Gastrointestinal helminth parasites of the threatened Australasian crested grebe (Podiceps cristatus australis, Gould 1844) in New Zealand, with descriptions of Baruscapillaria kamanae n. sp. (Nematoda: Trichuridae) and Cryptocotyle micromorpha n. sp. (Trematoda: Opisthorchiidae)

The Australasian crested grebe Podiceps cristatus australis, Gould 1844 is restricted to Australia and New Zealand, where it is listed as Threatened and Nationally Vulnerable. For the first time in New Zealand, we report on the parasitic helminths infecting three individuals from Lake Wanaka, Otago, using morphological and molecular tools. Seven helminth species were found in the gastrointestinal tract: 2 nematodes (Contracaecum ovale and Baruscapillaria kamanae n. sp.), 4 trematodes (Australapatemon minor, Cryptocotyle micromorpha n. sp., Tylodelphys darbyi and Neopetasiger neocomensis), and 1 cestode (Confluaria pseudofurcifera). Except for T. darbyi, all are new records for New Zealand. A change of orthography is proposed for Neopetasiger neocomensis and N. pseudoneocomensis. Cryptocotyle micromorpha n. sp. (Opisthorchiidae) is distinguished from similar species by its small size, wholly extracaecal vitellaria and anteriorly looped uterus. Baruscapillaria kamanae n. sp. (Trichuridae) is distinguished from other freshwater species by a combination of vulva and spicule morphology. The helminth parasites found here are mostly the same as those from the grebe in the northern hemisphere, indicating that they have been carried with the host species in its spread to Australasia. However, the parasite fauna may be depauperate due to a diminishing reservoir of intermediate hosts in that geographical migration.


Introduction
The great crested grebe (Podiceps cristatus Linnaeus) (Podicipediformes) has a broad distribution in Europe, Asia and Africa, but the subspecies P. cristatus australis Gould occurs only in Australia and New Zealand, where it is listed as Threatened and Nationally Vulnerable under the New Zealand Threat Classification System. In addition, the species is considered taonga (''treasure'') by Maori and is fully protected. Crested grebes arrived in New Zealand over 10,000 years ago (Jensen and Snoyink, 2005), and were once widespread throughout the country. However, they are now restricted to a number of lakes in the South Island, and a survey of 2005 found the largest numbers in Lake Hayes, Otago, and Lake Heron, Canterbury (Jensen and Snoyink, 2005). In New Zealand grebes face threats due to predation by introduced mammals, loss of shore line nesting habitat and disturbances from human activities (Jensen and Snoyink, 2005). The population at Lake Wanaka, Otago, however, has increased by 300 birds in recent years (Darby, 2021), thanks to the skill and dedication of John Darby. The crested grebe is the only grebe species currently to occur in the South Island of New Zealand.
Despite its threatened status, knowledge of some aspects of Australasian crested grebe biology is still sparse. In particular, the parasite fauna of great crested grebes in New Zealand has never been examined. The benefits of discovery and identification of an animal's parasites are well established; including data pertaining to host health, feeding habits, distribution, potential competition with other host species, and to compare with other grebe species (see Storer, 2000). Parasites can be disease causing agents too, making their study especially important for threatened host species.
Great crested grebe parasites are well documented in northern hemisphere populations. Storer (2000) listed a total of 102 helminth species (Trematoda,46;Cestoda,31;Acanthocephala,2;Nematoda,23), since which can be added a further 5 records from the literature (2 trematodes; 2 cestodes; 1 nematode) (Fain, 1956;Vasileva et al., 2003;Mutafchiev & Georgiev, 2008). Australia has geographically the closest population and is the probable origin of the New Zealand crested grebes. There are nine helminth species recorded from Australia (4 trematodes; 1 cestode; 4 nematodes), although only 5 are identified to species level and all reports are from just two studies (Johnston & Mawson, 1949;Mawson et al., 1986), so the parasite fauna from Australia is also very little known.
Due to the protected and threatened nature of crested grebes in New Zealand, it has not been possible to investigate the parasite fauna of these birds until now. However, between January 2017 and January 2020 three dead individuals were collected from Lake Wanaka, allowing the first investigation of the parasites of the New Zealand population of this subspecies. This study represents a record of the helminths found in these three grebe specimens, all of which are new records for New Zealand, and two of which are described as new species. We present brief morphological data for known species, and molecular data where required to confirm species identity, as well as descriptions of two new species.

Morphological data
Three Australasian crested grebes (Podiceps cristatus australis Gould) were found dead near the marina breeding area at Lake Wanaka, Otago in January 2017, January 2018 and January 2021. Deceased birds were donated by John Darby, conservator and protector of Lake Wanaka crested grebes. A white-faced heron (Egretta novaehollandiae (Latham)) was also found freshly dead at a nearby site in June 2018. A blackbacked gull (Larus dominicanus Lichtenstein) with comparable helminth infections was one of many dead birds donated by the Dunedin Wildlife Hospital from the Otago region. Birds were dissected fresh, and helminths were collected and preserved in 70% ethanol for whole-mount, 96% ethanol for genetic analyses and 4% buffered formalin for later SEM imaging.
Nematode specimens were cleared in lactophenol as temporary mounts for light microscopy. Cestode and trematode specimens were stained using acetic acid iron carmine, dehydrated in an ethanol series, cleared in clove oil and permanently mounted with Canada balsam.
Measurements were made using ImageJ software (Wayne Rasband, NIH, USA) from photographs taken on an Olympus BX51 compound microscope mounted with DP25 camera attachment. All measurements are in micrometres unless otherwise indicated, and in descriptions are given as range, followed by mean in parentheses, where numbers permit. Drawings were made with the aid of a drawing tube mounted on an Olympus compound microscope, or from photographic series.
Parasites were identified to the lowest taxonomic level possible. Taxa unfamiliar to the authors were identified using morphological keys such as Khalil et al. (1994), Gibbons (2010), Gibson et al. (2002), Bray et al. (2008), and original species descriptions. Some of the material reported in this article was described by the first author in a previous paper (i.e. Tylodelphys darbyi, Presswell & Blasco-Costa 2020).
Specimens of each species, when available, were chosen for scanning electron microscopy (SEM) and preserved in 4% buffered formalin. These specimens were transferred to 2.5 % gluteraldehyde in 0.1 M phosphate buffer, then post-fixed in 1% osmium tetroxide and dehydrated through a gradient series of ethanols, critical-point dried in a CPD030 BalTec critical-point dryer (BalTec AG, Balzers, Liechtenstein) using carbon dioxide, mounted on aluminium stubs, and sputter coated with gold/palladium (60:40) to a thickness of 10 nm in an Emitech K575X Peltiercooled high-resolution sputter coater (EM Technologies, Ashford, Kent, UK). The specimens were viewed with a JEOL 6700 F field emission scanning electron microscope (JEOL Ltd., Tokyo, Japan) or Zeiss Sigma VP variable-pressure scanning electron microscope (Carl Zeiss Inc., Oberkocken, Germany) at the Otago Centre for Electron Microscopy (OCEM, University of Otago, New Zealand). Voucher specimens of new species were deposited in Te Papa Museum, Wellington, New Zealand, under accession numbers W.003609-3614.

Molecular data and analyses
Specimens of each species were chosen for DNA sequencing. Genomic DNA was extracted using the DNeasyÒ Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For cestodes and trematodes a partial fragment of 28S rRNA gene was amplified using T16 and T30 primers (Harper & Saunders, 2001) and conditions of 94°C for 5min, 38 cycles of 94°C for 30sec, 45°C for 30 sec and 72°C for 2min, and 72°C for 7min. Additionally, cox1 was amplified when possible, using primers JB3 (Bowles et al., 1992) and trem.cox.rrnl (Králová-Hromadová et al., 2001) and conditions of 95°C for 2min, 40 cycles of 95°C for 30sec, 48°C for 40sec and 72°C for 1min, and 72°C for 10min. For nematodes, either the 28S, 18S or ITS1 genes were targeted using 28SF and 28SR (Nadler & Hudspeth, 2000), Nem18SF and Nem18SR (Wood et al., 2013) and SS1 and NC13R (Shamsi et al., 2008) primers respectively. The 28S PCR conditions consisted of 94°C for 5min, 38 cycles of 94°C for 30sec, 52°C for 30sec and 72°C for 1min, and 72°C for 10min. The 18S and ITS PCR conditions followed Wood et al. (2013) and Shamsi et al. (2008). All PCR products were cleaned using EXOSAP-TMTM Express PCR Product Cleanup Reagent (USB Corporation, Cleveland, OH, USA), following manufacturer's instructions. Sanger sequencing by capillary electrophoresis was performed by the Genetic Analysis Service, Department of Anatomy, University of Otago (Dunedin, New Zealand).
Sequences were used in BLASTn searches on GenBankÒ to establish a preliminary identification. Sequences were imported into Geneious PrimeÒv1.2, trimmed using the trim function with default parameters, and manually edited for incorrect or ambiguous base calls. A contiguous sequence was assembled for each sequence, alignments were created separately for each species sequenced, and genetic divergences were calculated in MEGA v.10 comparing between closely related species to confirm identity. Newly generated sequences are listed in Table 1, with their associated GenBank accession numbers.

Results
In total, 7 parasite species were recovered from three grebe specimens: 2 nematodes, 4 trematodes and 1 cestode. All three grebe individuals were infected with at least one species of parasite. Except for the capillarid nematode, and trematode Australapatemon minor, each parasite species was found in all of the grebe specimens. The parasite with the highest mean intensity was a hitherto undescribed species of Cryptocotyle, found in many hundreds of individuals per host (infection data are summarised in Table 2).

Remarks
Although the speciose genus Contracaecum has a confused and somewhat unreliable taxonomic history, species found mainly in grebes have distinctive characters that set them apart from other, mainly marine, species (see Discussion). Our specimens were therefore compared against all other species found in grebes, most of which we recognise as synonyms of C. ovale. DNA sequences were obtained for the 18S and ITS1 markers. Closest BLASTn hits gave a larval Contracaecum species from a freshwater fish in India for 18S (99.63% similarity [MG696304]). It seems highly likely that the Indian specimens belong to the freshwater 'grebe' group of species (see Discussion). ITS1 produced a BLASTn result that included many available Contracaecum sequences but no other sequences were available for known 'grebe' species, leaving molecular identification and exploration of the relationships of the 'grebe' species in abeyance for the time being. The sequence of 18S was identical to those of worms from the white-faced heron. Filipjev, 1929Trichinelloidea Ward, 1907Trichuridae Ransom, 1911Baruscapillaria Moravec, 1982 Type species: Capillaria obsignata Madsen, 1945 by original designation.

Enoplida
A total of 16 specimens of a hair-like nematode was found in the stomachs of two grebes, of which 6 were male, 7 were female and 3 were indeterminate fragments. Two specimens of matching morphology were also found in the white-faced heron. Most of these delicate nematodes were broken, allowing some measurements, but two entire males and two entire females remained to allow body length measurements to be taken. In having a male with caudal alae absent, pseudobursa with two small, rounded lobes, a long single spicule with aspinous spicule sheath, and presence of bacillary bands, the specimens conformed to the diagnosis of genus Baruscapillaria (Gibbons 2010). A description is given below and comparative morphometrics are given for Baruscapillaria kamanae n. sp. and other species from freshwater bird hosts (Table 3).

General
Body filiform; very long and thin, tapering to anterior end, widest along length of intestine. Sexually dimorphic; males smaller than females. Lateral bacillary bands present but very indistinct, number and extent not clear. Head end narrow, rounded; oral aperture terminal, slit-like, oriented dorso-ventrally. Cephalic papillae arranged around mouth in two circles, each with six recessed papillae ( Fig. 3f) Muscular oesophagus long; nerve-ring indistinct. Stichosome formed by single row of stichocytes; stichocytes long, subdivided into numerous annuli. Two large glandular cells present at junction of stichosome and intestine (Fig. 2e).
Etymology. The species name, a noun in the genitive case, is derived from the Maori name for the crested grebe, kāmana. Partial sequence of 28S was obtained for a specimen from one grebe. In BLASTn searches, the sequence showed greatest similarity with Aoncotheca paranalis (92.76% similarity over 445bp), Capillaria plica (91.22%) and Pearsonema plica (88.85% similarity). There are no overlapping sequences for Baruscapillaria spp. on GenBank, so these results tell us little at this time except for confirming the familial placement. Attempts to amplify 18S were unsuccessful.

Remarks
There are currently 21 species of Baruscapillaria that appear valid in the literature (Baruš & Sergejeva, 1990). Of these, 7 species are found in mammals, 6 in terrestrial birds and 4 in marine birds. The remaining 4 species infect freshwater birds, namely B. herodiae (Boyd, 1966) Moravec, 1982 in Ardea spp., B. mergi (Madsen, 1945) Baruš & Sergejeva, 1990 in Gaviiformes, Podicipediformes, Ciconiiformes and Anseriformes, B. podicipitis (Yamaguti, 1941) Moravec, 1982 in Podicipediformes, Anseriformes and Lariformes, and B. ryjikovi (Borgarenko & Daiya, 1972) Moravec, 1982 in Podicipediformes. Compared to Baruscapillaria kamanae n. sp., B. herodiae is considerably smaller in body length and spicule length, has no vulval swelling and a very short distance between vulva and stichosome (Boyd, 1966). B. mergi also has a smaller body length, as well as a small 'cup-like' vulval appendage, and fine striations on the spicule (Madsen, 1945). B. podicipitis is notably smaller in body length and spicule length, with a very short distance between vulva and stichosome and lemon-shaped eggs (Yamaguti, 1941). Baruscapillaria ryjikovi is closest to B. kamanae n. sp. in size, as well as being a grebe-specific species, but it is longer than B. kamanae n. sp. with a longer spicule, and has no vulvar swelling (Baruš & Sergejeva, 1990). An unnamed species of Baruscapillaria was reported by Presswell and Bennett (2021) from black shag Phalacrocorax carbo novaehollandiae (Stephens) in Otago. Those specimens were smaller than B. kamanae n. sp. with a shorter spicule. In addition to the anterior loop of the vagina, the above characters distinguish the New Zealand grebe specimens from other known species.
In possessing a bipartite body, testes tandem and irregular, vitellarium confined to hindbody and genital cone large with long, rugose hermaphroditic duct, the specimens conform to the diagnosis of genus Australapatemon Sudarikov, 1959 as emended by Blasco-Costa et al. (2016). We include a brief description below for future comparison.

Remarks
The small size of these specimens makes them comparable most closely to A. minor, which was described from the mallard, Anas platyrhynchos L. but which has also been reported from grebes (Sitko & Heneberg, 2015). The measurements of our specimens fit generally within the ranges given by Yamaguti (1933) (body length 0.8-1.2mm, forebody length 250-460, hindbody length 540-750, forebody:hindbody ratio 1:2) although the size ranges of the oral and ventral suckers and the pharynx, overlap those given for A. minor. In addition, the eggs of our specimens are smaller than those reported for A. minor. It is possible that these constitute an unreported species of Australapatemon, but without DNA sequence support (amplification was unsuccessful), and with few specimens, it would perhaps be premature to allocate these specimens to a new species of Australapatemon. We therefore tentatively attribute them to A. minor until more specimens allow further investigation.  This species was found in the upper intestine of all three grebe specimens, numbering 2, 31 and 7 respectively. Four specimens of matching morphology were also found in the white-faced heron. The species was previously described from some of the material reported herein (Presswell & Blasco-Costa, 2020) and sequence of ITS1 was provided (KU588152). Here we provide 28S and cox1 as additional molecular markers (Table 1). This is the first report of T. darbyi since its description in 2020. Cryptocotyle micromorpha n. sp. Fig. 5, 6a-g Specimens of this very small trematode were found in their hundreds in all three grebe hosts, and also in the white-faced heron. In having linguiform body, tegumentary spines, ventrogenital sac with protractile bulge bearing rudimentary ventral sucker, and oblique testes they were assignable to genus Cryptocotyle Lühe, 1899 (Pearson, 2008). Comparison to described species of Cryptocotyle revealed morphological differences that indicated this was a hitherto undescribed species, and a formal description follows. Comparative morphometrics are given for C. micromorpha n. sp. and other linguiform species (Table 4).

Remarks
In their paper, Selbach et al. (2014) found four different cercarial or metacercarial genotypes of Petasiger spp., and documented morphological differences between them. The authors suggested that two of their cercarial stages could belong to Petasiger pungens (Linstow, 1893) and P. neocomensis (both species now in Neopetasiger -see Tkach et al. 2016). In addition to this genetic match, the morphological features of our specimens, and the fact that N. neocomensis was described from Podiceps cristatus and is the only species of Neopetasiger recorded from this host, we believe we can confirm that Petasiger sp. 1 of Selbach et al. (2014) is, indeed, N. neocomensis.
A partial 28S gene was sequenced for four individuals which were identical, and gave a BLAST result of assorted Hymenolepidids with a maximum similarity of 93.60%. There are currently no sequences of Confluaria spp. on GenBank, so no relationships can be inferred at this time.

Remarks
A number of works by Vasileva et al. (1999aVasileva et al. ( , b, 2000Vasileva et al. ( , 2001Vasileva et al. ( , 2008 have reviewed this genus and present excellent descriptions and a key (Vasileva et al., 2000) from which the New Zealand specimens can be identified as C. pseudofurcifera. Only in two species does the epiphyseal thickening attach to both the handle and the guard of the rostellar hook; C. pseudofurcifera and C. podicipina (Szymanski, 1905). The epiphyseal thickening of C. podicipina is conspicuously more massive. Although no specimen was found with everted cirri, the base of the inverted cirrus was undoubtedly like that of C. pseudofurcifera with a short, thick enlargement armed with rosethorn spines.

Discussion
We found a total of 7 helminth species infecting three crested grebes in Otago, New Zealand: 2 nematodes (Contracaecum ovale and Baruscapillaria kamanae n. sp.), 4 trematodes (Australapatemon minor, Cryptocotyle micromorpha n. sp., Tylodelphys darbyi and Neopetasiger neocomensis), and 1 cestode (Confluaria pseudofurcifera). Previous to this study there were no records of gastrointestinal cestodes or trematodes from the crested grebe in New Zealand. Apart from the two new species, and T. darybi, all the species reported herein have been recorded from Podiceps cristatus in other parts of the world, but all of them are new records for New Zealand. Below we discuss the taxonomic histories, known biology and life cycles of the parasites, and host specificity in the crested grebe.
Until now, no Contracaecum species has been reported from freshwater birds in New Zealand (McKenna, 2010). However, C. podicipitis (now C. ovale -see below) was originally described from Podiceps cristatus australis from Australia (Johnston & Mawson 1949). In addition to the specimens from the grebe, we also found over 100 specimens of the same species (confirmed by DNA identity) in the white-faced heron from Lake Wanaka. Although there are over 100 species in the genus Contracaecum (Shamsi 2019), most are found in marine environments, and there are few species found in Podicipediformes. Based on the literature, all those species reported from grebes have a distinctive head morphology, in that the interlabia are greatly reduced in size, usually reaching to about 1/3 the height of the lips, and lack any bifurcation. In addition, these species lack the conspicuous annulations of the cephalic collar, interrupted by a lateral groove seen in other, mainly marine, Contracaecum species. We therefore compared our specimens to only those found with these features from grebes, of which there are six nominal species: C. nehli Karokhin, 1949; C. ovale (Linstow, 1907); C. podicipitis Johnston & Mawson, 1949;C. praestriatum Monnig, 1923;C. ruficolle Vuylsteke, 1953 andC. spasskii Mozgovoi, 1959. The validity of all of these species has been called into question by various authors. Indeed, Macko (1961) and Hartwich (1964) regarded C. nehli, C. spasskii and C. ruficolle as synonyms of C. ovale and Baruš & Zajíček (1967) included C. podicipitis in its synonyms. The original description of C. ovale (Linstow 1907) was short on detail, and various descriptions of the species have been published since (i.e. Cram, 1927;Mozgovoi, 1953;Macko, 1961;Hartwich, 1964;Galeano & Tanzola, 2012). Those reports that gave morphometrics have greatly increased the range of measurements and interspecific variability in all features, notably the size of spicules and number of male papillae. This being the case then it seems sensible to accept the synonymisations of C. nehli, C. podicipitis, C. ruficolle and C. spasskii, which fall within the ranges given by the above authors. Contracaecum praestriatum, however, does appear to have some distinctive characters, not least the presence of a gubernaculum and thick lips with rounded outline, and an abrupt change in the nature of the cuticular striations at one third of the body length (Monnig, 1923). For the present the most parsimonious approach to the 'grebe' species is to follow previous workers (Macko, 1961;Hartwich, 1964;Baruš & Zajíček, 1967;Baruš et al., 1978) and include them all (except for C. praestriatum) in C. ovale, until such time as genetic data are available from each of the putative species to elucidate their distinctiveness or lack thereof. Further sequences from all Contracaecum from grebes, or other freshwater fish eating birds, should help elucidate the taxonomy and evolutionary history of this species group.
Apart from the unnamed species mentioned previously (Presswell & Bennett, 2021) the only records of Baruscapillaria species (reported as Capillaria) from New Zealand, are from terrestrial birds: B. emberizae (Yamaguti, 1941) in a yellowhammer Emberiza citrinella L. and B. obsignata (Madsen, 1945) in domestic fowl Gallus domesticus L. and rock pigeon Columba livia (Gmelin) (McKenna, 2010). What little is known of the life cycle of Baruscapillaria spp. suggests that their life histories are variable. Baruscapillaria obsignata has been shown to have a direct life cycle with no intermediate hosts (Moravec et al., 1987), but those species infecting grebes are believed to use oligochaetes as intermediate hosts (Storer, 2000). Although three species of Baruscapillaria are hosted in Podicipediformes (B. mergi, B. podicipitis and B. ryjikovi), only B. podicipitis is recorded from Podiceps cristatus. However, B. podicipitis is not host-specific, also occurring in other grebes and Anseriformes, Lariformes (Baruš et al., 1978) and Pelicaniformes (this study).
One other species of Australapatemon has been recorded from New Zealand; Australapatemon niewiadomski Blasco-Costa, Poulin & Presswell, 2016 from the mallard duck. This species is larger in all metrics than the specimens found in the grebes. The specimens of A. minor were found in the upper intestine. In the gizzard of the second two grebes, and in the heron, were found thousands of metacercarial cysts of the strigeid type, along with a few juvenile excysted individuals. It was clear that, despite the huge numbers of strigeid cysts found in the gizzards of these birds, only these three parasite individuals had successfully passed through into the intestine and matured. However, several questions remain concerning this phenomenon: 1) if this species is able to mature in the grebe intestine as evidenced by the three mature specimens, why did we not see 1000s of mature specimens in the intestine?, 2) could the three mature specimens belong to a different strigeid species that is able to mature in the grebe, whereas the thousands in the gizzard were able only to excyst before expiring? (unfortunately, DNA amplification did not work for the strigeid cysts, nor the three adults, so we are unable to say whether or not they are the same species), 3) Australapatemon spp. are known to infect leeches as their second intermediate hosts. Although it is quite feasible that grebes would eat the occasional leech, it seems unlikely that they would ingest enough to accumulate the thousands of metacercariae in the gizzard, especially considering that grebes void at least part of their gizzard contents daily as pellets (Jehl, 2017). This leaves the likelihood that the cysts belong to a species of Apatemon, (possibly Apatemon sp., ''jamiesoni'' that occurs in large numbers in bullies Gobiomorphus cotinianus MacDowall). This species, yet to be formally named, has been found in a mallard, and a black-backed gull Larus dominicanus Lichtenstein (Presswell unpubs. data). On the assumption that the cysts in the gizzard are not Au. minor, we have not included these cysts as part of the grebe parasite fauna, as it seems they are unable to persist in the grebe. There is no doubt that there are more strigeid species yet to be found in New Zealand (Blasco-Costa et al., 2016), so this is a puzzle that will have to await more evidence.
As well as the three grebe specimens, Tylodelphys darbyi was also found in a single white-faced heron from Lake Wanaka. The second intermediate host of this trematode species is the bully (Gobiomorphus cotidianus), in which the metacercariae inhabit the eyes. The initial report of this larval stage was from Lake Hayes, in Otago (Blasco-Costa et al., 2017). However, the species has since been found in bullies in Lakes Wanaka and Hawea (both lakes with grebe populations) and Lake Heron (Ashburton Lakes) (also with a healthy grebe population), considerably further north (Ruehle et al. in press). Despite extensive searching among the gastropod fauna of Lakes Wanaka and Hawea, the first intermediate host of this diplostomid has not yet been found.
Cryptocotyle micromorpha n. sp. is the first named species of opisthorchiid reported from a New Zealand bird; the only previous report was an unnamed species of Metorchis in a ''wild duck'' (Rind, 1974). The genus Cryptocotyle has traditionally been considered a member of family Heterophyidae (Pearson, 2008), but recent genetic studies convincingly place the genus into family Opisthorchiidae (Thaenkham et al., 2011(Thaenkham et al., , 2012Tatonova and Besprozvannykh, 2019;Sokolov et al., 2021), andSokolov et al. (2021) provided emended diagnoses of the family and its superfamilies to take these changes into account. However, genetic relationships within the genus Cryptocotyle are still relatively unknown and a larger range of taxa will be required to elucidate the interrelationships of the species, including our newly generated sequences. Caution is required when referring to records of C. lingua in the literature. Creplin's (1825) description included no measurements, except for body length, and no illustrations, so identification of the species in the literature has been speculative, leading to a range of measurements that surely exceed the original species. Therefore, it is a matter of conjecture which source is used as a substitute for comparison. In this case we have used data from Jägerskiöld (1898) who confirmed identity of his specimens directly with those of Creplin. Specimens of C. micromorpha n. sp. have also been found by the authors in Southern black-backed gulls (Larus dominicanus), confirmed by DNA sequence identity. Morphologically comparable specimens were found too in the white-faced heron (Egretta novaehollandiae) from the same locality as the grebes. Cryptocotyle species are found in both freshwater and marine ecosystems, and gulls (Laridae) in particular are regular definitive hosts. In New Zealand, black-backed gulls (known as Kelp gulls elsewhere in the southern hemisphere) occur both at the coast and inland on freshwater lakes and rivers. One of the black-backed gulls dissected was infected only with parasites associated with freshwater, including Cryptocotyle micromorpha n. sp.
We present the first record in New Zealand of species of either Neopetasiger or its synonym in part, Petasiger Dietz, 1909. Neopetasiger was elevated to generic rank by Tkach et al. (2016) to include those species of Petasiger with 19 collar spines, predominantly parasitising grebes. The known distribution of Neopetasiger neocomensis includes much of central and Eastern Europe, as well as China. The history of the species includes many instances of misidentification and confusion with other species, described in detail in the revision of genus Petasiger, by Faltýnková et al. (2008). Their thorough and detailed study included measurements of new specimens from various different geographical and host sources, and measurements of our specimens fit convincingly within their given ranges for the species. Specimens of this species were also found in the white-faced heron from the same locality as the grebes, which constitute the first record of this species in a member of the Ardeidae.
We also present the first record in New Zealand of any species of Confluaria, a cestode genus that infects podicipediforms almost exclusively. In terms of numbers, cestodes constitute the dominant group in the helminth communities from grebes (Storer, 2000). Sitko and Heneberg (2015) found Confluaria sp. to be the most prevalent species of helminth in P. cristatus (80% prevalence). Specimens were found in all three of our birds, so the life cycle of this cestode obviously facilitates high prevalence, although the intermediate hosts are not known. Other species of Confluaria are reported to use cladocerans as intermediate hosts, although Storer (2000) suggests that it seems likely that fishes may act as paratenic or second intermediate hosts. Vasileva et al. (2000) examined specimens from several different collections and concluded that C. pseudofurcifera was exclusive to P. cristatus as they found no specimens in other grebe species.
All of the helminth parasites reported here were from the gastrointestinal tract. However, because of the rarity of dead grebe specimens, we took the opportunity to check the nasal passages of the last specimen for the possible presence of nasal schistosomes (Trichobilharzia spp.), the cause of swimmer's itch, which are found as larval stages in Lake Wanaka, and in the nasal passages of New Zealand scaup Aythya novaeseelandiae (Norm Davis pers. comm.). The livers were also examined for this worm and any other parasites, as well as other internal organs and the eyes. No parasites were found in any of these organs in the bird that we examined.
According to Storer (2000) many helminth species are found largely or exclusively in podicipedforms, but few, if any, are known to be specific to any one species of grebe. In the northern hemisphere part of the crested grebe's range 25 digenean species (22% of all those reported from grebes) are known only from the grebe type-host, but this figure is probably highly influenced by imprecise taxonomy and identification, rather than reflecting a true estimate of host-parasite specificity. In terms of cestodes, which show the greatest degree of specialisation to podicipediform hosts, one family, the Amabiliidae, are grebe specialists, and 15 Hymenolepididae genera are also known only from grebe hosts, with 2 species known only from their grebe type-host (Storer, 2000). Nematodes show little specificity with just 6 species recorded solely from grebes of which 2 are only known from their type-host. Storer (2000) concludes that there is no evidence for a strong pattern of specificity to single definitive host species, and that those that are reported from single grebe species more likely reflect insufficient sampling effort or geographic isolation. As several of the species found in the New Zealand grebes are the same as those found in Podiceps cristatus elsewhere in the world, it would seem that the NZ population has retained much of its worm fauna in its colonisation of Australasia. There are no other sympatric grebe species with which to share its parasites (the dabchick Poliocephalus rufopectus (Gray) and Australasian little grebe Tachybaptus novaehollandiae (Stephens) do not occur in the South Island), but we do find parallel infections (five out of seven helminth species) in the white-faced heron, which lives alongside the crested grebes and has a very similar diet.
Previous studies of parasite assemblages within Podiceps cristatus have found a more diverse helminth fauna. For instance, Sitko and Heneberg (2015) found 28 species in their birds (n = 438) from the Czech Republic. That we found only 7 species in our three birds no doubt reflects the huge difference in host sample sizes, but the lack of diversity in New Zealand may be real, and a consequence of the geographical remoteness of New Zealand and limited diversity of intermediate hosts to perpetuate infections. As the species dispersed to Australasia via the Pacific, Australia and thence to New Zealand, perhaps their parasite diversity was filtered out by an ever decreasing availability of intermediate hosts. Once in New Zealand, there were no other grebes from which to acquire more of the relatively specialist groups of parasites (Storer, 2000) that infect the podicipediforms. Recording helminth diversity is important in that it provides information regarding host health, diet, behaviour and ecology, and ecosystem health as a whole. This article provides a baseline for future research on the helminth diversity of an ecologically important bird species of Threatened status.
Funding Open Access funding enabled and organized by CAUL and its Member Institutions.

Declarations
Funding Funded by Department of Conservation, BCBC Threatened species grant (WBS D406-976-002) to Bronwen Presswell, a Zoology Department PBRF Research Enhancement grant to Bronwen Presswell, a University of Otago Doctoral Scholarship to Jerusha Bennett, and indirectly by the Marsden Fund (Royal Society of New Zealand: UOO1718).

Conflicts of interest/Competing interests The authors declare no conflict of interest
Availability of data and material Not applicable Code availability Not applicable Zealand, and who kindly provided our bird specimens, and Robert Poulin for his comments and continued support. Thank you to Liz Girvan of the OCEM for assistance with scanning electron microscopy. In particular the authors would like to thank Dr. Clement Lagrue (DoC) for initiating and facilitating our relationship with the Department of Conservation, and enabling this work to be carried out.
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