Lyperosomum spp

Heneberg, Petr, Sitko, Jiljí, Casero, María & Rząd, Izabella, 2023, New molecular data help clarify the taxonomy of Central European avian Dicrocoeliidae Looss, 1899 (Trematoda: Plagiorchiida), International Journal for Parasitology: Parasites and Wildlife 22, pp. 276-299 : 280-285

3.1.1. Lyperosomum spp

The analyzed material contained several Lyperosomum spp . The molecular analyses revealed that the currently used morphological characteristics are highly problematic for differentiation between Ly. petiolatum and Lyperosomum turdia (Ku, 1938) . The molecular evidence supports the validity of both species [ Fig. 1 ; see also the 28S rDNA analysis by Hildebrand et al. (2019)] but these species were difficult to distinguish morphologically, particularly when comparing vouchers of these species that did not originate from identical host species. Ly. turdia is not limited to Turdus spp. , as we also collected it from Pica pica , Corvus monedula , and Scolopax rusticola . Lyperosomum petiolatum was collected from the corvids Corvus frugilegus , C. monedula , P. pica , and Garrulus glandarius , as well as Prunella modularis , Parus major , and T. merula . DNA sequencing also confirmed its presence in Falco tinnunculus (e.g., OR426412). The current descriptions of these two Lyperosomum species do not allow error-free species identification even when analyzing a large series of individuals, and further study is needed to identify new morphological characteristics (J. Sitko, pers. obs.). Relative to Ly. petiolatum , Ly. turdia has a thinner body, longer forebody, and longer distance between oral and ventral suckers. When applying molecular techniques, the differential diagnosis of Ly. petiolatum and Ly. turdia is possible using CO1 ( Fig. 1 , nodal support 99%) or ND1 loci ( Fig. 2 , nodal support 99%). The less variable loci, such as 28S rDNA ( Fig. 3A ) or 18S rDNA ( Fig. 3B ) had only low nodal support (60% and 67%, respectively). These species cannot be distinguished using the ITS2 locus ( Fig. 4 ). Note that individuals consistent with the morphological diagnosis of Ly. petiolatum have previously been also rarely collected from owls ( Strix aluco ) and birds of prey ( Falco tinnunculus ) ( Nicoll, 1915 sensu Macko, 1968; Sitko et al., 2006).

All the individuals with morphological characteristics matching those of Lyperosomum dujardini (Strom and Sondak, 1935) from Pr. modularis were genetically identical to Ly. petiolatum . We sequenced six such individualsin the present study, and another individual was sequenced previously by Hildebrand et al. (2019), with a similar outcome. Therefore, Ly. dujardini is synonymized with Ly. petiolatum .

A different situation was observed for Lyperosomum longicauda (Rudolphi, 1809) , a species characterized by the anterior edge of the vitellaria at the level of the posterior margin of the anterior testis, nearly round ovary and testes, and one row of vitelline follicles. When fresh material is analyzed, Ly. longicauda can be differentiated from other Lyperosomum spp . by a longer body, longer hindbody, higher body width/length ratio, higher forebody/hindbody ratio, and larger ventral sucker. However, the molecular analysis revealed that nearly all individuals (originating from frozen material), which were consistent with the morphological diagnosis of Ly. longicauda , represented Ly. petiolatum . However, Ly. longicauda is a valid species; one individual of this species was confirmed in C. frugilegus , but previous studies possibly included individuals representing different species. The egg size appears to be smaller in Ly. longicauda than in Ly. petiolatum ( Dolfus, 1957; Macko and Mackov´a, 1995; Hildebrand et al., 2019) but it is unclear, whether egg size could be used for the differential diagnosis of the two species. The long posterior part of the body represents another diagnostic characteristic of Ly. longicauda . However, this characteristic is problematic because the caudal part of the body of closely related Ly.

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petiolatum is elongated when isolated from hosts ≥24h post-mortem (J. Sitko, unpubl.). This diagnostic feature cannot be used for identification if materials are softened and degraded. Therefore, particularly large old adult individuals of Ly. petiolatum can be potentially misidentified as Ly. longicauda and likely were misidentified in some previous descriptions from European T. merula and Corvidae .

Lanius spp. serve as hosts to two Lyperosomum spp . First, we note that Ly. collurionis is recognized as a valid species and its synonymy with Ly. petiolatum proposed by Aldhoun et al. (2018) is rejected. See ON 695709 in Fig. 4 for an illustration of the position of this species within the ITS2 maximum likelihood tree. It is possible that the previously published CO1 sequence (KU212192) of Ly. collurionis originated from an erroneously identified Lyperosomum . We, however, did not succeed with the amplification of the CO1 locus from the present Ly. collurionis voucher. The analyzed 18S and 28S rDNA loci did not allow distinction between Ly. petiolatum and Ly. collurionis ; therefore, the sequences from likely misidentified individuals include KU212193, AY222259 (both 28S rDNA), and AY222143 (18S rDNA) aligned together with those of the two species ( Fig. 3 ). Note that the ITS2 sequence of Ly. collurionis is closely related to Skrjabinus , and that the ND1 sequence of Ly. collurionis even fall between Skrjabinus spp. The molecular delineation of Lyperosomum and Skrjabinus needs to be addressed in future studies. The host range of Ly. collurionis is unknown and may extend beyond Lanius spp.

Lanius collurio was revealed to serve as host to another undescribed species that is closely related to the Ly. turdia / petiolatum complex ( Lyperosomum sp. n. 5, described below as Lyperosomum atricapillae , Fig. 1 ). This new species has a broad host range. In addition to specimens from La. collurio , this newly described species was collected multiple times from Sylvia atricapilla , less frequently from Emberiza schoeniclus and even Scolopax rusticola and Dryocopus martius . One individual of this species was previously sequenced by Hildebrand et al. (2016), who erroneously identified it as Ly. collurionis . The differential diagnosis of this species is possible using the CO1 ( Fig. 1 , nodal support 94%) or ND1 ( Fig. 2 , nodal support 100%) loci. The 18S rDNA locus analyzed in this study did not allow distinction from Ly. petiolatum ( Fig. 3B ). The ITS2 locus of this species is identical to those of Ly. petiolatum and Ly. turdia ( Fig. 4 ).

Ly. alagesi has recently been proposed as a synonym of Ly. petiolatum based on specimens collected from P. pica (Hildebrand et al., 2019) . Here, three individuals with morphological characteristics that matched those of Ly. alagesi were sequenced, two from C. monedula , and one from P. pica . All three were identified as Ly. petiolatum based on the molecular markers ( Fig. 1 ), supporting the synonymy of Ly. alagesi with Ly. petiolatum .

Lyperosomum clathratum (Deslongchamps, 1824) was also sequenced. The molecular markers support the recognition of this species within Lyperosomum in agreement with previous reports (Hildebrand et al., 2019).

Lyperosomum sp. n. 1 (described below as Lyperosomum tenori ) was isolated from Acrocephalus schoenobaenus . Phylogenetic analysis suggested that it is closest to Ly. longicauda , Ly. collurionis and to another undescribed Lyperosomum sp. from Delichon urbicum (NCBI Acc. No. MK391431) (Hildebrand et al., 2019) ( Figs. 1–4 ). Some morphologically similar individuals were previously identified as Lyperosomum oswaldoi (Travassos, 1917) ( Table 3). The type material of Ly. oswaldoi from Brazil consisting of three individuals collected from Myospiza humeralis , coll. Freitas No. 13916 (kindly provided by Instituto Oswaldo Cruz, Rio de Janeiro, Brazil) was checked. Based on this comparison, it was concluded that the individuals analyzed here and those found previously in Europe cannot be identified as Ly. oswaldoi . They were somewhat in accordance with the original description but did not match the parameters of the fixed type material provided from the Instituto Oswaldo Cruz. The type material of Ly. oswaldoi provided from the Instituto Oswaldo Cruz has a larger body, a shorter forebody and hindbody, a higher forebody/hindbody ratio, spines on the body, smaller oral and ventral suckers, higher ratios of oral/ventral suckers dimensions, larger

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testes and vitellaria, and smaller eggs than Lyperosomum sp. n. 1 ( Table 3). The species identification by Jaron´(1969) in D. urbicum and Hirundo rustica of Polish origin is likely incorrect, as spines were not noted (but may be lost), and the trematode was partially softened and degraded, which resulted in body elongation and ventral sucker shrinkage ( Table 3). In contrast, Kopˇriva and Tenora (1961) described one trematode from D. urbicum of Czech origin that fits the variability observed in the material of Lyperosomum sp. n. 1. They identified it as Ly. collurionis , similar to Lyperosomum specimens from corvids. The dimensions provided by Kopˇriva and Tenora (1961) ( Table 3) suggest that the body length, the forebody/hindbody ratio, vitellaria length, and size of the ovary and testes do not fit within the variability of Ly. collurionis . The original specimen no longer exists. Based on the variability of the examined material, the difficulty in identifying this group of species, and the host spectrum, it was assumed that the individual examined by Kopˇriva and Tenora (1961) from D. urbicum represents Lyperosomum sp. n. 1. In contrast, the individuals examined by Kopˇriva and Tenora (1961) from C. monedula represent Ly. petiolatum . The latter Lyperosomum species is commonly hosted by corvids (see the text above for the discussion on difficulties associated with correctly identifying Ly.

petiolatum). The spectrum of hosts ( A. schoenobaenus , D. urbicum ) may be broader, as morphologically similar trematodes were also collected from Muscicapa striata . Nevertheless, as an ethanol-fixed specimen was not available, it was not possible to confirm the identification of these trematodes from M. striata at the molecular level.

Swallows host another undescribed Lyperosomum species. The sequences of this species were labeled as Lyperosomum sp. n. 4 ( Table 1; Figs. 1–4 ; described below as Lyperosomum hirundinidis ). Phylogenetic analyses revealed that it is closely related to Lyperosomum sp. n. 1, to an undescribed Lyperosomum sp. From Delichon urbicum (NCBI Acc. No. MK391431) (Hildebrand et al., 2019), and to L. collurionis ( Figs. 1–4 ).

Figures

Fig. 1. Maximum likelihood analysis of sequences of the CO1 DNA locus of Dicrocoeliidae. Bootstrap values (n = 1000) are indicated for nodal support. Black circles indicate new sequences. The scale-bar indicates the number of substitutions per nucleotide site.

Fig. 2. Maximum likelihood analysis of sequences of the ND1 DNA locus of Dicrocoeliidae. Bootstrap values (n = 1000) are indicated for nodal support. Black circles indicate new sequences. The scale-bar indicates the number of substitutions per nucleotide site.

Fig. 3. Maximum likelihood analysis of sequences of nuclear DNA loci (28S rDNA (A) and 18S rDNA (B)) of Dicrocoeliidae. Bootstrap values (n = 1000) are indicated for nodal support. Black circles indicate new sequences. The scale-bars indicate the number of substitutions per nucleotide site.

Fig. 4. Maximum likelihood analysis of sequences of the ITS2 DNA locus of Dicrocoeliidae. Bootstrap values (n = 1000) are indicated for nodal support. Black circles indicate new sequences. The scale-bar indicates the number of substitutions per nucleotide site.