a history of host colonization in the evolutionary radiation among ancylostomatid hookworms. Further, phylogenetic relationships among bears and a history of ecological and geographical isolation for giant pandas may be consistent with two independent events of host colonization in the diversification of Ancylostoma among ursid hosts. A history for host colonization within this assemblage and the relationship for A. ailuropodae n. sp. demonstrate the potential of this species as a zoonotic parasite and as a possible threat to human health. The cumulative morphological, molecular and phylogenetic data presented for A. ailuropodae n. sp. provides a better understanding of the taxonomy, diagnostics and evolutionary biology of the hookworms.

(Continued from previous page) Conclusion: Ancylostoma ailuropodae n. sp. is the fourth species of hookworm described from the Ursidae and the fifteenth species assigned to the genus Ancylostoma. A sister-species association with A. ceylanicum and phylogenetic distinctiveness from the monophyletic Uncinaria Frölich, 1789 among ursids and other carnivorans indicate a history of host colonization in the evolutionary radiation among ancylostomatid hookworms. Further, phylogenetic relationships among bears and a history of ecological and geographical isolation for giant pandas may be consistent with two independent events of host colonization in the diversification of Ancylostoma among ursid hosts. A history for host colonization within this assemblage and the relationship for A. ailuropodae n. sp. demonstrate the potential of this species as a zoonotic parasite and as a possible threat to human health. The cumulative morphological, molecular and phylogenetic data presented for A. ailuropodae n. sp. provides a better understanding of the taxonomy, diagnostics and evolutionary biology of the hookworms.
Recent molecular-based genetic and epidemiological investigations have shown that among certain wild or domestic animal-derived species of Ancylostoma, A. ceylanicum is becoming the second most common hookworm found to infect and complete its life-cycle in humans [12,[14][15][16][17][18]. Similar transmission and crossinfection cases have been reported for other congeneric species, notably A. caninum [12,19,20] and A. braziliense [12]. Such situations highlight the public health significance of hookworm infection and the necessity to assess their prevalence and distribution, and to identify their wildlife hosts. This has become especially important for wildlife hosts that may have recently adapted to the human environment due to rapid urbanization [14,21] leading to increased interactions with people in conservation centers and zoological gardens constructed for endangered and valuable animals [22]. Regrettably, little attention has been broadly paid to the species of Ancylostoma because of a limited understanding of their diversity, abundance and distribution and the difficulty in etiological and epidemiological sampling in the wild [12,14].
The giant panda, Ailuropoda melanoleuca (David), one of the most endangered and rare species of China, is regarded as one of the preeminent species for wildlife conservation in the world. Higher taxonomic status for these enigmatic carnivorans had been unresolved, until relatively recent decisions that unequivocally placed giant pandas among the Ursidae (e.g. [23][24][25][26]). Wild giant pandas currently inhabit six small mountain ranges of China i.e. Qinling, Minshan, Qionglai, Daxiangling, Xiaoxiangling and Liangshan (Fig. 1), with an estimated population size of~1,864 [27][28][29][30]. Since the 1950s, numerous natural reserves, conservation centers, research bases and zoological gardens were specifically established by the Chinese government to protect this threatened species [31]. Some of these wild giant pandas have become closely associated with humans as they are housed for artificial breeding and conservation and biological investigations. Also, some pandas have been displayed publically as the 'messenger of peace and friendship' around the world [32]. Although ecological, genetic and etiological studies have shown that the panda faces the threat of extinction due to habitat loss, poor reproduction and low resistance to infectious diseases [33,34], recent surveys strongly indicate that parasitic infections represent the leading health threat to giant pandas of China [35][36][37][38][39][40].
Hookworm parasites have been frequently observed in the intestines of wild dead giant pandas since 1995 [28] and the first record, attributed to a species of Ancylostoma, was reported by Zhang et al. in 2005 [41]. However, detailed morphological descriptions, determination of taxonomic status and indicators of pathogenicity of the Ancylostoma sp. derived from giant panda are lacking. The recent collection of parasites from a wild giant panda that died in the Fengtongzai Natural Reserve in Sichuan Province of China resulted in the recovery of fresh Ancylostoma specimens and provided an opportunity to fill some of these gaps in our knowledge. We have used DNA sequence and morphological analysis, applying clear species criteria established in a phylogenetic context [42], to recognize and describe a previously unknown hookworm species from the giant panda. A putative sister-species relationship with the 'anthropozoonotic' A. ceylanicum suggests a possible zoonotic risk for transmission and infection to humans.

Parasite collection and microscopic examination
In November 2013, a wild female giant panda was found dead in the Fengtongzai Natural Nature Reserve, Sichuan Provence of China (Fig. 1). After a routine necropsy, seventeen hookworm specimens (seven males and ten females) were collected from the small intestine under the Scientific Procedures Premises License for the College of Veterinary Medicine, Sichuan Agricultural University (Sichuan, China). In addition, parasite eggs were isolated from the intestinal content by the centrifuge-flotation method using saturated MgSO 4 [43]. After washing in physiological saline, the hookworm specimens were either directly fixed in Berland's fluid (95% glacial acetic acid and 5% formaldehyde) for morphological analysis or stored in 70% ethanol for subsequent molecular profiling. For morphology, the hookworms were identified to the genus level on the basis of the existing taxonomic keys and descriptions of Ancylostoma spp. (e.g. [44]). In brief, the worms (n = 15; 6 males and 9 females) were prepared as temporary whole mounts in glycerin after clearing in lactophenol and examined under both dissecting and light microscopy at magnifications of 10-40× and 40-200×, respectively; male and female specimens were characterized morphologically including photo-micrographic imaging and morphometrics. Measurements are given in micrometres (μm) unless specified otherwise and presented with the range followed by the mean within parentheses. In addition, some key characteristics of the adults were drawn with the aid of serial photographs for morphological comparison and differentiation from other related species. These specimens including the type-series and vouchers for molecular analyses have been deposited in the Department of Parasitology, Sichuan Agricultural University (accession numbers code GYY-XY).

Molecular profiles and phylogeny
For molecular analysis, two adult specimens of Ancylostoma sp. (one female and one male; sample codes FTZ1 and FTZ2, respectively) preserved in 70% ethanol were air-dried and their mid-body regions (~1 cm) were excised individually for extraction of genomic DNA using the Universal Genomic DNA Extraction Kit (TaKaRa, Dalian, China) according to the manufacturer's protocol. The cephalic and caudal extremities of each specimen were retained as archived vouchers. The DNA extract was used as template for PCR amplifications at the nuclear internal transcribed spacer ITS1-5.8S-ITS2 region (734 bp) and mitochondrial cytochrome c oxidase subunit 1 (cox1) locus (393 bp) using primer pairs designed based on the alignments of the relatively conserved regions of the congeneric species A. ceylanicum, A. caninum, and A. duodenale in GenBank. Two PCR primer sets were as follows: ITS1-5.8S-ITS2, forward: 5′-GTC GAA GCC TTA TGG TTC CT-3′ and reverse: 5′-TAA CAG AAA CAC CGT TGT CAT ACT A-3′; cox1, forward: 5′-ATT TTA ATT TTG CCT GCT TTT G-3′ and reverse: 5′-ACT AAC AAC ATA ATA GGT ATC ATG TAA-3′. The PCR reactions contained~20 ng of genomic DNA were performed in 50-μl reaction volumes containing 25 μl 2× Phusion High-Fidelity PCR Master Mix (Finnzymes OY, Espoo, Finland), 3 μl gDNA, 3 μL of each primer and 16 μl of ddH 2 O. PCR cycling conditions carried out in a Mastercycler Gradient 5331 thermocycler (Eppendorf, Germany) were an initial denaturation at 95°C for 5 min; then for ITS1-5.8S-ITS2, 35 cycles of 95°C for 30 s, 39.8°C for 30 s, and 72°C for 45 s; but for cox1, 35 cycles at 95°C for 30 s, 44.1°C for 30 s, and 72°C for 30 s; followed by a final step at 72°C for 10 min. For each amplification, samples without parasite gDNA and host DNA as negative controls were also included. All PCR products were examined on agarose (1%) gels to verify that they represented the target bands. The corrected gel-isolated amplicons were column-purified and sequenced in both directions using terminator-based cycle sequencing with BigDye chemistry (Applied Biosystems, Foster City, CA, USA) on an ABI 3730 DNA sequencer (Applied Biosystems) in TaKaRa Biotechnology Co. Ltd. (Dalian, China). To ensure maximum accuracy, each amplicon was sequenced three times independently. The consensus sequences were utilized for the following bioinformatic analyses and added to GenBank under the accession numbers KP842923 (FTZ1) and KP842924 (FTZ2) for ITS1-5.8S-ITS2 and KP842921 (FTZ1) and KP842922 (FTZ2) for cox1.
Sequences of ITS1-5.8S-ITS2 and cox1 of Ancylostoma sp. in the present study were separately aligned with reference sequences from closely related species (Table 1) [46], Uncinaria sp., and Bunostomum phlebotomum (Railliet, 1900), using the Clustal X 1.83 program [47]. During the procedure, the nucleotide alignment of cox1 was further adjusted by a codon-guided protein alignment. Given the presence of the ambiguous regions within these alignments, an online version of GBlocks (http://molevol.cmima.csic.es/castresana/Gblocks_server. html) was also introduced here. After refining the alignments using Gblocks, the sequence datasets were used for phylogenetic analyses using both maximum parsimony (MP) (PAUP* 4.10b [48]) and Bayesian inference (BI) methods (MrBayes 3.2 [49]). In the MP analysis, heuristic searches were executed by branch-swapping utilizing tree-bisection-reconnection (TBR) algorithm and 1,000 random-addition sequence replicates with 10 trees held at each step, and finally the optimal topology with bootstrapping frequencies (BF) was obtained using Kishino-Hasegawa, as described previously [50]. For the BI analysis, the nucleotide substitution model GTR + I + G was determined using the Bayesian Information Criteria (BIC) test in jModeltest v. 2.1.6 [51], and the trees were constructed employing the Markov chain Monte Carlo (MCMC) method (chains = 4) over 100,000 (cox1) or 1,000,000 (ITS1-5.8S-ITS2) generations with every 100th (cox1) or 1000th (ITS1-5.8S-ITS2) tree being saved; when the average standard deviation of the split frequencies reduced to less than 0.01, 25% of the first saved trees were discarded as "burn-in" and the consensus (50% majority rule) trees were inferred from all remaining trees and further plotted in TreeviewX (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html), Ancylostoma duodenale - A. duodenale - Hu et al. [67]  with nodal supports expressed as posterior probabilities (PP). The livestock hookworm B. phlebotomum was used as outgroup reference and included in each phylogenetic analysis. Paralleled to the phylogenies, among the genus Ancylostoma the new hookworm species coupled with A. ceylanicum, A. caninum, A. duodenale and A. tubaeforme was also subjected to detection of synonymous and non-synonymous mutations in the mitochondrial cox1 gene using their corresponding protein sequences, followed by determination of genetic distances between them using a distance matrix based on the maximum composite likelihood model in MEGA [52].

Description
General. Slender, relatively small nematodes of white coloration in life (Fig. 2a). Body cylindrical, tapering toward cephalic and caudal extremities with fine transversely striated cuticle; head oriented dorsally in males and females. Buccal capsule widening posteriorly to prominent oral aperture, possessing two pairs of ventrolateral teeth and two pairs of triangular dorsolateral teeth (Fig. 2b-f). Ventrolateral teeth vary in size and shape, with small, subaduncate inner and large triangular outer teeth extending dorsally. Dorsal gland well developed, associated with rodlike oesophagus, slightly swollen posteriorly, terminating in a lobed valve at junction with intestine ( Fig. 4a, b). Nerve-ring at midlevel of oesophagus. Cervical papillae well developed, conical, situated posterior to level of nerve-ring. Excretory pore opens at level between cervical papillae and nerve-ring (Fig. 5a1, 2).  (Fig. 5a6).

Remarks
Ancylostoma ailuropodae n. sp. is established based on comparisons to available descriptions among congeners in the global fauna [6,7,9,10,[54][55][56][57][58][59][60][61][62]. Ancylostoma ailuropodae is unequivocally differentiated from congeners by structural characteristics of male and female specimens including body size, arrangement, number and dimensions of buccal teeth and shape of the buccal capsule, and in males by the configuration of the dorsal ray and bursa and lengths of spicules and gubernaculum, respectively (see Table 2 and Fig. 5b  Furthermore, the shape of the dorsal rays appears to be another potential species-specific morphological indicator (Fig. 5b). Specimens of A. ailuropodae n. sp. vary from A. tubaeforme by differences in cleft length of two digitations in each branch ( Fig. 5b9 and 10) and further from A. taxideae, A. duodenale, A. paraduodenale, A. caninum, A. malayanum, A. kusimaense, A. ceylanicum and A. braziliense by the absence of a third digitation in each branch ( Fig. 5b1-8 and 10). Verified specimens of A. genettae, A. protelesis and A. somaliense have not yet been described and these three species were not included in the comparison above. Notably, the adults of both A. pluridentatum and A. buckleyi can be distinguished from the new species by the number of ventrolateral teeth, given that A. pluridentatum has only one pair while A. buckleyi has three pairs according to the original descriptions (e.g. [60,63]). Based on these morphological attributes, A. ailuropodae is considered to be a previously unrecognized species within the genus Ancylostoma.

Molecular characterization
To further probe the taxonomic position of A. ailuropodae, both nuclear ITS1-5.8S-ITS2 and mitochondrial cox1 sequences from two representative specimens (codes FTZ1 and FTZ2, respectively) were obtained and subjected to sequence characterization and phylogenetic analyses.

Sequence characterization
For  Further, we located these sites and determined if there were non-synonymous substitutions apparent via comparison of their protein sequences, and the results are shown in Fig. 6. Out of 78 variable base sites, 13 were unique for A. ailuropodae (in red); 16 were identical between A. ailuropodae and one of A. ceylanicum, A. duodenale, A. caninum and A. tubaeforme (in orange); and 49 were shared between A. ailuropodae and any two or three of these four congeneric species (in yellow). Among the 49 variable sites, however, the nonsynonymous substitutions A/G 250 in A. ceylanicum and T/A 251 in A. caninum led to their amino acid changes: I (Ilu) → V (Val) in the former and I (Ilu) → N (Asn) in the latter (see Fig. 6). In addition, analysis of genetic distances using maximum composite likelihood estimates placed A. ailuropodae close to A. ceylanicum with the minimum interspecific evolutionary divergence (0.084), compared with 0.121 evolutionary divergence to A. tubaeforme, 0.127 to A. duodenale, and 0.151 to A. caninum (not shown).

Phylogenetic characterization
Phylogenetic relationships between A. ailuropodae and other species were inferred from the respective sequences of ITS1-5.8S-ITS2 and cox1 using both MP and BI algorithms and their corresponding tree topologies are shown in Fig. 7. Although the two consistent structures (MP/BI) topologically varied from each other due to the different reference species included, both trees provided an identical, robust phylogenetic resolution for A. ailuropodae within the genus Ancylostoma and for the genus Ancylostoma within the family Ancylostomatidae. Specifically, (i) the two A. ailuropodae specimens clustered together as a monophyletic group that was separated from the other Ancylostoma species. (ii) When the congeneric species A. ceylanicum, A. caninum A. duodenale and A. tubaeforme were considered in our cox1-based analysis (Fig. 7a), A. ailuropodae and A. ceylanicum were more closely related to each other than to A. caninum, A. tubaeforme and A. duodenale, with robust support for tree topology (BP = 95 and PP = 0.99).
(iii) When another species, A. braziliense, was added to re-construct this phylogenetic relationship using the ITS1-5.8S-ITS2 data (Fig. 7b), A. ailuropodae remained as the putative sister of A. ceylanicum, regardless of isolate origins (one from the UK and another from India; see Table 1), with high statistical support (BP = 89 and PP = 0.91), which was in agreement with the inferences from  and N. americanus in the genus Necator, demonstrated phylogenetic stability of these monophyletic groups, with the current analyses being consistent with previously proposed molecular phylogenies of the hookworms based on the nuclear ribosomal and mitochondrial DNA data [64][65][66][67][68][69][70][71][72][73][74][75].

Discussion
Hookworms in the genus Ancylostoma cause significant medical and veterinary disease (ancylostomiasis) in various hosts including humans and domestic and wild mammals [2,71]. Recent epidemiological surveys revealed that some wild animal-derived species of Ancylostoma are emerging as important helminthic zoonotic agents because of rapid urbanization and increased human-wildlife interactions [11,[13][14][15][16][17][18][19][20][21]. The giant panda, for example, is an endangered and rare wild species in China that has been artificially protected and

Giant panda This study
Abbreviation: nr not reported; the source paper presented no data on the species under consideration a Only the length of gubernaculum was found in the original description [7] even partially housed for decades due to habitat loss [33]. Clinically unidentified specimens of Ancylostoma in giant pandas had been confirmed by veterinarians and wildlife biologists since the last century, but their potential zoonotic importance remains to be defined [41]. In the present study, A. ailuropodae n. sp. was isolated from the giant panda, morphologically characterized and demonstrated to be closely related to the anthropozoonotic A. ceylanicum by molecular analysis. In general, morphological identification is a conventional and authoritative approach to define a new nematode parasite species. Concerning the genus Ancylostoma, several common species can be morphologically differentiated by key characters such as body size, teeth of the buccal capsule and shape of bursal rays (see Table 2 and Fig. 5b; cf. [9]). Similarly, specimens of A. ailuropodae from giant pandas are separated from other hookworms on the basis of either ventrolateral and dorsolateral teeth or dorsal rays, supporting the previous conclusions that teeth and rays were reliable morphological indicators in the differential diagnosis of Ancylostoma spp. [55,56]. Among this assemblage, it is important to note that A. ailuropodae is clearly structurally distinct from A. malayanum, the only other species of Ancylostoma known in ursid hosts (e.g. Ursus thibetanus) ( Table 2), with the implication that each of these species endemic to China may be more closely related to other congeners within the genus. Specimens upon which the description and differentiation of A. ailuropodae n. sp. was based were restricted to fully developed adults and eggs. Further work, using a combined laboratory-egg cultivation and Baermann technique, to describe the morphology of developmentally advanced larval stages is needed to complement morphological characteristics of the new species, and to provide valuable information assisting in species identification and differentiation in this genus [55,76].
Following our morphological evidence, A. ailuropodae from giant pandas was further confirmed as an independent species by molecular analysis. For example, the internal transcribed spacer region (ITS1-5.8S-ITS2) of the nuclear ribosomal DNA is regarded as an appropriate genetic marker to resolve nematode relationships at the species level [77]. Pairwise comparisons of ITS1-5.8S-ITS2 in A. ailuropodae with congeneric species available in the GenBank database revealed a species-specific sequence feature (containing 59 variable informative sites) and overall identity of 92.6-99.6% among A. ceylanicum, A. tubaeforme, A. caninum and A. braziliense. Furthermore, high bootstrap support was evident, based on phylogenetic analysis of ITS1-5.8S-ITS2 that demonstrated monophyly of A. ailuropodae as the putative sister of A. ceylanicum (see Fig. 7b).
Critically, similar conclusions were reinforced by analysis of the mitochondrial cox1 gene. It should also be noted that cox1 analysis was included because recent studies of the substitution patterns for nematode mitochondrial genes (e.g. cox1 and nad4) revealed that they have utility in identifying and differentiating novel or cryptic species among closely related taxa due to assumed faster evolutionary rates than nuclear genes, features of maternal inheritance and absence of recombination [78][79][80]. Compared to the nuclear ITS, the cox1 of A. ailuropodae appeared to have more variable informative sites (n = 78, including 13 unique loci). Nevertheless, results based on cox1 were consistent with inference from ITS, in revealing a sister-species relationship with A. ceylanicum among a broader assemblage of congeners in the genus. Phylogenetic analysis of cox1 data (Fig. 7a) also supported the contention that A. ailuropodae n. sp. is an independent species which is clearly differentiated from A. ceylanicum, A. caninum, A. tubaeforme and A. duodenale.
Based on the results from integrated molecular and morphological comparisons, we propose that A. ailuropodae of giant pandas is a previously unrecognized and separate species that is closely related to the anthropozoonotic A. ceylanicum within the genus Ancylostoma. Additional information regarding the ultrastructure and genomics of this species and other related hookworms is still required. Broader taxonomic comparisons can provide an increasingly precise morphological and molecular basis for species recognition among hookworms. In addition, there were two non-synonymous base substitutions detected in cox1 genes of A. ceylanicum (A/G 250 ) and A. caninum (T/A 251 ) (Fig. 6) that were confirmed to be fixed and species-specific after homologous comparisons with other A. ceylanicum or A. caninum isolates from two sites in the same geographic area.
Ancylostoma ailuropodae identified here is the fourth hookworm to be described from the Ursidae. Previously, the hookworm Uncinaria yukonensis (Wolfgang, 1956) was characterized in black bears and Uncinaria rauschi (Olsen, 1968) in grizzly and black bears [81,82]. On the basis of comparisons of morphometric and distribution data of ursine hookworms as well as the historical biogeography of bears, Catalano et al. [13] proposed that there was a relatively recent host-switching event of U. rauschi from black bears to grizzly bears.
The occurrence of A. ailuropodae appears consistent with speciation following a host colonization event to giant pandas apparently from a carnivoran source in sympatry, and further indicates a history of independent association with ursine hosts for the broader a b ancylostomatid hookworm assemblage. The timing and geographic source for these hookworms cannot be elucidated based on the currently available data and the reduced and relictual distribution for pandas, but a history of host colonization is compatible with the current tree topology (for parasites and hosts) and distribution of carnivore hosts for other species of Ancylostoma (e.g. [24]). We suggest that acquisition of Ancylostoma by giant pandas likely occurred prior to 7 million years ago (MYA) when a shift from an omnivorous diet to one dominated strictly by bamboo (by 2.4 MYA) was underway [25]. Divergence of A. ailuropodae appears to have occurred prior to acquisition of A. ceylanicum by humans in Southeast Asia, and prior to the intense bottlenecking of giant panda populations that has characterized the past century (cf. [33,83] for details about the history of giant pandas). This interpretation is significant, as it would relate to the historical independence of A. ailuropodae and A. ceylanicum before the current intensified conservation campaign for maintaining giant pandas, and the potential for cross-transmission of both hookworm species when infected humans are in contact. The unique niche and specialized bamboo-feeding habits of giant pandas suggest that colonization in ecological time, related to the source or origin of A. ailuropodae, was unlikely given relative isolation with respect to a sympatric assemblage of carnivorans or other mammals that may serve as hosts for species of hookworms [25,26,33]. Parasitological inventory among potential carnivoran hosts in Sichuan and nearby regions remains necessary to demonstrate that A. ailuropodae has a narrow host range and may now be limited to the giant panda [84]; apparent narrow host range, however, does not preclude the potential or capacity for contemporary host switches to humans as a zoonotic parasite given opportunity due to permissive ecological circumstances [85][86][87][88].
Phylogenetic and historical isolation of giant pandas from the broader assemblage of ursids and ursine bears (e.g. [23,24]) in conjunction with apparent structural divergence (e.g. teeth and configuration of the dorsal ray; Table 2 and Fig. 5b5 and 10) of A. ailuropodae and A. malayanum suggests that independent events of host colonization, separated in space and time, were essential in the process of speciation for these hookworms; molecular data, particularly from A. malayanum, is still needed to explore this hypothesis. Moreover, phylogenetic hypotheses for the Ursidae have placed giant pandas distantly from species of Ursus (and other ursines) near the base of an extensive radiation for bears that unfolded across the late Miocene and Pliocene [24]. Among ursine hosts for Ancylostoma, U. thibetanus (Asiatic black bear) is regarded as the sister of U. americanus (American black bear) and placed among crown species in ursid phylogenies [23,24]. These relationships alone would serve to refute a coevolutionary hypothesis for Ancylostoma hookworms among bears, conversely supporting a history of independent events of host colonization that have structured this fauna.
Unlike U. yukonensis and U. rauschi in bears, the hookworm from giant panda is genetically similar to other Ancylostoma species (Fig. 7). These respective genera are referred to two independent subfamilies within the Ancylostomatide, namely Ancylostomatinae Looss, 1905 for Ancylostoma and Bunostominae Looss, 1911 for Uncinaria, consistent with extended evolutionary trajectories for these taxa among the hookworms. This suggests the independent origin of A. ailuropodae, supporting monophyly of A. ailuropodae and congeneric species A. ceylanicum, A. duodenale, A. tubaeforme, A. caninum and A. braziliense, and strengthens the close relationship between the giant panda hookworm and A. ceylanicum within the clade. Concurrently it suggests that Uncinaria spp. from pinnipeds and ursids are a distinct monophyletic group in the family Ancylostomatidae [70]. The apparent genetic differences of A. ailuropodae n. sp. in pandas and U. rauschi and U. yukonensis in bears, coupled with their divergent biogeographic and ecological histories suggest this system as a good model for exploring the complexities of diversification and faunal assembly in the evolution of host range and associations among hookworms (e.g. [85][86][87][88]).
The potential for genetic partitioning among possible disjunct populations of hookworms in giant pandas should be considered, as it will reflect information about the timing of colonization to giant pandas and the duration of the history of association. Further, the history of fragmentation and isolation for giant pandas across now isolated mountain systems in southwestern China suggests a complex relationship among hosts and hookworms in this region. Such history could be explored through fecal-based approaches in conjunction with molecular diagnostics to examine occurrence and the extent of genetic diversity and distribution for hookworm parasites among populations and subspecies of giant pandas.

Conclusions
This study is the first to describe and define a new member of the genus Ancylostoma, A. ailuropodae, in the wild giant panda using morphological and molecular criteria. Morphological characters (e.g. ventrolateral (two pairs) and dorsolateral (two pairs) teeth and dorsal rays) distinctly separate A. ailuropodae n. sp. from other congeneric species in the genus Ancylostoma. Further, nuclear ITS1-5.8S-ITS2 and mitochondrial cox1-based genetic distance analysis and phylogenies supported the assertion that A. ailuropodae is independent and shares a sister-species relationship with the anthropozoonotic A. ceylanicum. Although additional molecular evidence is warranted, this finding should enhance public awareness of parasitic hookworms in giant pandas, especially in captive populations that have frequent contact with breeders, veterinarians and even tourists. Moreover, the morphological and molecular data presented here enhances the information on species within the genera Ancylostoma, Uncinaria, and Necator and contributes to a more complete understanding of the taxonomy, diagnostics and evolutionary biology of hookworms.