Pasipha liviae Marques & Leal-Zanchet 2022, sp. nov.
Creators: name: Marques, Alessandro Damasceno
name: Hartmann, Alef
name: Valiati, Victor Hugo
name: Leal-Zanchet, Ana Maria
Publisher: name: Zenodo
Year: 2022
Subjects: Biodiversity
Taxonomy
Animalia
Platyhelminthes
Tricladida
Geoplanidae
Pasipha
Pasipha liviae

description: Pasipha liviae Marques & Leal-Zanchet, sp. nov.
urn:lsid:zoobank.org:act: 9217826F-C114-4691-83DB-9FB1ED329BDC
Etymology: The specific name honours Dr Livia Medeiros Cordeiro, for her valuable contributions to this study and the zoology and biospeleology of Brazil.
Type material. Holotype: MZUSP PL. 2287: leg. A. Marques, 23 May 2019, Bonito (surroundings of “ Gruta das Pitangueiras ”), state of Mato Grosso do Sul, Brazil —anterior tip: transverse sections on 13 slides; anterior region at the level of the ovaries: sagittal sections on 21 slides; pre-pharyngeal region: transverse sections on 13 slides; pharynx: sagittal sections on 40 slides; copulatory apparatus: sagittal sections on 64 slides.
Paratypes: MZU PL. 00321: leg. G. Iturralde, 23 May 2019, Bonito (surroundings of “ Gruta das Pitangueiras ”), state of Mato Grosso do Sul, Brazil —preserved in 70% ethanol. MZU PL. 00322: leg. T. Dolacio, 23 May 2019, Bonito (surroundings of “ Gruta das Pitangueiras ”), state of Mato Grosso do Sul, Brazil —preserved in 70% ethanol. MZU PL. 00323: leg. G. Iturralde, 23 May 2019, Bonito (surroundings of “ Gruta das Pitangueiras ”), state of Mato Grosso do Sul, Brazil —preserved in 70% ethanol. MZU PL. 00324: leg. G. Iturralde, 23 May 2019, Bonito (surroundings of “ Gruta do Mimoso ”), state of Mato Grosso do Sul, Brazil —preserved in 70% ethanol. MZU PL. 00325: leg. L. Cordeiro, 23 May 2019, Bonito (surroundings of “ Gruta das Pitangueiras ”), state of Mato Grosso do Sul, Brazil —preserved in 70% ethanol. MZU PL. 00326: leg. T. Dolacio, 23 May 2019, Bonito (surroundings of “ Gruta das Pitangueiras ”), state of Mato Grosso do Sul, Brazil —preserved in 70% ethanol. MZU PL. 00327: leg. T. Dolacio, 23 May 2019, Bonito (surroundings of “ Gruta do Mimoso ”), state of Mato Grosso do Sul, Brazil —prepharyngeal region: transverse sections on 11 slides; copulatory apparatus: sagittal sections on 30 slides. MZU PL. 00328: leg. T. Dolacio, 23 May 2019, Bonito (surroundings of “ Gruta do Mimoso ”), state of Mato Grosso do Sul, Brazil —preserved in 70% ethanol. MZU PL. 00329: leg. G. Iturralde, 23 May 2019, Bonito (surroundings of “ Gruta do Mimoso ”), state of Mato Grosso do Sul, Brazil —pre-pharyngeal region: transverse sections on 12 slides; pharynx and copulatory apparatus: sagittal sections on 27 slides. MZU PL. 00330: leg. A. Castro, 23 May 2019, Bonito (surroundings of “ Gruta do Mimoso ”), state of Mato Grosso do Sul, Brazil —preserved in 70% ethanol.
Diagnosis. Species of Pasipha with dorsal surface brownish, becoming reddish at the anterior tip, and a narrow pale-yellow median stripe bordered by greyish pigmentation; dorsal eyes with clear halos; glandular margin absent; pharynx collar-shaped; prostatic vesicle showing two portions, a pear-shaped proximal portion and a globose distal portion separated by a constriction; female atrium ample and funnel-shaped; male and female atria with independent muscle coats and separated by a dorsal fold.
Type-locality: Bonito, state of Mato Grosso do Sul, Brazil
Distribution: Known only from areas of semideciduous Forest from its type-locality.
Description
External features. Body elongate and flat with parallel margins; anterior tip rounded and posterior tip pointed (Figs. 28–32). Maximum length reaches 78 mm when creeping and 68 mm after fixation. Mouth and gonopore located in the posterior third of body in mature specimens (Table 3).
Dorsal surface of live animals brownish, becoming inconspicuously reddish at the anterior tip in some specimens, with a pale-yellow median stripe bordered by greyish pigmentation (Figs. 28–31) that may spread onto the dorsal surface, giving a greyish colour to the entire dorsal surface in some specimens (Fig. 29–31). The median stripe begins close to the anterior tip and ends close to the posterior tip. Ventral surface pale yellow with some small greyish dots and an inconspicuous whitish median stripe (Figs. 30, 32). After fixation, dorsal colour fades (Fig. 31).
Eyes, initially uniserial and monolobate (pigment cups of about 20–30 µm in diameter), contour anterior tip and, after that, become pluriserial. Eyes become trilobate (pigment cups of about 16–20 µm in diameter) behind the cephalic region (approximately 7% of the body length), being surrounded by clear halos, and spread onto the dorsal surface, occupying almost the whole body width (Fig. 33).
Sensory organs, epidermis and body musculature. Sensory pits, as simple invaginations (30–33 µm deep), contour cephalic region in a single row (Figs. 34–35). Creeping sole occupies the whole body width (Table 3).
Three types of gland discharge through dorsal epidermis and creeping sole of pre-pharyngeal region: rhabditogen cells with xanthophil secretion, cyanophil glands with amorphous secretion, as well as xanthophil glands with coarsely granular secretion. Cyanophil glands with amorphous secretion, xanthophil glands with finely granular secretion, erythrophil glands with coarsely granular secretion and scarce rhabditogen glands with small rhabdites open through the creeping sole. Glandular margin absent (Fig. 36).
Cutaneous musculature with the usual three layers (circular, oblique and longitudinal layers), longitudinal layer with thick bundles (Figs. 34–38). Mc:h 9–13% (Table 4). The musculature is thicker medially, about two times thicker than the epidermis, becoming progressively thinner towards the body margins. The ventral musculature is thicker than the dorsal musculature at the sagittal plane in the pre-pharyngeal region of the holotype (Table 4). Thickness of cutaneous musculature gradually diminishes towards anterior tip.
Mesenchymal musculature (Figs. 36–38) mainly composed of three layers: (1) dorsal subcutaneous decussate oblique muscles (about 3 fibres thick); (2) supra-intestinal transverse muscles (about 4–5 fibres thick); (3) subintestinal transverse muscles (about 4 fibres thick). In addition, there are scattered dorsoventral fibres and ventral subcutaneous oblique fibres. The mesenchymal musculature of the cephalic region of the body is less developed than in the pre-pharyngeal region (Fig. 34–35).
Pharynx. Pharynx collar-shaped, about 7% of body length. The mouth is situated in the median third of the pharyngeal pouch. Pharynx occupies almost whole length of pharyngeal pouch. Oesophagus absent (Fig. 39).
Pharynx and pharyngeal lumen lined by ciliated, columnar epithelium with insunk nuclei. Pharyngeal glands constituted by four gland types: numerous xanthophil glands with coarse granules and cyanophil glands of two types (with coarse granules and amorphous secretion), besides less abundant erythrophil glands with coarse granules. Outer pharyngeal musculature (1–4 µm thick) comprised of a subepithelial layer of longitudinal muscles, followed by some circular fibres. Inner pharyngeal musculature (11–40 µm thick) composed of a thick subepithelial layer with circular fibres, mixed with longitudinal fibres.
Reproductive organs. Testes arranged biserially on either side of the body, located beneath the dorsal transverse mesenchymal muscles (Figs. 36, 38). Testes begin slightly anteriorly to the ovaries, in the anterior third of the body, and extend to near the root of the pharynx (Table 4). Sperm ducts dorsal to ovovitelline ducts, medially displaced (Fig. 37), forming spermiducal vesicles laterally to the pharynx. Spermiducal vesicles extend laterally to the penis bulb and recurve to open terminally into the forked portions of the prostatic vesicle (Figs. 41–43). Large prostatic vesicle extrabulbar, with folded walls, adjacent to the pharyngeal pouch. This vesicle shows two portions separated by a narrow canal: proximal portion forked, pear-shaped, and arranged ventroposteriorly; distal portion unforked, spacious and globose (Figs. 41–43). The distal portion narrows, enters the muscle coat, and opens into the sinuous ejaculatory duct. This duct opens into the proximal part of the male atrium (Fig. 41). Penis papilla of the eversible type. Male atrium long, with two differentiated main regions (Figs. 41–42). Proximal region, about anterior half (43%) of the male atrium length, obliquely orientated and laterally expanded, presenting numerous small folds. Distal region of the male atrium shows larger folds with an ampler lumen close to the gonopore (Figs. 41–42).
The prostatic vesicle is lined with ciliated tall columnar epithelium. Proximal portion of vesicle receiving numerous erythrophil glands with finely granular secretion and sparse cyanophil glands with amorphous secretion. Distal portion of vesicle receiving abundant erythrophil glands with coarsely granular secretion and scarcer erythrophil glands with finely granular secretion. The muscularis of the prostatic vesicle is thick (10–130 µm thick), being constituted of interwoven longitudinal, circular and oblique fibres. The ejaculatory duct is lined with ciliated, columnar epithelium, receiving scarce openings from glands with cyanophil granular secretion. The muscle coat of the ejaculatory duct is constituted of interwoven circular and longitudinal fibres (30–45 µm thick).
The male atrium is lined with a cuboidal to columnar epithelium, densely ciliated in the proximal region. At least three types of glands open through the almost entire surface of the male atrium: two types of cyanophil glands (with coarsely granular and finely granular secretion) and glands with xanthophil, coarsely granular secretion, the latter being more abundant in the proximal region of the atrium. The muscularis of the male atrium is mainly comprised of circular fibres mixed with longitudinal fibres (10–30 µm thick).
Vitelline follicles well developed, situated between the intestinal branches. Ovaries oval-elongate (Fig. 40), about four times longer than wide, measuring about 0.4 mm in length; they are located in the anterior third of the body (Table 3), dorsally to the ventral nerve plate. Ovovitelline ducts emerge dorsally from the median third of the ovaries (Fig. 40) and run posteriorly immediately above the nerve plate. Posteriorly to the female atrium, these ducts run medially and unite to form a short and ascending common glandular ovovitelline duct, which enters the common muscle coat in a postflex posterior approach to open into the ental portion of the female canal (Figs. 41–42, 44). Female canal almost horizontal and located posteriorly to the female atrium. Female atrium oval-elongate, showing a length of about one-third of the male atrial length. The female atrium shows an ample lumen, narrowing towards the gonopore.
Ovovitelline ducts and common ovovitelline duct lined with ciliated, cuboidal epithelium and covered with intermingled circular and longitudinal muscle fibres. Shell glands with erythrophil secretion empty into the common glandular ovovitelline duct as well as into the ovovitelline ducts up to the male atrium level (Fig. 41–42, 44). The female canal and atrium are lined by a ciliated, pseudostratified epithelium, exhibiting irregular height and multilayered aspect in the proximal portion of the female atrium (12–54 µm thick). Three types of secretory cells open through the epithelium of the female atrium and canal: cyanophil glands of two types (with finely and coarsely granular secretions) and scarcer erythrophil gland with finely granular secretion. The muscularis of the female atrium and canal is thin (about 7–27 µm), thinner on the proximal portion, comprising interwoven circular and longitudinal fibres.
Male and female atria with independent muscle coats. A large dorsal fold separates male and female atria. Gonoduct inclined forward at the sagittal plane (Figs. 41–42) and lined with ciliated, columnar epithelium containing lacunae. Muscularis of the gonoduct consisting of a subepithelial layer of circular fibres, followed by a longitudinal layer (10–20 µm thick). Common muscle coat thin, with circular, longitudinal, and oblique intermingled fibres, thicker around the male atrium (Figs. 41–42).
Molecular results and phylogenetic analyses. In the alignment of 43 sequences of the COI gene, with lengths between 410 and 930 bp, we detected 270 (29%) variable nucleotide sites. The results showed that the mean divergence among 32 specimens of Pasipha was 8.5% (0.0% to 13.1%), as high as the other interspecific divergences. The mean intraspecific divergences varied between 0.0% and 0.2% (Table 5), except for P. chimbeva (Froehlich, 1955), the specimens of which showed 5.4% of mean divergence. The lowest interspecific divergence was found between P. albicaudata Amaral & Leal-Zanchet, 2018 and P. variistriata Amaral & Leal-Zanchet, 2018 (4.6%), whereas the highest divergence occurred between P. paucilineata Amaral & Leal-Zanchet, 2018 and P. chimbeva (13.1%). Regarding P. liviae., the lowest interspecific divergences occurred between it and P. hauseri (Froehlich, 1959) (7.4%), and the highest was between it and P. pasipha (Marcus, 1951) (14%). The mean divergence between P. liviae and the other species of Pasipha included in our analysis was 9.1%.
* only one specimen for the species. ** higher intraspecific divergence than that recorded for planarian species.
Maximum likelihood (RAxML) and Bayesian (MrBayes) phylogenetic trees, which were constructed based on the partial sequences of the COI gene, show a comparable topology (Figs. 45–46). Both analyses resulted in similar, highly supported trees (bootstrap = 98%; PP = 1), recovering the monophyly for Pasipha and pointing out the existence of two internal clades. One clade (bootstrap = 99%; PP = 1) contains the type-species, P. pasipha and the species P. paucillineata, P. albicaudata, P. brevillineata Leal-Zanchet et al., 2012, P. varistriata and P. chimbeva. A second clade (bootstrap = 99%; PP = 0.99) is formed by P. liviae and P. hauseri. The phylogenetic analyses were unable to assign the position of P. tapetilla (Marcus, 1951), P. pinima (Froehlich, 1955) and P. rosea (Froehlich, 1955) in relation to other species of the genus (Figs. 45–46).
In total, 32 specimens from 10 recognized species of Pasipha plus P. liviae were tested for molecular delimiters of species using a concatenated data matrix of the COI gene. The maximum likelihood of the null model (logL NULL = 131.3706), in which all sequences belong to a single species, was significantly lower than the maximum likelihood of the GMYC model (logL GMYC = 132.2362) (p-value <0.001), suggesting that the number of putative species and MOTUs is 12. Similar results were obtained by ABGD and mPTP, indicating that P. liviae corresponds to a new evolutionary lineage of Pasipha and the two analysed specimens of P. chimbeva represent two different species (Fig. 43).
Comparative discussion. Pasipha liviae Marques & Leal-Zanchet, sp. nov. can be easily assigned to the genus Pasipha, by presenting its diagnostic characteristics, such as a slender body with parallel margins; extrabulbar prostatic vesicle; an ejaculatory duct opening directly into a long and folded male atrium and female canal flexed ventrally, arising from the posterior region of the female atrium (Ogren & Kawakatsu 1990; Carbayo et al. 2013).
The new species shares some resemblances with six other species of Pasipha, namely P. carajaensis Amaral & Leal-Zanchet, 2019, P. ferrariaphila Leal-Zanchet & Marques, 2018, P. penhana (Riester, 1938), P. plana (Schirch, 1929), P. rosea (EM Froehlich, 1955) and P. velutina (Riester, 1938) (Schirch 1929; Riester 1938; EM Froehlich 1955a; b; Leal-Zanchet & Marques 2018a; Amaral et al. 2019). The shared characteristics are the following: eyes spreading over the dorsal surface, a collar-shaped pharynx, and a prostatic vesicle with two distinct portions, separated by a canal or constriction. Therefore, the following comparative discussion will be carried out considering these species.
Regarding the colour pattern, by showing a brownish dorsal surface with a narrow pale-yellow median stripe bordered by greyish pigmentation, the new species can be distinguished from P. ferrariaphila, which shows an almost homogeneous dorsal surface with a nearly imperceptible median stripe (Leal-Zanchet & Marques 2018b). Pasipha liviae also differs from P. penhana, which has four longitudinal dorsal stripes, and P. carajaensis with a yellow median dorsal band, bordered by two dark paramedian stripes (Riester 1938; Amaral et al. 2019). In addition, the new species can be differentiated from P. plana and P. velutina, which have a marbled dorsal pattern, and P. rosea that has a lighter dorsal surface covered by dark pigmentation (Schirch 1929; Riester 1938; EM Froehlich 1955a; b; Silva & Carbayo 2020).
Considering the copulatory apparatus, by having a prostatic vesicle with a pear-shaped proximal portion and a globose distal portion, P. liviae differs from P. penhana and P. velutina, which have a prostatic vesicle with tubular portions (Riester 1938). The new species can also be distinguished from P. carajaensis by the shape of the prostatic vesicle since P. carajaensis shows a small, ovoid proximal portion and an ample globose distal portion in its prostatic vesicle (Amaral et al. 2019). Pasipha liviae has a similar prostatic vesicle to those of the remaining species, namely P. ferrariaphila, P. plana and P. rosea. However, P. liviae has no differentiation along the musculature of the female and male atria, whereas P. plana and P. rosea have a distinct circular musculature in the distal portion of the male atrium and P. ferrariaphila shows a strongly developed muscularis in the proximal part of the female atrium (Riester 1938; EM Froehlich 1955a; Leal-Zanchet & Marques 2018a; Silva & Carbayo 2020).
It is worth noting that, considering the results of the phylogenetical analyses, P. liviae and P. hauseri constitute a well-supported clade. However, despite having similar external features, both species can be easily distinguished by the shape of their prostatic vesicle. In contrast to the prostatic vesicle with pear-shaped or globose portions separated by a canal in P. liviae, P. hauseri shows a tubular prostatic vesicle with long proximal portions and an Sshaped distal portion, both more elongate in the holotype, from southern Brazil, than in the specimen later found in north Brazil (Froehlich 1959; Froehlich & Froehlich 1972). The specimens of P. hauseri used in the present analysis were sampled close to its type-locality. Further analyses are necessary to validate the conspecificity of specimens from north Brazil analysed by Froehlich & Froehlich (1972) with the original specimens of P. hauseri from southern Brazil.
Notes on ecology and distribution. Paraba aurantia occurred in areas of semideciduous forest from the municipalities of Bonito and Bodoquena, showing low abundance (n=3). The areas have different levels of anthropic impact since the type locality is a preserved area and the other sampling site is a municipal leisure area that receives many visitors. In contrast, Pasipha liviae occurred in areas of semideciduous forest located in the surroundings of two caves from the municipality of Bonito, showing high abundance (n=16). Despite a considerable searching effort inside the caves, no specimens of P. liviae were found there. One specimen of P. aurantia and most specimens of P. liviae were in an immature stage.
description: Published as part of Marques, Alessandro Damasceno, Hartmann, Alef, Valiati, Victor Hugo & Leal-Zanchet, Ana Maria, 2022, Two new land planarian species (Platyhelminthes: Tricladida) from the Cerrado biome in southwestern Brazil, pp. 301-330 in Zootaxa 5205 (4) on pages 314-324, DOI: 10.11646/zootaxa.5205.4.1, http://zenodo.org/record/7306976