New records of free-living microturbellarians from the Chillón River, Peru

Abstract Microturbellarians are all mostly small free-living Platyhelminthes that do not belong to either the Polycladida or Tricladida order. This group includes species of the clades Catenulida and Rhabditophora. Species of microturbellarians are known to live in marine and continental waters such as rivers, where they are diverse and abundant. However, there are few records of microturbellarians in most of the rivers of the Pacific slope of Peru. Here, we report eight species of microturbellarians from the Chillón River, in the central region of Peru. Of the total species recorded, five (Gieysztoria cuspidata, G. bellis, Myostenostomum vanderlandi, Stenostomum tuberculosum and S. saliens) represent the first reports on the Pacific slope of the Neotropical region in Peru, thereby increasing the diversity of microturbellarians in this country.


Introduction
Catenulida and Rhabditophora are the clades of Platyhelminthes containing species of microturbellarians that are generally short in length [1,2]. The genital systems of some species of these hermaphroditic flatworms are important for species identification, with the male copulatory apparatus containing sclerotic stylets, spines and/or cirrus [3]. These organisms live in different environments such as marine, brackish and freshwater (rivers, lagoons and temporary pools) habitats [3][4][5][6]. They have also been found in moist environments (e.g. moss, vascular plants, wood snags, detritus, gravel, or sand) [7].
With the aim of increasing the knowledge of the microturbellarian fauna, we surveyed a hydrographic basin in the center of Peru, the Chillón River. Photographs and schematic representations of the species reported are shown to facilitate recognition in subsequent assessments in other hydrographic basins.

Material and methods
The study area was the Chillón River, between the provinces of Lima and Canta, in the central region of Peru. This river flows from east to west for about 126 km (11° 22ʹS-11°56ʹS; 76°26ʹW-77°08ʹW), originating in the Viuda mountain range (~4500 meters above sea level [m.a.s.l]) and flowing towards the Pacific Ocean [32] ( Figure 1). Samples from the riverbanks at an altitude of ~500 m.a.s.l were taken almost monthly from February 2013 to November 2017. The sites sampled were characterized by muddy substrates and stones, with herbaceous vegetation and algae. Water temperature and pH of each locality were measured.
Microturbellarians were collected from aquatic herbaceous vegetation and algae using a net with a mesh size of 80 µm, and the samples were carried alive to the laboratory. All specimens were measured and described based on external and internal characteristics. Some microturbellarians were relaxed using MgCl 2 7% and examined under light microscopy. When sclerotic structures (e. g. copulatory stylets) were observed, whole-mounted preparations were performed using polyvinyl-lactophenol. Stylets and soft body tissues were measured in micrometers (μm) using the ImageJ software [33]. The material studied was deposited in the Departamento de Protozoología, Helmintología e Invertebrados Afines, Museo de Historia Natural (MUSM), Peru. Some specimens deteriorated during the observations were not deposited.

Material studied
Observations of live individuals, three of which were studied in squashed preparations.

Other localities in Peru
Pacaya Samiria National Reserve in the Loreto region [34].

Description and remarks
The color of the body is whitish, being darker in the intestinal region ( Figure 2A). The body size of worms formed by two or three zooids ranges between 797 and 2404 μm long and 108-324 μm wide. The anterior and posterior brain lobes, light-refracting bodies, and pharyngeal glands, are characteristic of the species [21] ( Figure 2B-E). The body length of specimens from Peru is within the species range (730-2000 μm) [12,13,21,26,36,37]. Kepner & Carter, 1931 ( Figure 3A)

Material studied
A single individual, studied in a squashed preparation.

Description and remarks
Live animal is yellow-whitish and fusiform. The body is 246.7 μm long and 45.7 μm wide; no zooids were observed. The anterior region has two small ciliated lateral pits, with six pairs of anterior brain lobes associated with these. Light-refracting bodies absent. The intestine has scarce excretophores with no particular arrangement, and the posterior region of the body is free of intestine. These characteristics are in accordance with the diagnosis of the species [36]. The single-zooid specimen from Peru is similar in length to those found in Argentina (150-620 μm) [21], but smaller than those in Poland (600-800 μm) [42], USA (620 μm) [36] and Brazil (400-600 μm) [13,26]. Interestingly, Kepner & Carter [36] did not mention the presence of excretophores in the intestinal region, although Larsson & Willems [43] reported that excretophores in Stenostomum saliens from Sweden are absent. Specimens from Peru present scarce excretophores, suggesting that these structures are a variable feature of the species. In addition, S. saliens has not previously been recorded in Peru, thereby expanding the distribution range of the species to the occidental coast of the Neotropical region.

Material studied
Two individuals, studied in squashed preparations.

Description and remarks
Single-zooid animals are whitish. The body is 475 μm long and 66 μm wide, is covered by long semi-rigid sensory cilia, which are more abundant at the anterior and posterior end of the body. The anterior tip of the body has a notorious tubercle. The intestine is lobulated, and the posterior body region is free of intestine. In comparison with single-zooid specimens found in other regions, animals from Peru are larger than those recorded by Nuttycombe & Waters [37] (200 μm) and Marcus [13] (200-500 μm). However, specimens of four zooids are larger than Peruvian worms [13,37], and those of two zooids are similar in length [42,44] or longer [21]. The finding of S. tuberculosum, which represents the first report in Peru, expands its distribution to the occidental border of the Neotropical region.

Material studied
Two individuals, one animal studied in squashed preparations.

Description and remarks
Single-zooid animals are whitish in color. The body is 1103 μm long and 182 μm wide. The anterior brain lobe has 7 paired metameric ganglia. The posterior brain lobe has a triangular shape. The blind-sac intestine has a muscular belt, 78.6 μm long and 155.2 μm wide at its anterior portion. Excretophores are randomly arranged along the intestine. These features agree with the specimens of M. vanderlandi from Suriname [41], Brazil [26] and Russia [46]. In addition, the body length of the individuals from Peru is within the range of the species (500-1300 μm) [26,41,46]. This is the first record

Description and remarks
Live mature animals are 900-1600 μm long. The body has dark-brownish anastomosed dots (Figures 4A-B) not mentioned in specimens studied in other regions [48,49]. In the male reproductive system, the globular seminal vesicle shows the same size as the prostatic vesicle. The penis stylet has five claw-like spines similar in length, with digitiform projections at the proximal region, without a real girdle. Each spine has a skirt-like projection for muscular insertion (Figures 4C-D). The tips of the spines are slightly curved outward. Spines are 20.5-32.1 µm long (48.5-56.7 µm long including the proximal digitiform projections). The egg is 252.7-164.8 µm long and 108.1-153 µm wide ( Figure 4A-B). Two tubular browngreenish vitellaria branches extend from the ventral posterior zone to the anterior dorsal zone, reaching the pharynx level ( Figure 4A). This is the first record of Gieysztoria cuspidata in the Neotropical region.

Description and remarks
Live mature animals are 300-700 µm long and 67-162.2 µm wide. Reddish-brown pigmentation present throughout the body ( Figure 5A). In the male reproductive system, the thick-walled seminal vesicle is 28.5-36.1 µm long and 31.2-37.1 µm wide and is completely filled with spermatozoa ( Figure 5C). The seminal vesicle is partly separated from the prostatic vesicle by a septum (Figure 5C). The "U"-shaped prostatic vesicle is 20.6-21.8 µm long and 26.7-27.3 µm wide, with granular prostatic glands ( Figure 5C). The 37-45.1 µm-long crown-like penis stylet has a smooth girdle, 2.7-8 μm high. In some stylets, the girdle has two weak distal rings (Figures 5B, E). The girdle has 20-21 fang-like spines of similar size and shape. Spines are 31.2-38.9 µm long and 2.5-3.7 µm wide, directed slightly outwards ( Figure 5E, F). The egg is 60.9 µm long and 37.3 µm wide. A pair of dark green vitellaria with finger-like projections run from the posterior end of the pharynx to the end of the intestine ( Figure 5A). The body length and the number of spines of the penis stylet of individuals from Peru are within the species range, with some differences regarding specimens from Brazil. Peruvian individuals have a shorter and smooth non-fenestrated girdle, not reported by Marcus [14]. In addition, Marcus [14] described ~12 bridges between the girdle and the 19 spines of the stylet, and the spines were not projections of the bridges. In this sense, individuals from Peru have no bridges between the girdle and the 20-21 spines, but two weak rings were present in some individuals. Also, individuals from Peru have larger fang-like spines, while Marcus [14] reported shorter and slender spines with cone-like distal tips. The girdle, could be a variable structure within species of Gieysztoria since observations in other species suggest a remarkable variation in the height of the girdle during maturity [50]. Weak distal rings observed in Peruvian individuals could suggest that the girdle presents intraspecific differences. Morphological variations of the penis stylet within species populations are usual within Gieysztoria [47,49], but the number and size of spines are usually stable [50]. This is the first record of G. bellis in Peru and the second for the Neotropical region. Prorhynchus stagnalis Schultze, 1851 (Figure 6A-B)

Material studied
A single individual observed alive, studied on a wholemounted preparation.

Description and remarks
Live mature individual ~3200 µm long and slender ( Figure 6A). The hollow spine of the male reproductive system is ~77.2 µm long, and concentric bars are ~33 µm long. The female reproductive system is at the middle of the body and is composed of a germovitellarium. The hollow spine of the penis stylet and the concentric bars of the individual studied from Peru are within the species range (50-123 µm and ~51 µm, respectively) [6,11,26,52,53]. These findings extend the distribution of this species to the central coast of Peru.

Material studied
A single individual observed alive, studied on a wholemounted preparation.

Description and remarks
Live mature, whitish-colored individual, ~2230 µm long and flattened. The anterior end is flabelliform, while the posterior end is rounded. Two brownish eyes at both sides of the pharynx. Features observed in the single individual from Peru agree with descriptions of Geocentrophora applanata [6,11,31,53]. This is the first record in Lima, on the central coast of Peru.

Discussion
Peru presents great invertebrate diversity [54][55][56][57], but microscopic aquatic invertebrates such as microturbellarians are not usually considered in assessments and inventories of biodiversity [5,30]. In the last two decades, sporadic investigations on microturbellarians were mainly performed in the rainforest region of this country [5,22,24,29,30,34]. These investigations demonstrated the potential high diversity of microturbellarians with the description of six new species [22,24,29]. However, a vast territory in which these organisms are very likely to occur remains unexplored. In this sense, the central coastal region of Peru has rivers, such as the Chillón River, which has the following features: it is a few km long, with altitudinal gradient and with notorious seasonal water level fluctuations [32]. These features create heterogeneous environments in which microturbellarians can be expected to inhabit. Nevertheless, there are only three records of microturbellarians (Macrostomum quiritium Beklemischev, 1951; M. tuba Graff, 1882 and Yagua lutheri du Bois-Reymond Marcus, 1958) living in these rivers [5,28].
Stenostomum is a rich and widely distributed genus [3,24], but species identification can be difficult to discern. Diagnostic features need to be examined in live individuals [43]. The species of Stenostomum reported here show most of the diagnostic features mentioned in their original descriptions and posterior reviews [14,21,36,37]. Individuals of Myostenostomum from Peru present a muscular belt, in addition to features on the anterior and posterior brain lobes that correspond to descriptions for M. vanderlandi [26,46,58].
The species of Gieysztoria from Peru are only represented by the Inaequales group, that is, species with spines of different shapes and stylet sizes (G. complicata (Fuhrmann, 1914) [22]. Thus, the two new records (G. cuspidata and G. bellis) represent the first record of an Aequales group species (specimens with spines of similar shape and stylet sizes) in Peru. In addition, evidence suggests that the morphological complexity of the penis stylet in the species of Gieysztoria is greater in South America [17,20,22,59]. However, consolidated information on species diversity, distribution and dispersal patterns makes it difficult to observe clearer traits in the penis stylet configuration among biogeographical regions [60]. The finding of G. cuspidata in the Neotropical region provides greater support to the hypothesis that certain groups of species (e.g. cuspidata-isoldeae) could represent an ancient lineage, with G. cuspidata already well established in the Mesozoic [60].
The species of Prorhynchidae found in Peru are wellknown and are found worldwide [6,11,34,[51][52][53]. Unfortunately, few individuals of P. stagnalis were collected. These animals are known to be fragile when studied alive and are also sensitive to temperature variations [61] thereby preventing further information gathering.
Microturbellarians play important roles in continental aquatic environments. For instance, experiments have demonstrated that species of Stenostomum prey on cladocerans and rotifers and can affect population densities and structured microinvertebrate communities [62, [63]]. Similarly, species of Gieysztoria prey on microinvertebrates as well as individuals of the same species [64]. Therefore, understanding the population dynamics of microturbellarian species in freshwater ecosystems is necessary to obtain better understanding of the trophic chains in Peruvian microinvertebrates.
In conclusion, the diversity of microturbellarians in Peru is far from being properly cataloged, with only 31 species having been recorded to date. More data is needed to provide a better panorama of the freshwater invertebrate communities and to obtain clearer evidence to assess species richness in continental waters, such as those in rivers of the Pacific slope in Peru. The results of the present study increase the number of microturbellarian species in Peru to 36 species, five of which were recorded for the first time. Likewise, one species (G. cuspidata) is recorded for the first time in the Neotropical region, providing evidence to establish a biogeographical hypothesis of distribution from geological eras.