Morphology, genetic characterization and molecular phylogeny of the poorly known nematode parasite Cissophyllus leytensis Tubangui & Villaamil, 1933 (Nematoda: Ascaridida) from the Philippine sailfin lizard Hydrosaurus pustulatus (Eschscholtz, 1829) (Reptilia: Squamata)
Authors: Ni Xue-Feng , Chen Hui-Xia , Xu Zhen , Gu Xiao-Hong , Li Liang 
Published: April 2022

Abstract
Background
The genus Cissophyllus (Cosmocercoidea: Kathlaniidae) is a rare group of nematodes parasitic in turtles and lizards. To date, only four species have been reported in Asia and North America. However, most of them are inadequately described. The species Cissophyllus leytensis has never been reported since it was originally described by Tubangui and Villaamil in 1933 from the Philippine sailfin lizard Hydrosaurus pustulatus (Eschscholtz, 1829) (Reptilia: Squamata). Furthermore, the systematic status of Cissophyllus/Cissophyllinae in the family Kathlaniidae of the superfamily Cosmocercoidea remains under debate.

Methods
The detailed morphology of C. leytensis was studied using light microscopy (LM) and, for the first time, scanning electron microscopy (SEM), based on newly collected specimens from the type host H. pustulatus. Six different genetic markers, including nuclear sequences [small ribosomal subunit (18S), internal transcribed spacer (ITS) and large ribosomal subunit (28S)], plus mitochondrial genes [cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2) and 12S small subunit ribosomal RNA gene] of C. leytensis were sequenced. Additionally, in order to test the validity of the subfamily Cissophyllinae and clarify the phylogenetic relationships of Cissophyllus and the other genera in the family Kathlaniidae, phylogenetic analyses based on 18S + 28S and ITS sequence data were performed using maximum likelihood (ML) and Bayesian inference (BI) analyses, respectively.

Results
Our observations using LM and SEM revealed some previously unreported morphological features, necessitating the redescription of this poorly known species. The presence of remarkable morphological variation in the isthmus and the position of excretory pore among different individuals was found. Molecular analysis showed no intraspecific nucleotide divergence detected in the 18S, ITS, 28S, cox2 and 12S regions among different individuals of C. leytensis, but a low level of intraspecific genetic variation was found in the cox1 (0.52%). Our phylogenetic results showed the representatives of the Cosmocercoidea divided into four large clades (Cosmocerca + Aplectana + Cosmocercoides representing the family Cosmocercidae, Cruzia representing the subfamily Cruzinae of Kathlaniidae, Falcaustra + Cissophyllus + Megalobatrachonema representing the subfamily Kathlaniinae of Kathlaniidae, and Orientatractis + Rondonia representing the family Atractidae). The genus Cissophyllus clustered together with the genus Megalobatrachonema in both the ML and BI trees using ITS sequence data, but displayed a sister relationship to the genus Falcaustra in the ML tree and to the genera Falcaustra + Megalobatrachonema in the BI tree using 18S + 28S sequence data.

Conclusions
Molecular phylogenetic results further confirmed that the family Kathlaniidae is not a monophyletic group. The subfamily Cruziinae should be moved from the hitherto-defined family Kathlaniidae and elevated as a separate family Cruziidae. The present phylogenetic results also negated the validity of the subfamily Cissophyllinae and supported the genus Cissophyllus assigned in the subfamily Kathlaniinae. Molecular analysis indicated that the morphological variation in the isthmus and position of excretory pore among different individuals should be considered as intraspecific variation. Moreover, some characters important for the specific diagnosis of C. leytensis are reported for the first time: the number of acuminate denticles (lamellae) on each lip, the chitinized pharynx with three flabellate pharyngeal plates, the presence of single medioventral precloacal papilla and the detailed morphology of caudal papillae. The present study is only the second record of C. leytensis.

Graphical Abstract