New specimens and molecular data provide validation of Apatemon jamiesoni n. sp. (Trematoda: Strigeidae) from water birds in New Zealand

A study published in 2016 reported on an undescribed species of Apatemon (Strigeidae) from New Zealand that was previously well known from its larval stages. Only a single specimen from a mallard duck was available at the time, which was described and given the provisional name Apatemon sp. “jamiesoni”. Specimens also obtained from a spotted shag were not in good enough condition to form the basis of a new species description. A black-backed gull has since been discovered with specimens of this strigeid, their identity confirmed by genetic similarity, allowing formal description and naming of this species. This paper provides a description of the new specimens from the black-backed gull, along with a comparison with the specimens from other bird hosts, reprises some data from Blasco-Costa et al. (Parasitol Res 115:271–289, 2016) and presents formally the name Apatemon jamiesoni n. sp. This species differs from all other species of Apatemon in its small size, particularly that of the ovary and testes. It is most similar to A. jamesi from which it differs in the size of the oral and ventral suckers.


Introduction
A study published in 2016 (Blasco-Costa et al. 2016) reported on an undescribed species of Apatemon Szidat, 1928 (Strigeidae) from New Zealand, that was nevertheless previously well-known from its larval stages (references listed under Synonyms). Unfortunately only a single specimen from a mallard was available at the time, which was described and figured, and given the provisional name Apatemon sp. ''jamiesoni'' until more specimens were found in order to establish the species. At the time specimens were also reported from a spotted shag, but these were not in sufficiently good condition to form the basis of a new species description. A third bird species, a blackbacked gull, has since been discovered to harbour specimens of this strigeid, their identity confirmed by genetic similarity, and the author is now in a position to complete the description and formally name the species.
This species was originally familiar in New Zealand as a distinctive metacercarial cyst found in large numbers in common (Gobiomorphus cotidianus McDowall) and upland (G. breviceps (Stokell)) bullies (Actinopterygii: Eleotridae), and as cercariae from the freshwater snail, Potamopyrgus antipodarum (Gray) (Gastropoda: Tateidae). These hosts inhabit the fresh to brackish waters of coastal and inland lakes and streams of South Island, New Zealand. Cercariae have been reported from P. antipodarum in North Island (Hechinger 2012), although identity with the South Island species has not been confirmed genetically.
This paper provides a description of the new specimens from the black-backed gull, along with a comparison with the specimens from other bird hosts that match genetically, reprises some data from Blasco-Costa et al. (2016) and presents formally the name Apatemon jamiesoni n. sp. A phylogenetic tree is presented that confirms the identity of the specimens from the different hosts described herein, in the context of known strigeid sequences.

Materials and methods
Collection of material. An adult mallard duck, Anas platyrhynchos L., was found as roadkill in the Mount Watkin region of Otago (2013), a spotted shag, Phalacrocorax punctatus (Sparmann), was found dead in Otago Harbour (2014) and a black-backed gull, Larus dominicanus Lichtenstein, was donated by the Dunedin Wildlife Hospital where it had died (2019). Viscera were dissected and intestinal worms were preserved in either 96 % ethanol for molecular analyses or 70 % ethanol for morphological study.
Morphological analysis Trematodes were stained using iron acetocarmine, dehydrated through a graded ethanol series, cleared in clove oil and mounted in 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. Drawings were made with the aid of a drawing tube mounted on an Olympus compound microscope. Note that in the descriptions and tables the terms ''prosoma'' and ''opisthoma'' are used to replace the formerly used ''forebody'' and ''hindbody'', following Achatz et al. (2021).
Molecular analysis. New DNA sequence of this Apatemon species was obtained from a black-backed gull and two common bullies. Sequences were also generated from two specimens of Diplostomum, one from the eye of a common bully (''humour metacercaria'' of Ruehle et al. 2021) and one from a Caspian tern from Otago (D. spathaceum (Rudolphi, 1819)), which were collected during a different study, and used here as additional diplostomid data. Genomic DNA was extracted using the Dneasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The D2 domain of the large subunit ribosomal DNA (28S) was amplified using primers T16 (5 0 GAGACCGATAGC GAAACAAG-TAC 3 0 ) and T30 (5 0 TGTTA GACTCCTT GGTCCGTG 3 0 ) (Harper & Saunders, 2001) and conditions of 94°C for 5 min, 38 cycles of 94°C for 30 sec, 45°C for 30 sec and 72°C for 2 min, and 72°C for 7 min. All polymerase chain reaction (PCR) products were cleaned using EXOSAP-TM TM 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 imported into Geneious PrimeÒ version 1.2, trimmed using the trim function with default parameters and manually edited for incorrect or ambiguous base calls. They were aligned, together with published sequences of strigeids from GenBank (Table 1), using MAFT alignment tool in Geneious Prime. Bayesian inference analysis was conducted with MrBayes version 3.2.6 using the online interface Cyberinfrastructure for Phylogenetic Research (CIPRES) Science Gateway (Miller et al. 2010). Each analysis used random starting trees for 2 runs (each 1 cold and 3 heated chains), employing a Markov chain Monte Carlo (MCMC) approach for sampling the posterior probability distribution across 10,000,000 generations, sampling every 1,000 generations. The first and last 25% of samples were discarded as ''burn-in''. Mixing and convergence of each run were monitored through statistics provided in Mr. Bayes and Tracer v1.6.0 (Rambaut et al., 2014). The resulting phylogenies were summarized in a 50% majority-rule consensus tree with clade credibility support values (Bayesian posterior probability, bpp) and branch length information; bbp higher than 0.95 was considered strong support for nodal positions. Genetic divergence was estimated in Geneious. Newly generated sequences were submitted to GenBank under accessions OM949805-9.
formed a trichotomy with Apatemon cf. hypseleotris from Australia, and A. gracilis from Norway and an unnamed species of Apatemon from Japan, support for which was strong. Sister to these were an unknown species from a mallard duck in New Zealand, and another from Norway. This clade combined with one containing Australapatemon spp., and one with Apharyngostrigea spp., to form an unresolved trichotomy. This phylogeny further supports the standing of Apatemon and Australapatemon as distinct genera (Blasco-Costa et al. 2016). Parastrigea brasiliana which sits uncomfortably within the Apharyngostrigea clade, has recently been moved to Apharyngostrigea (López-Jiménez et al. 2022), but we have retained the original GenBank name in the tree for ease of reference.
A single A/G dimorphism produced internal structure in the Apatemon clade, in which the sequences were otherwise identical. Interspecific divergence between Apatemon jamiesoni n. sp. and other Apatemon species ranged from 0.4% to 1.6%. Average genetic divergence between species of Apatemon and Australapatemon was 4.9% for the 28S marker, which fell within the range of variation of other strigeid genera (1.7-9.0%). Unfortunately, attempts to sequence internal transcribed spacers (ITS1&2) and cytochrome oxidase 1 (cox1) markers were unsuccessful, so it is not possible to present phylogenies for these markers at this time. However, the new 28S phylogeny presented here includes additional samples to that of Blasco-Costa et al. (2016) and demonstrates the genetic identity of the new specimens and their relationship to other genetically characterised species.   Specimens collected. Thirty-two from black-backed gull, 40 from spotted shag, 1 from mallard. Prevalence. In one black-backed gull out of 50; in one spotted shag out of 35; in one out of 16 mallard; 100 % of 77 common bully from Lake Waihola; in 2 out of 3232 P. antipodarum from Lake Waihola (0.06 %). Intensity in second intermediate host: 9-800 individuals, mean intensity 204, in common bully from LakeWaihola. Etymology. The species is named in memory of the late Professor Ian Jamieson, friend and colleague, who through his research contributed enormously to bird conservation in New Zealand. Description (Fig. 2) [Based on holotype specimen from Larus dominicanus Lichtenstein. Measurements of all other specimens given in Table 2. Measurements in micrometres].
Metacercaria and cercaria Full descriptions, illustrations and measurements of metacercariae from common bully Gobiomorphus cotidianus McDowall and cercariae from Potamopyrgus antipodarum (Gray) are given in Blasco-Costa et al. (2016).

Remarks
In having a bipartite body, cup-shaped prosoma, subcylindrical opisthoma, vitellarium confined to opisthoma, tandem testes, a small genital cone poorly delimited from parenchyma, and ringnapf absent (Niewiadomska 2002), these specimens conformed to the genus Apatemon. A full comparison with other species of Apatemon is given in Blasco-Costa et al. (2016), including a table of measurements. The new specimens have increased the range of metrics for this species description (see Table 2), but the new ranges have little altered the comparative diagnosis when all eight valid Apatemon species and subspecies are considered.
The new specimens were compared against all congeners by consulting the original descriptions. Apatemon jamiesoni n. sp. is smaller in body size than Apatemon gracilis (Rudolphi, 1819) (as re-described by Dubois (1968)), A. hypseleotris Negm-Eldin & Davies, 2001, A. somateriae somateriae (Dubois, 1948), A. s. fischeri Dubois, 1968, A. vitelliresiduus Dubois & Angel, 1972, A. buteonis (Yamaguti, 1933 and A. fuligulae Yamaguti, 1933. Notably, the length of the ovary and size of the testes of Apatemon jamiesoni n. sp. lie below the range of all the above species. Likewise, Apatemon jamiesoni n. sp. measurements lie outside of those for the suckers and pharynx of A. s. fischeri. In addition, A. vitelliresiduus has vitellaria that extend into the prosoma and A. buteonis has post-equatorial testes and a ringnapf. Moreover, A. gracilis has metacercarial cysts that are lemon shaped as opposed to the egg-shaped cysts of Apatemon jamiesoni n. sp. and A. hypseleotris and A. fuligulae have notably smaller cysts.  139-180 respectively). Furthermore, A. jamesi is unusual in having a leech second intermediate host. -Costa et al. (2016) took the prudent step of not publishing a formal naming of the Apatemon specimens found during their study due to a lack of suitably well conserved specimens. However, a full description of their single specimen was provided, along with descriptions and illustrations of the larval stages, and the provisional name Apatemon sp. ''jamiesoni'' was given, pending discovery of further specimens. In finding new specimens, defined by genetic identity with the original specimens from the mallard and the spotted shags, the author has hereby been able to complete the erection of the new species as Apatemon jamiesoni n. sp.

Blasco
The new specimens described here raise a number of questions. Generally thought of as marine birds, the spotted shag and black-backed gull seem unlikely hosts of a parasite whose first and second intermediate hosts are found in fresh to brackish water. However, spotted shags are known to forage and roost in estuaries, and black-backed gulls in New Zealand Data from a single bird host in each case. n = number of worms from which data were taken are found anywhere from coastal waters to highcountry farms, particularly during the breeding season (Szabo 2013;Miskelly 2013). An interesting observation is worthy of note here: Apatemon jamiesoni n. sp. specimens were found in only a single blackbacked gull, which was also infected with Cryptocotyle micromorpha Presswell & Bennett, 2022a, 2022b, a gastrointestinal trematode only known from freshwater habitats. This was the only bird out of fifty that the author has examined to have parasites of a freshwater origin, underlining that, despite their cosmopolitan occurrence, black-backed gulls remain preferentially foragers of the sea shore. A further puzzle that occurs is the apparent rarity of Apatemon jamiesoni n. sp. in birds frequenting freshwater and brackish habitats. We have examined a large number of water fowl individuals from localities where the metacercariae are found in huge numbers in common bullies, and yet only in these three disparate birds have they been discovered as adults. The mallard was found in a terrestrial environment and the shag and gull are preferentially seabirds. A recent investigation into three specimens of Australasian crested grebe and one white-faced heron from Lake Wanaka (Presswell & Bennett 2022b) found thousands of metacercarial cysts and freshly excysted juveniles in the stomachs of both species, but none in the intestine, and no adults at all. Although DNA sequencing was not possible from those specimens it was proposed that they were the metacercariae of Apatemon jamiesoni n. sp. which were unable to mature in these two hosts. Which leaves the question, through which definitive host is this species of strigeid completing its lifecycle in large numbers, considering the high prevalence and intensity in bullies? Further collection of freshwater birds is needed to answer this question. Finally, this new species, confirmed and named in this paper, represents only one more strigeid in the trematode list for New Zealand. Undoubtedly, as stated in Blasco-Costa et al. (2016) there are more discoveries to be made. The author's collection includes a genetically distinct Apatemon sp. from another mallard found in Mackenzie District (included in phylogeny, Fig. 1) and a further morphological variant from New Zealand scaup, along with a species of Strigea from Australasian harriers, all of which have yet to be described.