Bird cestodes from Huinay (Comau Fjord), Chilean Patagonia: several species of the family Dilepididae (Platyhelminthes, Cyclophyllidea), with the erection of two new genera

Abstract Birds in the Huinay area, Los Lagos region, Chile, were studied for parasites. Here we report 2 new genera and species of the family Dilepididae (Cyclophyllidea) found in common local passerines: Janinelliapeebeehigen. n., sp. n. was found in Elaeniaalbiceps (Tyrannidae) and Huinaylepiselegansgen. n., sp. n. was found in Aphrasturaspinicauda (Furnariidae). Other dilepidid parasites are reported for the first time from Xolmispyrope (Tyrannidae) and from 2 species of Rhinocryptidae. Cotylorhipissureshi Jadhav & Shinde, 1981 is considered a species inquirenda. The very high diversity and endemism of the observed cestode fauna in the Valdivian temperate rain forests is noted.


Introduction
Chile is home to a very rich avifauna comprising more than 460 species (Jaramillo et al. 2003). However, apart from a few isolated reports limited to a small number of hosts (i.e. Babero et al. 1981;Torres et al. 1991;González-Acuña et al. 2017), very little is known of the cestode fauna of birds in this country. In the frame of a global and coordinated effort to improve our knowledge of the diversity of the Cestoda (Platyhelminthes) (Caira and Jensen 2017), the authors spent two weeks collecting bird tapeworms in coastal Valdivian temperate rain forests around the Huinay Scientific Field Station (HSFS) (Chile) in November and December 2008. A total of 87 birds belonging to 19 species were examined for parasites during this trip and 31 of them (36%) belonging to 12 species (63%) were found infected with one or more species of cestode. The diversity and novelty of this parasitic fauna is surprisingly high: all the parasite taxa recovered are new for Chile and several are new for science. Two earlier contributions based on this material described a new hymenolepidid genus from Trochilidae (Widmer et al. 2013) as well as a new species of the genus Anonchotaenia (Paruterinidae) from the tyrant flycatcher Elaenia albiceps (Tyrannidae) (Phillips et al. 2014).
This paper reports on a series of tapeworms of the family Dilepididae found in hosts belonging to the Furnariidae, Tyrannidae and Rhinocryptidae, including 2 new genera and species.

Methods
Hosts were caught with mist nets, kept in fabric bags for a short time, then killed with an overdose of diethyl-ether, and autopsied immediately after their death. The digestive system was entirely removed, cut in sections if necessary, and searched for parasites under a dissecting microscope. Cestodes were fixed in hot 4% formalin after a small fragment was preserved in ethanol for DNA studies. Fixed specimens were transferred to 70% ethanol for storage. Further treatment included staining with Mayer's hydrochloric carmine, dehydrating in an ethanol series, clearing in eugenol and mounting in Canada balsam. Some scoleces were prepared in Berlese's medium for examination of their rostellar armament.
Drawings and photographs were made respectively with a drawing tube and a digital camera on a Nikon 80i microscope. Unless otherwise stated all measurements are in micrometers. Minimum and maximum values are reported followed by the mean and number of observations in parentheses when applicable. Conventions for dilepidid descriptions follow Bona (1994). All material is deposited in the Invertebrates Department of the Museum of Natural History of Geneva (MHNG). The nomenclature of birds follows the latest available edition of Avibase (Lepage 2018). Partial COI (cox1) sequences were generated according to the methodology described in Scholz et al. (2013). These sequences are deposited in Genbank under accession versus a longer straight one in our specimens. Furthermore, Pseudochoanotaenia is presently restricted to Apodiformes and has never been reported from South America. In consequence, we consider the present material as belonging to a new genus. Etymology. The species name phonetically reminds one of the acronyms of the NSF program that was sponsoring the project (Planet Biodiversity Inventory, PBI). Description. Body of small to medium size, up to 34 mm long (inferred from fragmented specimen). Strobila with almost parallel margins, gradually expanding in posterior direction: immature, mature, pregravid and gravid proglottides up to 250, 550, 700 and 1025 wide, respectively. Maximum width achieved at level of early gravid proglottides. Most complete specimens consisting of up to 157 proglottides. Proglottides weakly craspedote, wider than long except for very last gravid ones, which can be up to twice longer than wide. Two pairs of osmoregulatory canals. Ventral canals up to 50 in diameter, with posterior transverse anastomosis in each proglottis. Dorsal ones up to 11 in diameter. Scolex ( Fig. 1) not clearly separated from neck, 210-295 (258, n = 3) wide at middle of suckers; anterior part of scolex conically tapering; apex may form pipettelike protrusion. Suckers weakly muscular, oval 110-137 × 82-105 (120 × 96, n = 12). Apical apparatus consisting of oval rostellar pouch, 127-142 × 65-75, thin-walled, densely filled with glandular tissue, reaching to level of middle to posterior half of suckers. No rostellum. Neck straight, 225-325 wide. Genital ducts passing between osmoregulatory canals. Genital pores at border of first third of lateral proglottis margin, often forming genital papilla, irregularly alternating. Genital atrium simple, inconspicuous, thin-walled, tubular, with infundibular orifice. Testes 13-17 (14.8, n = 25) in number, disposed in two dense layers, forming a continuous field filling most of posterior two thirds of median field of proglottides; posterior, lateral and dorsal to vitellarium, overlapping posterior parts of ovary; in younger proglottides, testes may occupy most of dorsal parenchyma (Fig. 2); testes 75-100 (89, n = 15) in diameter. External vas deferens very developed, forming multiple coils at extremity of cirrus-sac, filling the antero-poral and central part of median field. Cirrussac 130-182 × 25-38 (157 × 32, n = 30), oblique, straight, thin-walled, reaching or crossing poral osmoregulatory canals. Internal vas deferens coiled, making several loops in proximal half of cirrus-sac (Fig. 3). Evaginated cirrus short (up to 44 long), almost cylindrical, thin, 8-9 in diameter at its basal part, slightly tapering distally; unarmed.
Remarks. The new species is the type species of the newly erected monotypic genus Janinellia gen. n.
Diagnosis. Dilepididae, Dilepidinae. Small strobila. Rostellum armed with two rows of hooks with peculiar and irregular 2-1 alternation. Rostellar pouch glandular. Suckers armed on anterior half, with largest antero-central hooks and progressively shorter ones laterally. Genital pores irregularly alternating. Genital ducts dorsal to osmoregulatory canals. Cirrus sac reaching osmoregulatory canals. Cirrus armed with strong spines. Testes in one field extending bilaterally and often also anteriorly to form circle reaching anterior proglottis margin. Ovary small. Uterus labyrinthic. Parasite of South American passerines (Furnariidae).
Etymology. The genus name (feminine) derives from the name of the locality and the Latin suffix -lepis (scales).
Remarks. Dilepidids with armed suckers, especially armed with true hooks and not merely spines, are very uncommon. The present material can only be compared with Cotylorhipis Blanchard, 1909, which is also found in South American furnariid birds. Although the general aspect of C. furnarii (Del Pont, 1906) and our specimens is similar because of the obvious sucker armament and despite an incomplete description of the former taxon [based on Del Pont (1906) as reported and completed in Blanchard (1909)], a number of characters easily separate them. Cotylorhipis specimens do not have any rostellum, their sucker armament is complete (all around the suckers circumference), and the testes never reach the anterior proglottis margin whereas our material shows an obvious armed rostellum in a large and distinctive rostellar pouch; sucker armament is restricted to the anterior half of suckers and testes are often far found anterior. Furthermore, the terminal gravid proglottides contain thousands of eggs and their length reaches up to 4-5 times their width in Cotylorhipis, while there are only a few hundreds of eggs and they are twice as long as wide in our specimens. These characters are sufficient to separate our material from Cotylorhipis and we propose to place it in the new genus Huinaylepis. It is, however, very likely that Cotylorhipis and Huinaylepis are closely related given the general appearance of their genital anatomy, presence of armed suckers, and shared host and geographical distribution. Description. Body of small size, up to 18 mm long and with maximum width 600 at level of gravid or late mature proglottides. Up to 36 acraspedote proglottides (observed; maximum number of proglottides inferred from various fragments is about 44). Proglottides initially wider than long, progressively becoming as long as wide at level of male proglottides, then up to about twice as long as wide (terminal gravid proglottides). Development of strobila with marked steps instead of being progressive (similar to serial maturation sensu Spasskii 1951), i.e. with maturation of cirrus sac, testes or eggs sudden from one proglottis to next. Two pairs of osmoregulatory canals. Ventral canals with posterior transverse anastomoses in each proglottis. Scolex (Fig. 6) rounded. Rostellum musculo-glandular, mushroom-shaped, elongate, 67-112 × 40-55 (96 × 48, n = 18), with maximum diameter at level of crown of hooks. Rostellar pouch oval, 105-142 × 65-92 (126 × 78, n = 17), densely filled with glandular masses, reaching but usually not exceeding posterior margin of suckers. Rostellar hooks 29-35 in number, 16-18 long, in two rows, with particular 1-2 arrangement (1 hook on anterior row alternates with two on posterior row) but with recurring irregularities (i.e. 1-1-1, 2-2-2, 1-3-1) or sometimes intermediate positions (Fig. 11C-E). Hooks with long handle with small epiphyses, short blade and massive guard (Figs 7, 11E, F). Suckers round to slightly oval, with maximum diameter 60-90 (76, n = 79), bearing highly visible and very typical anterior semicircle of 12-17 (14.3, n = 109) hooks of similar shape as those of rostellum but with proportionally longer handle (Fig. 11A, B); epiphyses of sucker-hooks not always well marked but maybe conspicuous on handle (Fig. 11F); hooks of suckers up to 40 long, larger centrally and becoming gradually shorter (20-25) laterally (Fig. 11A B). Neck very short, formation of proglottides appears immediately behind scolex. Genital ducts dorsal to osmoregulatory canals. Genital pores alternating irregularly in short series, at border of first fifth of lateral proglottis margin.
Testes 16-22, rarely only 14 or up to 24 (19, n = 84) in number, disposed in entire median field reaching anterior proglottis margin and forming U-shaped field, with converging branches, often forming circle with one or two anterior testes linking the two lateral fields (Fig. 8), essentially in one layer, although some posterior testes may be superimposed; testes reaching up to 85 in diameter in late mature proglottides. Vas deferens coiled just at antiporal end of cirrus sac, often poorly visible but occasionally well-developed (up to 12 in diameter) and filling antero-central space. Cirrussac ( Fig. 9) 90-137 × 50-77 (119 × 62, n = 55), ovoid, thin walled, anterior, with proximal extremity often touching anterior limit of proglottis, usually crossing poral osmoregulatory canals. Cirrus massive, armed with clearly visible spines (about 2 long) except on apex, 22-25 in diameter and reaching up to 145 long when evaginated. Internal vas deferens forming several coils.
Remarks. The new species is the type species of the newly erected monotypic genus Huinaylepis gen. n.
In 2005, the late Prof. Franco V. Bona (Torino) left his tapeworm collection to the MHNG. Among this rich material we found 35 slides of worms collected in Aphrastura spinicaudata and Pygarrhichas albogularis. All material was collected in the Valdivia Forest (about 250 km N of our study area) on 19-20 January 1985. On his accompanying notes, Bona noticed the interest of this material and indicated that it belonged to a new genus on some slides. Most of Bona's specimens are juveniles, a few are mature and only a couple of proglottides are pregravid. We have examined all these slides and this materiel fully corresponds to Huinaylepis elegans sp. n. as described above. The only minor variations we noticed were two specimens with a slightly higher number of hooks than in the type series (36 and 38). These observations imply that H. elegans is probably more widespread in southern Chile than the few occurrences reported here may suggest. Over 30 species of Furnariidae are known in the country and, so far, have never been investigated for their parasites.
Cotylorhipis was a monotypic genus until Jadhav and Shinde (1981) described C. sureshi in domestic fowl from Aurangabad in India. Both the description and illustrations of this species are extremely succinct and of substandard quality. It is not even clear whether hooks reported by Jadhav and Shinde (1981) as "fanlike outgrowth", and illustrated by asterisks, are present on suckers or not! In addition, and according to B. Jadhav (personal communication to M. Oros), there is no type material for C. sureshi. The disjoint geographic distribution, unrelated host, as well as the few morphological details given by Jadhav and Shinde (1981) make it highly dubious that their material belongs to Cotylorhipis. There is no possibility of checking the validity or taxonomic position of the material described as Cotylorhipis sureshi, which must consequently be considered a species inquirenda.
Vitellarium immediately anterior to testes field, central, slightly V-shaped. Ovary lobulated and elongate transversely, anterior (poorly visible in our material). Vagina in same plane as, and posterior to, cirrus sac; wide, straight, opening in simple genital atrium about 15 deep (Fig. 14). No gravid proglottides and no early uterine development visible.
Remarks. This material is likely to represent a new species. However a complete description is not possible without observations of the uterine development. Its generic position remains uncertain. According to Bona's (1994) keys, it could belong to one of two very similar genera: Monosertum Bona, 1994or Kintneria Spaskii, 1968, both parasitic in passerine birds. These genera differ from one another essentially by the structure of their uterus, with or without capsules, a character, which we cannot determine in the present material due to the lack of gravid proglottides. A few other diagnostic characters are given by Bona (1994). Among them, the small size of the body of our specimens rather resembles Monosertum; however, this character can often vary among species in many dilepidid genera. On the other hand, it corresponds to the diagnosis of Kintneria because of its larger rostellar hooks, the ovary that doesn't reach the anterior proglottis margin and its cirrus armament consisting of short spines. For these reasons, we consider that it most likely belongs to the genus Kintneria. This genus was erected by Spasskii (1968) for a parasite of introduced European sparrows Passer domesticus (L.) in North America. The type species of this genus was identified as Choanotaenia passerina (Fuhrmann, 1908) by Kintner (1938). Spasskii (1968) considered it a distinct species, differing from the Palaearctic Monopylidium passerinum (Fuhrmann, 1908); he also placed it in the newly erected subgenus Kintneria Spasskii, 1968 within the genus Monopylidium as M. (Kintneria) capsulata Spasskii, 1968. Bona (1994 eventually elevated Kintneria to the generic rank and its validity was accepted by Mariaux et al. (2017). Kintneria is known from the Nearctic and members of Xolmis are restricted to the Neotropics; however, other tyrant-flycatchers are known to migrate between North and South America, suggesting that genera of avian cestodes may have rather Pan-American than restricted distributions. This is the first cestode ever reported from the genus Xolmis.
To our knowledge the genus is monotypic and K. capsulata can easily be separated from our material by its shorter rostellar hooks and longer cirrus sac. Thus, should the observation of gravid segments confirm the placement of the present material into Kintneria, it would belong to a new species.
The two species belonging to the other similar genus, Monosertum parinum (Dujardin, 1845) and M. mariae (Mettrick, 1958), are known from European passerine birds only; furthermore, they are characterized by an osmoregulatory system forming a complicated reticular formation in the scolex and the neck (Komisarovas and Georgiev 2007). Such complicated network of osmoregulatory canals has not been observed in the present material.

Dilepididae from Rhinocryptidae
A few specimens were retrieved from 2 species of terrestrial Rhinocryptidae with a limited distribution. Unfortunately, our material does not allow for complete descriptions of these worms. The limited available characters are briefly reported below.
Dilepididae. gen. sp. 1: Three fragments of 2 or 3 incomplete specimens, no gravid proglottides. Max length 2.6 mm for 20 proglottides weakly craspedote and wider than long, max width 560. A single scolex 312 wide. Powerful suckers 195-205 in diameter. Rostellar pouch indistinct, about 135 long. Rostellum muscular 107 × 40 with hooks mostly lost. Remaining hook fragments suggest 2 rows and a length of about 12. Neck short. Very rapid development of strobila with testes appearing in 7 th proglottides and mature proglottides in 12 th to 14 th . Genital pores regularly alternating and genital ducts passing between osmoregulatory canals. 10-14 testes (12, n = 10) in one posterior field and 1 to 2 layers, not extending past osmoregulatory canals.

Remarks
It is the first record of tapeworms in this family of birds. Despite the limited material available, key characters confirm that at least 3 different species of cestodes are present. Thus an interesting diversification of dilepidids has occurred, at least locally, in these rather elusive hosts. It would obviously be very interesting to collect more of them in various areas of their range, which encompasses most of South America.

Discussion
It is widely accepted that biodiversity is particularly important in tropical rainforests (Mittermeier et al. 2003). However, our observations in Chile serve as a reminder that other biodiversity hotspots might be of great interest for parasitologists. This is particularly the case of Mediterranean-climate regions in general, and those with unique ecological conditions like the Chilean coastal forests (i.e. Ormazabal 1993;Smith-Ramirez 2004). In the humid temperate Chilean forests we have explored, the local diversity of birds is relatively poor, and we could only sample a small number of hosts. Nevertheless, not only the global prevalence (at 36% vs about 20% on average for similar studies according to our experience) and diversity of tapeworms in these hosts was high but their originality was also exceptional. Elaenia chilensis for example was previously found to harbour another new and, until now, endemic cestode species, Anonchotaenia prolixa Phillips, Georgiev, Waeschenbach & Mariaux, 2014(Phillips et al. 2014. The presence of several new endemic lineages (that may even be more numerous once the study of the remaining material from this mission is achieved) is remarkable for the region, and should stimulate more research in similar non-tropical areas. Although more observations are desirable before generalizing these results, they already confirm the interest of the Chilean Valdivian Temperate Forest as a biodiversity hotspot as recognized by Myers et al. (2000). Although some of these forests are relatively well protected compared to those in other parts of the world, parts of them remain highly vulnerable to progressive fragmentation and other human driven influence (Wilson et al. 2005;Echeverria et al. 2008;Schmitt et al. 2009;Gillespie et al. 2012). This is also an opportunity to point out that active research programs on parasites should be part of all studies involving the collection of vertebrate specimens in such areas.