Systematic revision of the family Pleioplanidae Faubel, 1983 (Polycladida, Acotylea): new genus and combinations

New morphological information, reconsiderations and the fi rst combination of a generic name based on a previously established species are presented in the current revision of the family Pleioplanidae (Polycladida, Acotylea). Species belonging to this family are briefl y presented and Laqueusplana bocki gen. et sp. nov. is described. An identifi cation key to all valid species of Pleioplanidae is provided. Furthermore, results from phylogenetic analyses of the species treated herein are discussed.


Introduction
The family Pleioplanidae belongs to the superfamily Leptoplanoidea within the suborder Acotylea (Order Polycladida). Faubel (1983) established Pleioplanidae based on the anatomical reconstruction of Notoplana atomata (O.F. Müller, 1776) and the inner organization of its prostatic vesicle, which is divided into numerous tubular chambers that are not directly attached to the central ejaculatory duct. The prostatic vesicle organization of N. atomata ("atomata-type") is considered the main apomorphy justifying the formation of Pleioplanidae. Therefore, N. atomata was designated as the type species of the family and renamed Pleioplana atomata.
The genus Notoplana Laidlaw, 1903 (Acotylea, Polycladida) is one of the largest and most revised genera of Polycladida. Notoplana, traditionally included within the family Notoplanidae, comprised more than 70 species prior to Faubel's (1983) revision. Following this revision, the genus was reduced to 34 species. As mentioned, one of Faubel's major actions was to combine and delimit a specifi c group of Notoplana species into the family Pleioplanidae.
Two attempts to reorganize the numerous species of Notoplana were made prior to Faubel (1983). The fi rst attempt was carried out by Bock in 1913 who established three clades: group A with Notoplana evansii Laidlaw, 1903 as the main species, group B based on N. atomata (O.F. Müller, 1776) and group C with N. alcinoi (Schmidt, 1861) as the primary species. Later, Marcus & Marcus (1966) divided the genus into nine groups of species based on the presence or absence of nuchal tentacles and specifi c characteristics of the male copulatory system. However, tentacles in preserved specimens may be poorly defi ned or not visible or recognizable.

Material and methods
The examined material includes specimens collected, with the aid of the Grupo de Estudo do Medio Mariño (GEMM), from the north-western coasts of Galicia (Spain), mainly from the Ría de Arousa. Specimens were collected mainly by scuba diving, covering a bathymetric range that extends from the intertidal zone to the maximum accessible depth for scuba diving at approximately 40 metres. Most species were found under stones and rocks or on algal stalks and empty mussel shells, all of which are characteristic habitats for polyclads. Specimens were fi rst photographed in their natural surroundings, then collected by hand using a brush or net and stored in containers according to specimen size. One specimen of Pleioplana atomata found in the intertidal zone at Los Chalanos Beach, Muros de Nalón (Asturias, Spain), was also studied, as were specimens of Pleioplana megala collected from the northern coast of Ubatuba, São Paulo (Brazil), kindly provided by the Swedish Museum of Natural History.
Specimens were fi rst anaesthetized in a 7.5% magnesium chloride solution, then fi xed with temperate Bouin following the Newman & Cannon method (2003) or placed directly on frozen fi xative (Bouin), which causes them to become lethargic and immobile, and thus easily positioned. As the solution melts, specimens become submerged in the fi xative. Prior to fi xation, a lateral piece of tissue was extracted from each specimen and stored in absolute ethanol intended for future molecular studies.
Specimens were subsequently embedded in paraplast, serially sectioned between 7 and 10 μm and stained with AZAN (trichrome staining method). Reconstructions of the internal anatomy were derived from serial sagittal sections. Measurements were obtained from both living and preserved material.
In the present article, the literature was extensively revised. Using morphological information taken from this review and the analysis of new specimens, a list of morphological characters and states was compiled with the Delta software (Dallwitz et al. 1993) to produce a key, update the descriptions and generate the matrix (Table I)

Phylogenetic analysis
To better understand the evolutionary relationships and observed similarities between the different genera and species of the family Pleioplanidae, a phylogenetic analysis was performed using the following methodology and parameters: optimality criterion = parsimony; bootstrap with heuristic search; number of bootstrap replicates = 1000; number of characters resampled in each replicate = 45. Character-status summary of 45 total characters: all are of type "unordered", and all have equal weight. Of these, 19 characters are constant; 6 variable characters are parsimony-uninformative; 20 characters are parsimony-informative; and gaps are treated as "missing". Starting tree(s) obtained via stepwise addition. Addition sequence: simple (reference taxon = Pleioplana atomata; number of trees held at each step = 1. Branch-swapping algorithm: tree-bisection-reconnection (TBR) with reconnection limit = 8; initial 'Maxtrees' setting = 100; branches collapsed (creating polytomies) if maximum branch length is zero; no topological constraints in effect. Leptoplana tremellaris was designated as the outgroup (Fig. 7).
Also a neighbour joining (NJ) analysis was performed using character-state optimization with accelerated transformation (ACCTRAN).

Diagnosis
Pleioplanidae with tentacular and cerebral eye clusters; without tentacles. Male copulatory apparatus directed forward. Seminal vesicle and/or spermiducal bulbs present; penis duct forms a loop between prostatic vesicle and pharynx. Penis stylet long and pointed. Female apparatus usually with vagina bulbosa; Lang's vesicle present.

Etymology
The name Laqueusplana refers to the term "laqueus", Latin for loop. This is in reference to the loop that the stylet makes between the prostatic vesicle and the pharynx in this genus.

Other species of the genus
Pleioplana megala (Marcus, 1952) also has a forward directed male copulatory system and shows a loop between the prostatic vesicle and the pharynx. Therefore, it must be transferred to the new genus Laqueusplana gen. nov.

Etymology
The name of the species is dedicated to Sixten Bock, famous Swedish invertebrate zoologist .

Description
Living worms 25 mm long and 7 mm wide. Body shape elongated, of fl eshy consistency, broader anteriorly, then stretching to form a slightly pointed posterior end; few smooth marginal undulations ( Fig. 1A-C). Dorsal surface with pale beige ground colour and evenly distributed dark brown spots,  except in pharyngeal and genital regions; body margin transparent and spot-free (Fig. 1A). Tentacles absent; instead tentacular eyes present in two rounded clusters. Cerebral eyes form two elongated groups (between 15 and 17 eyes) next to tentacular eyes; marginal or frontal eyes lacking (Fig. 1A). Ventral surface pale brown, almost transparent. Bilobated brain with two symmetrical lobes located ventral to eyes. Pharynx occupies middle third of body, with oral pore opening ventrally in its medial region. MALE REPRODUCTIVE SYSTEM. Male copulatory organ consists of an interpolated prostatic vesicle, a true seminal vesicle and a penis papilla with a long, slender stylet, and is located almost immediately behind pharyngeal cavity (Fig. 2C). Testes located ventrally, sometimes observed between intestinal branches. Vasa deferentia run ventrally along both sides of posterior body region, swelling in spermiducal vesicles before entering seminal vesicle proximally. Seminal and prostatic vesicles forward directed ( Fig. 2A). Seminal vesicle with thick muscular walls, connected to prostatic vesicle by short and narrow ejaculatory duct (Fig. 2D). Ejaculatory duct projects proximally into prostatic vesicle, crosses it and opens near distal end. Prostatic vesicle oval-shaped, enlarged and covered with strong muscular layers. Prostatic vesicle internally subdivided into at least eight parallel tubular chambers extending from proximal end, giving characteristic citrus-like appearance (Figs 2D, 3A). Extravesicular glands can be found lining proximal end of prostatic vesicle ( Fig. 2A). Long coiled stylet emerges from distal end of prostatic vesicle, forms characteristic spiral-like loop between prostatic vesicle and pharynx, turns backwards and opens dorso-ventrally into male atrium (

Differentional diagnosis
Due to the presence of a prostatic vesicle fi lled with tubular chambers (atomata-type), separated gonopores and a vagina bulbosa, the genus Laqueusplana gen. nov. belongs to the family Pleioplanidae. However, the presence of a long stylet with a spiral loop and a of forward directed male copulatory system justifi es the erection of a new genus within this family.
Species of the genera Izmira Bulnes, 2010, Melloplana Faubel, 1983and Persica Maghsoudlou et al., 2015 share some characteristics of the Pleioplanidae family, mainly the presence of an atomata-type prostatic vesicle. However, these genera can be differentiated from the genus Laqueusplana gen. nov. et by the following: Izmira lacks cerebral eyes and Lang's vesicle and has a backwards directed male copulatory system and a penis rod; Melloplana is characterized by the presence of an unarmed, muscular penis papilla and Persica presents distinct characteristics such as the presence of nuchal tentacles, a male copulatory organ wrapped in a muscular bulb and an elongated stylet.
Laqueusplana gen. nov. and Pleioplana Faubel, 1983 are morphologically most closely related to each other, which is refl ected in a number of shared characteristics. Externally, both have an elongated body with pale ground colour, two clusters of both tentacular and cerebral eyes and lack tentacles. They also possess a ruffl ed pharynx that occupies the middle third of the body and a copulatory system that begins posterior to the pharynx. Laqueusplana gen. nov., unlike Pleioplana, has the seminal and prostatic vesicles directed forward and a longer, coiled stylet situated between the pharynx and prostatic vesicle. The female copulatory system of the genus Laqueusplana gen. nov. consists of a well-developed vagina bulbosa and an enlarged Lang's vesicle, which sometimes reaches the posterior end of the animal. In general, the female system in this genus is larger than those of other Pleioplanidae genera. Pleioplana and Melloplana show a similar female system as Laqueusplana gen. nov., although smaller in size, while Izmira and Persica present a shorter female track and a complete absence of Lang's vesicle.

Biology
Live specimens have rapid scrolling movements and, when stressed, are able to swim by rotating the body, orienting the ventral body surface towards the water surface, and shaking the body while waving its edges.

Remarks
During the study and analysis of Laqueusplana bocki gen. et sp. nov. and similar species we discovered that in the description of Notoplana atomata of Bock (1913: 202), this author mentioned that some individuals in his material showed a long stylet forming a loop. However, in his reconstruction of Notoplana atomata (Bock 1913: 201, fi g. 39) this author did not draw a stylet with a loop or a prostatic vesicle directed forward, but he photographed a specimen (Bock 1913: table XX, fi g. 3) with these characteristics. This fact means that Bock took note of the outstanding features of the new genus Laqueusplana gen. nov., but probably due to the scarcity of material did not give it any importance and considered the differences as exceptions or artefacts of fi xation that did not allow the establishment of new species. Nevertheless, it is clear that of Laqueusplana gen. nov. was present in the study of Bock but it wasn't described; therefore, we dedicate the new species to this great specialist of Polycladida. Laqueusplana megala (Marcus, 1952) (Fig. 1D). Marginal eyes absent. Tentacles absent; instead tentacular eyes present in rounded and compact clusters. Dorsal and ventral frontal eyes absent. Cerebral eyes in two clusters (Fig. 1E). Pharynx located in second or last body third, strongly ruffl ed and ventrally oriented. Oral

Biology
Specimens found on algae from the upper littoral zone.

Diagnosis
Pleioplanidae with tentacular and cerebral eye clusters; without tentacles. Male copulatory apparatus directed backwards. Seminal vesicle and/or spermiducal bulbs present. Penis duct continuous with a short (thick or thin) penis stylet. Female apparatus usually with vagina bulbosa; Lang's vesicle present.

Biology
Commonly found along coastal shores, mainly under stones.

Type locality
THAILAND: Gulf of Thailand.

Biology
Found along the coast during the low tide.

Biology
Found during low tide between rocks and sand.

Distribution
California, USA.

Note
Pleioplana inquieta Heath & McGregor, 1912, considered synonymous with P. californica by Hyman (1953). According to Hyman, P. inquieta not only shows the same morphological features as P. californica, but also shares the same habitat and distribution, namely Monterey Bay, California. Therefore, in this review, P. inquieta is considered a synonym of P. californica.

Biology
Found on a hard bottomed mussel bed of Mytilus galloprovincialis.

Diagnosis (after Bulnes 2010)
Pleioplanidae, oval to elongated, without tentacles. Tentacular eyes present; cerebral, frontal and marginal eyes absent. Male apparatus oriented backwards with a true seminal vesicle, interpolated true citrus-type prostatic vesicle, without stylet and with a penis rod. Female apparatus with vagina bulbosa; Lang's vesicle absent.

Type locality
TURKEY: Izmir Bay, Aegean Sea.

Biology
Found in sublittoral mussel beds of Mytilus galloprovincialis.

Biology
Found in the intertidal zone along the Iranian coast of the Persian Gulf.

Distribution
Iran, Persian Gulf.

Diagnosis (after Faubel 1983)
Pleioplanidae with tentacular and cerebral eye-spots; tentacles lacking. Male copulatory apparatus oriented backwards with seminal vesicle, without stylet and with penis papilla. Female apparatus usually with vagina bulbosa. Lang's vesicle present.

Biology
Found under stones and on algae, e.g., Halimeda.

Biology
Specimens were collected from the undersurface of stones deeply embedded in the sand at low tide during spring and summer.

Distribution
Japan.
The recovered topologies from both trees (MP and NJ) support the genus Persica as the sister taxon of all other family members (Fig. 7). In addition, the genera Melloplana, Pleioplana and Laqueusplana gen. nov. group together in a well-supported clade (BP=100), with Izmira as the sister group of this clade. Furthermore, based on the NJ tree, Pleioplana atomata and the two species belonging to Laqueusplana gen. nov. show greater similarity to the Melloplana species than to the other Pleioplana species.

Discussion
Following a systematic revision of the family Pleioplanidae, a new genus Laqueusplana gen. nov. is established and described. Laqueusplana gen. nov. is characterized by a very long stylet that forms a spiral-shaped loop and a forward oriented male copulatory organ (likely due to the manner in which the stylet loops). Phylogenetic analyses, used to elucidate the relationship between this new genus and other genera of Pleioplanidae, notably show the early divergence/differentiation of the genus Persica.
In contrast to other genera of Pleioplanidae, Persica has nuchal tentacles. In addition, its reproductive system is distinct, having prostatic glands distributed over the entire surface of the prostatic vesicle, a male copulatory organ wrapped in a muscular bulb and an elongated stylet.
The phylogenetic analyses also recovered the two species of the genus Izmira as a monophyletic group, which form the sister group of all the other species of the family, with the exception of Persica. The genus Izmira is differentiated from the other genera by the absence of cerebral eyes, gonopores located closely together, a backwards-directed vagina and an unarmed male copulatory organ with a penis rod.
Our results support a close evolutionary relationship among Pleioplana, Melloplana and Laqueusplana gen. nov. The species of these genera are characterized by, in Pleioplana, the presence of a straight, wide or narrow, stylet, in Melloplana by an unarmed male organ without stylet or rod and in Laqueusplana gen. nov. by a long, spiral-shaped stylet. Although Laqueusplana bocki gen. et sp. nov. and L. megala comb. nov. have previously been described as belonging to the genus Pleioplana, and specifi cally in the case of L. bocki as P. atomata, both Laqueusplana gen. nov. species differ from P. atomata in the length and shape of the stylet and the orientation of the copulatory organ.
would like to thank Melinda Modrell for kindly reviewing the English and the two anonymous reviewers for the careful corrections and valuable comments they made on the manuscript.