Polycladida (Platyhelminthes, Rhabditophora) from Cape Verde and related regions of Macaronesia

The systematics and distribution of the order Polycladida within the Macaronesian archipelagos are analysed. New species (Marcusia alba sp. nov., Prostheceraeus crisostomum sp. nov., Parviplana sodade sp. nov., Euplana claridade sp. nov., Stylochus salis sp. nov. and Distylochus fundae sp. nov.), new variety (Pseudoceros rawlinsonae var. galaxy), new records and records of shared species among different archipelagos are studied to compare the marine flatworm biodiversity of each island. The complex of archipelagos known as Macaronesia (including Madeira, Selvagens Islands, Canary Islands, Azores and Cape Verde) share a volcanic origin and European political influence. The five archipelagos are located along the eastern coast of the Atlantic Ocean and are subject to similar trade winds, streams (like the Gulf Stream) and cold currents. The term Macaronesia has suffered several changes throughout the years and it still is a topic of discussion in present times. The new delimitation of Macaronesia is mainly based on systematic studies on the invertebrate fauna of the islands. The resulting analyses shed new light on the differences and similarities among these archipelagos. In addition, molecular analyses employing 28S nuclear gene sequences are compared to verify relationships among anatomically similar species of marine polyclads. European Journal of Taxonomy 736: 1–43 (2021) 2


Introduction
Exhaustive information about the external features was carefully recorded with notes, photographs and drawings. Information about pigmentation, color patterns, movement, size, and presence or absence of tentacles or eyes was gathered. Dorsal structures like papillae, warts or any type of epithelial or dermal formations were compiled.
Most of the photographs were taken on a black background with transmitted light using a Nikon D300 camera fi tted with a Micro Nikon 60 mm lens, a Kenko extension tube and two wireless R1 speed lights.

Histological processing
For fi xation, the individuals were previously anesthetized with seawater/magnesium chloride (7%). A small piece of tissue was removed for molecular analysis and the whole individual was fi xed in Bouin solution (0.8 gr of picric acid in 80 ml, 20 ml of formaldehyde and 2 ml of acetic acid) for histological studies. Histological sagittal series from 6 to 12 micrometres thick were stained with AZAN trichrome. The histological preparations of the studied specimens were deposited in the collections of the Nacional Museum of Natural Sciences (MNCN), Madrid.
For the defi nitive identifi cation of genus/species internal anatomic reconstructions, particularly of the reproduction apparatus, were made using a Zeiss Axio Scope A1 microscope.

DNA extraction, amplifi cation and sequencing
Tissues for molecular studies were fi xed in absolute ethanol. Total genomic DNA was extracted from each sample following the phenol-chloroform protocol (Chen et al. 2010). DNA concentration and purity of the extraction was measured using a NanoDrop Fluorospectrometer (Thermo Fisher Scientifi c). Sequences of the ribosomal gene 28S of the investigated Polycladida species were studied. All PCRs were performed using Taq DNA polymerase of Mastermix (Invitrogen, Carlsbad, CA) following the manufacturer's protocol in a total volume of 25 μl.
Sequences of approximately 1100 bp of the 28S gene were amplifi ed with degenerated primers designed de novo by the fi rst two authors: forward primer (5´-AGCCCAGCACCGAATCCT3-´) and reverse (5´-GCAAACCAAGTAGGGTGTCGC-3´). The PCR consisted in an initial denaturation step at 95°C (3 min), followed a pre-cycle of 5 cycles of denaturation at 96°C (30 sec), annealing at 55°C (30 sec) and extension at 72°C (1 min), followed by 40 cycles of denaturation at 95°C (30 sec), annealing at 59°C (30 sec) and extension at 72°C (1 min), with a fi nal extension of 10 min at 72°C.

Sequence alignment and phylogenetic analyses
A comparative analysis with both newly obtained sequences and those obtained from the NCBI GenBank database was carried out. A total of 147 sequences were aligned and edited using Geneious R6 (ver. 6.1.5). Forty-one of them were new sequences (NCBI accession numbers in Table 1).
The newly obtained sequences of 1100 bps were adapted to the length of those gathered from GenBank. The alignment was generated using the program MAFFT ver. 7 (Katoh & Standley 2013). Ambiguously aligned and variable regions were recognized and excluded using the program Gblocks ver. 0.91b (Castresana 2000) with relaxed parameters (smaller fi nal blocks, gap positions within the fi nal blocks, and less strict fl anking positions allowed). Thus, a matrix of 744 bp was obtained.
Bayesian inference (BI) analyses were carried out using MrBayes ver. 3.2.2 (Ronquist et al. 2012) (-mset option). Two independent runs of 1 000 000 generations and four chains (one cold, three heated) were run. Trees were sampled every 1000 generations. Convergence of chains was diagnosed using a deviation of standard frequencies below 0.05 and of the 1001 sampled trees, 250 trees were discarded as burn-in. A majority-rule consensus tree was constructed from the remaining 751 trees to approximate posterior probabilities.

Etymology
The name of the new species, Marcusia alba, comes from the Latin 'albus' (white), and refers to the ivory white coloration this species shows. MALE REPRODUCTIVE SYSTEM. Male copulatory organ backwards oriented, with a muscular penis papilla, very muscular seminal vesicle (Fig. 2C, E) and without a prostatic vesicle, instead a simple glandular epithelium leads into the penis papilla ( Fig. 2D-E). Seminal vesicle rounded, frontal oriented and with thick muscular walls, opening into the ejaculatory duct. Short ejaculatory duct opens into the penis papillae. The male atrium is small and thin, connected to the common atrium.  (Fig. 2C, E). The vagina runs from the common genital atrium and continues dorsally into a narrowed duct that widens into a chamber, the cement pouch. The vagina continues dorsally, then curves posteriorly and ventrally and ends with the entry of the oviducts.

Remarks
Marcusia alba sp. nov. belongs to the genus Marcusia due to the presence of cerebral, frontal and marginal eyes, male copulatory organ enclosed in a muscular bulb, the absence of prostatic vesicle and the common male and female atrium genital, as well as the common gonopore. The genus Marcusia contained only one species, Marcusia ernesti Hyman, 1953, known from the coast of the Gulf of California (Hyman 1953). Marcusia ernesti and M. alba sp. nov. can be easily distinguished by their coloration patterns. Marcusia ernesti is black or grey with darker splotches and dotted with white spots, only visible in preserved individuals after Hyman (1953), M. alba sp. nov. is ivory white with brownish dots and stripes. The penis papilla is spherical in M. alba and elongated in M. ernesti, with the male atrium being tube-like and longer in the Californian species.
Another difference lies in the eyes' presence and distribution. Marcusia ernesti presents marginal, frontal and cerebral eyes as well as two characteristic eye clusters with diagnostic value (Hyman 1953). Marcusia alba sp. nov., on the other hand, has cerebral, marginal and tentacular eyes, but not frontal eyes or eye clusters. FEMALE REPRODUCTIVE SYSTEM. Atrium elongated and highly ciliated, continues dorsally into the long but not ciliated vagina externa. The vagina externa narrows into a non-ciliated small cavity that continues in the vagina interna. It presents a widened epithelium and ends with the entry of the oviducts (Fig. 3H). Cement and shell glands lie around the female atrium, vagina externa and distal region of the vagina interna, but opens into the small cavity (pouch) between both vaginas.

Remarks
Prostheceraeus crisostomum sp. nov. belongs to the genus Prostheceraeus due to the presence of cerebral, frontal and marginal eyes, true anterior tentacles, bell-shaped pharynx, the male copulatory system with prostatic vesicle, penis armed whit stylet and the presence of multiple uterine vesicles.
The genus Prostheceraeus comprises 10 species, mainly characterized by coloration pattern, with colorful pigmentations and dorsal longitudinal lines of different widths, as in P. fuscolineatus Dixit, Raghunathan & Chandra, 2017, P. roseus, P. pseudolimax Lang, 1884, P. giesbrechtii, P. vittatus (Montagu, 1815 and P. zebra (Hyman, 1955) or with fi ne, transversal lines as in P. crozieri (Hyman, 1939). Three other species of Prostheceraeus show a color pattern free of lines or bands: P. albocinctus Lang, 1883, P. moseleyi andP. rubropunctatus Lang, 1884. These three species, together with P. crisostomum sp. nov., have a dotted pattern, but the background colors are different in the four species: caramel brown background with white or whitish spots and white marginal line in P. albocinctus, blue-gray or cream background with black dots and yellow marginal band in P. moseleyi and fi nally P. rubropunctatus with a pink to reddish background color, white dots and without marginal band. The base coloration of P. crisostomum is similar to P. albocinctus, but much clearer and almost ivory; the dorsal points are black like in P. moseleyi and lacks a marginal line or band similar to P. rubropunctatus. All these differences delimit P. crisostomum sp. nov. as a new species of the genus Prostheceraeus.

Etymology
The name 'galaxy' comes from the pattern of the small white spots on the dorsal surface, which resemble a star galaxy. Pseudotentacles constitute two simple folds that present each of them a small cluster of tentacular eyes in their margin. Round cluster of cerebral eyes present and surrounded by a spot of white pigment. Pharynx ruffl ed, butterfl y-shaped and located at the anterior third of the body. Oral pore, female and male gonopore close to each other and located at the anterior end (Fig. 3G). Male and female genital pores located after the pharynx in the anterior half of the body (Fig. 3H-I).

CAPE VERDE • São
MALE REPRODUCTIVE SYSTEM. Male genital pore between the posterior lobes of the ruffl ed pharynx. Male copulatory organ dorso-ventrally orientated consists in a prostatic vesicle and a very muscular seminal vesicle, as well as a penis papilla armed with a stylet (Fig. 3H-I). Vasa deferentia open separately into the seminal vesicle. Seminal vesicle rounded, frontally oriented and lined with a thick muscular wall. Prostatic vesicle rounded, muscular and smaller than the seminal vesicle. Sperm duct muscular and long, extends frontally to join the prostatic duct inside the proximal end of the conical stylet. The short ejaculatory duct appears surrounded by the stylet cone and the penis sheath. The male atrium is wide and tetra-folding (fork-like) as characteristic of the genus (Fig. 3I). FEMALE REPRODUCTIVE SYSTEM. With a short muscular vagina, backwards oriented and surrounded by cement glands.

Remarks
The genus Pseudoceros comprises approximately 89 species with similar copulatory organs, but bright and unique coloration patterns. However, within these patterns some taxa share evident similarities. Pseudoceros rawlinsonae var. galaxy shares with P. bicolor Verril, 1902, P. mororum and P. rawlinsonae Bolaños, Quiroga & Litvaitis, 2007 the brown background and one whitish, broad marginal band, but in P. bicolor the marginal band is wide with inner waves (Litvaitis et al. 2010: fi g. 4a-i); P. rawlinsonae shows, in addition to the wide band, a thin orange line (Litvaitis et al. 2010: fi g. 4j-p); in P. mororum the whitish band is interrupted and drop-shaped and additionally, two orange marginal stripes border the entire body (Cuadrado et al. 2017: fi g. 6a-b); fi nally, the Cape Verdean species shows, together with the drop-shaped white band, two black and orange thin lines (Fig. 3E-F).
Although the four previously mentioned species can be clearly differentiated due to their coloration, this is not the case in the molecular analysis (Fig. 8). In both the Bayesian and Maximum Likelihood analyses, individuals from Cape Verde appear closely related to P. rawlinsonae, so much so that the separation of both populations (the Cape Verdean population and the Caribbean population) is only possible at the level of variety, not of species. Therefore, we determined the individuals from Cape Verde as a variety within the species P. rawlinsonae.
Nonetheless, we want to emphasize that the decision to maintain this population (organisms) as a 'variety' of the species P. rawlinsonae is the sole and exclusive responsibility of the authors. We are aware that 'variety' is not a taxonomic category (according to ICZN) and that therefore it will remain a non-existent species until molecular analyses allow us to consider it as such.

Etymology
The name of the new species, Parviplana sodade, comes from 'sodade' the Cape Verdean expression for saudade and regional song with rhythms of 'coladeira'.

Description
BODY. Shape oval elongated. Length 0.8 cm. Smooth dorsal surface. Background pigmentation light white, transparent where the intestinal braches can be appreciated (Fig. 4A-B). Four clusters of cerebral eyes, two anterior with few eyes and more elongated than the posterior two. In sum around 50 cerebral eyes (Fig. 4B). Ruffl ed pharynx. Male and female genital pores located in the posterior half of the body.
European Journal of Taxonomy 736: 1-43 (2021) 20 MALE REPRODUCTIVE SYSTEM. Directed backwards and with a dorso-ventrally oriented penis papilla. With elongated prostatic vesicle, tall granular lining included in the muscular penis bulb (Fig. 4C). The vasa deferentia enter the seminal vesicle separately. Seminal vesicle rounded, below the penis bulb and connected with a sort seminal duct to the prostatic vesicle (Fig. 4C). The male atrium is small and thin, with an internal fold that surrounds the distal part of the penis bulb like a penis sheath (Fig. 4C). Parviplana comprises 3 species, P. hymani Faubel, 1983, P. jeronimoi Pérez-García, Noreña & Cervera, 2018and P. lynca (Du Bois-Reymond Marcus, 1958. Parviplana lynca can be easy and clearly distinguished from the other two species by the presence of nuchal tentacles, exclusive of this species.
Parviplana hymani can be distinguished from P. sodade sp. nov., by the vas deferens which opens together into the seminal vesicle, and the prostate vesicle not included into de penis bulb.
Parviplana sodade sp. nov. possesses more similarities with P. jeronimoi. Both species share the penis sheath and more than 25 cerebral eyes, but clear differences separate them. Parviplana jeronimoi has a fl eshy appearance and amber pigmentation. The size is also noticeable different, P. jeronimoi can reach lengths of 20 mm, while P. sodade in full mature state does not reach 8 mm. Parviplana jeronimoi also presents vasa deferentia joined in a single vas deferens, a small female atrium and a corrugated surface between the two genital pores, characteristics not present in P. sodade.

Etymology
The name of the new species, Euplana claridade, comes from "Claridade", a journal of literary review that revolutionized Cape Verdean culture during the fi rst half of the twentieth century.

Description
BODY. Shape oval. Length 1.1 cm. Smooth dorsal surface. Background pigmentation ivory white, denser along the middle dorsal region of the body and in the intestinal braches (Fig. 4D). Two clusters of 16 cerebral eyes each (Fig. 4E). Ruffl ed pharynx. Male and female genital pores located in the second half of the body close behind the posterior end of the pharynx. CUADRADO D. et al., New species and records from Macaronesia and Cape Verde 21 MALE REPRODUCTIVE SYSTEM. Male copulatory organ backwards oriented, englobed in a muscular bulb with a small penis papilla. Vas deferens opens proximally into the ejaculatory duct ( Fig. 4F-H). Without prostatic or seminal vesicle. Male atrium deep and thickened in the point of union with the penis (Fig. 4F-H).

Remarks
Euplana claridade sp. nov. belongs to the genus Euplana due to the absence of tentacles, prostatic vesicle and Lang´s vesicle, and presence of a true seminal vesicle and elongated coiling ejaculatory duct.
The genus Euplana encompasses 3 species, E. carolinensis Hyman, 1940, E. gracilis Girard, 1853and E. hymanae Marcus, 1947. The three species can be differentiated through the eyes number and disposition of them. E. gracilis and E. carolinensis present four cluster of eyes, two cerebral and two tentacular; E. hymanae and E. claridade sp. nov. only show two groups of cerebral eyes.

Etymology
The name of the new species, Stylochus salis refers to the type locality, Sal, a Cape Verdean Island.  (Fig. 5A). Few cerebral and marginal eyes, scattered between the tentacles and anterior end (Fig. 5C). Two small nuchal tentacles with abundant basal eyes (Fig. 5B). Ruffl ed pharynx in the middle of the body and the oral pore in the end of the pharynx pouch and close to the gonopores. Male and female gonopores located close together in the posterior end of the body. MALE REPRODUCTIVE SYSTEM. Male copulatory organ backwards oriented and provided with an inconspicuous unarmed penis papilla. Prostatic vesicle muscular with granular lining (polyglandulartype after Bulnes et al. 2005) (Fig. 5D). Seminal vesicle elongated, empties at the distal end of the prostatic vesicle. The short penis papilla and ejaculatory duct emerge in a small male atrium (Fig. 5D). FEMALE REPRODUCTIVE SYSTEM. Shows the characteristic confi guration of the genus. A tubiform canal with s-shaped ending in a small widening.

Remarks
Stylochus salis sp. nov. belongs to the genus Stylochus due to the presence of gonopores separate and arranged in the second body half. With large and much ruffl ed pharynx. Tentacular, cerebral, marginal, and often frontal eye-spots present. Male copulatory apparatus with seminal vesicle and papillate penis. Lang's vesicle lacking (after Faubel 1983).
Stylochus salis sp. nov. clearly differs from other known species of Stylochus Ehrenberg, 1831 by its peculiar cream pigmentation bordered with the internal orange outline and the white/creamy outer band. The color of the eastern Atlantic known species (S. alexandrinus, S. castaneus Palombi, 1939, S. neapolitanus, S. plessissii Lang, 1884, and S. suesensis Ehrenberg, 1831 varies between brown, light brown, reddish or beige and spotted as in S. neapolitanus. None of them present a continuous (or discontinuous) marginal line.
The most conspicuous anatomical feature is the location of the oral pore, very close to the gonopore, clearly different from the central position of the oral pore in this genus. The peculiar location of the oral and genital pores distinguishes S. salis sp. nov. from the remaining species. Such a close location of the pores could only be found in the genus Latocestus Plehn, 1896 (Latocestidae, Stylochoidea).   5E). Cerebral and tentacular eyes, scattered between the small tentacles (Fig. 5F). Ruffl ed pharynx, well developed, extending along ⅔ of the body. Male and female genital pores located in the posterior half of the body, together, but clearly separated. MALE REPRODUCTIVE SYSTEM. Male copulatory system backwards oriented, with a small penis papilla. Prostatic vesicle surrounded by muscular layers and lined with fi ngered granular lining, most probably polyglandular (Fig. 5G-H). Seminal vesicle divided into two sections. A muscular and elongated proximal section, and a more rounded distal section provided with a thin wall. Both regions separated by muscle narrowing (Fig. 5H). The distal section leads to the seminal duct that opens into the prostatic duct and forms a short ejaculatory duct. The two vasa deferentia dilated to form spermatic vesicles, open into the proximal section. Male atrium small, englobing a short penis papilla (Fig. 4H). FEMALE REPRODUCTIVE SYSTEM. Apparatus simple and backwards oriented. Comprises an elongated tube without clear differentiation between external and internal vagina and ends in a small widening, without Lang's vesicle.

Remarks
The new species belongs to the genus Distylochus due to the presence of few scattered marginal, tentacular and cerebral eyes. Gonopores together and are located near the posterior end. Male apparatus with a short papilla and unarmed. Seminal vesicle confi gured in two regions, following the "doublevesicle-system" after Faubel (1983) and female apparatus simple, without Lang's vesicle.
There are currently only three known species for the genus Distylochus: D. pusillus (Bock, 1913) recorded for Hong Kong, D. martae (Marcus, 1947) in Brazil and D. isifer (Du Bois-Reymond, 1955) also from Brazil. These species were described on fi xed specimens, therefore the colors are unknown, but apparently and after the original descriptions, they have pale pigmentation that contrast sharply with the orange-vermilion colors of the new species.
The most conspicuous difference of the new species is the disposition of female and male gonopore. In Distylochus fundae sp. nov. the gonopores are clearly separated, while in the Brazilian species are common and in the Chinese species are very close together.

New records
Following the known species that are captured in the study area. All of them have been studied through photographs and histological sections, currently in RCCN.

New record
São Vicente Island, Cape Verde.

Description
Body shape oval. Length 0.5 cm. Smooth dorsal surface; background color red-orange, with white dots scattered over the dorsal surface ( Fig. 6A-C). Ventral sucker in the posterior half of the body (Fig. 6F). Small tentacles. Few tentacular eyes distributed frontally and at the base of the tentacles. Cerebral eyes fused in a single elongated oval cluster (Fig. 6B). Ruffl ed pharynx located at the anterior third of the body. Oral pore posterior to the cerebral ganglion.
The reproductive system coincides with the original description, presenting the characteristic fold in front of the female genital pore mentioned by Lang (1884) for some specimens (Fig. 6D-F

Remarks
The specimens of Cycloporus gabriellae captured in Cape Verde summarize the original description of C. gabriellae published by Marcus (1950), but differ externally and in coloration from C. gabriellae of Cabo Frio, Brazil (Bahia et al. 2014). This is the fi rst record in the eastern region of the Atlantic Ocean for the species.

Distribution
Tyrrhenian Sea (Lang 1884); Ria de Arosa, Spain (Noreña et al. 2015). This species has also been cited by DORIS (Données d'Observations pour la Reconnaissance et l'Identifi cation de la faune et la fl ore Subaquatiques) for the English Channel and the North Sea, from the south of the United Kingdom to the Bay of Biscay (Spain) (http://doris.ffessm.fr/Especes/Prostheceraeus-moseleyi-Planaire-tachetee-716).

Remarks
The specimen collected in the Azores presents similar coloration to the specimen photographed by Wirtz & Debelius (2003)

Remarks
Anonymous ruber is characterized by reddish brown tones, but some individuals from Cape Verde are paler and presented cream tonalities. From an anatomical (internal or external) and molecular point of view, the species found in Cape Verde does not differ from the species in the Canary Islands (Fig. 8).

Remarks
Pseudoceros velutinus is known for its velvety black pigmentation, but the coloration varies with transmitted or refl ected light. Fig. 7D shows the appearance of P. velutinus with transmitted light.

New records
Baía das Gatas, São Vicente Island, Cape Verde. Sidi Ifni, Morocco. These are the fi rst records outside the Mediterranean Sea.

Description
Body oval and elongated sometimes pear-shaped, with wavy margins and thickened dorsal midline. Length 2.3 cm. The background color varies with transmitted or refl ected light. With refl ected light, the dorsal surface is chocolate brown, speckled with white patches. Under transmitted light, the pigmentation looks milky brown with dark margins and a dark dorsal midline that delimited the characteristic bulge (Fig. 7E). Dorsal surface smooth. Tentacles formed by simple folds and cone-like in shape. Cerebral eyes form a single large rounded cluster behind the tentacles; frontal eyes scattered between the tentacles. Compact ruffl ed pharynx located directly behind the cerebral eyes.

New record
Madeira.

Remarks
Planocera pellucida, together with Pseudoceros wirtzi, Pseudoceros cf. maximus and Prostheceraeus giesbrechtii are the three polyclad species currently recorded for Madeira. As can be seen in Fig. 7J. The specimens of Planocera pellucida from Madeira do not differ molecularly from those captured in the Canary Islands.

Remarks
Gnesioceros sargassicola was limited to the Antilles and the Caribbean Sea until the record of Laidlaw (1903) for the Cape Verde Islands. The new record of G. sargassicola for the Canary Islands shows a progressive 'colonisation' of the Atlantic east coast.

Molecular analysis
The main purpose of the 28S analysis was to confi rm the determinations made from the morphological study and verify the relationships among similar species.
After Anonymidae, we fi nd a well-supported branch (BPP = 0.9, BS = 95) that encompasses the families Prosthiostomidae Lang, 1884, Euryleptidae and Pseudocerotidae. This branch is, in turn, divided into two main branches (both supported by maximum values: BPP = 1, BS = 100) where Prosthiostomidae is separated from Euryleptidae and Pseudocerotidae, families with a clear relation.

Discussion
After the present study and the results obtained, it is evident that the knowledge on polyclad biodiversity in Macaronesia is clearly uneven. Polyclads from Cape Verde, Madeira and Canary Islands are mostly well studied, while information from the Azores and Selvagens Islands is irregular. Consequently, a comparison of the biodiversity of the different archipelagos is biased, but not impossible.  (Tyler et al. 2006(Tyler et al. -2020. The four remaining species Anonymus ruber, Latocestus plehni Laidlaw, 1906, Cycloporus gabriellae and Pericelis cata, are currently only recorded for Canary Islands and also known for the coasts of Brazil. Stylochus neapolitanus is described for the Mediterranean and has also been recorded for Cape Verde by Laidlaw (1906). Considered by the same author as doubtful because according to this author the species of this genus (Stylochus) are diffi cult to distinguish (Laidlaw 1906: 707).
This group of common species shows Cape Verde as an independent archipelago with unique biological and ecological characteristics and therefore, fauna. Regardless, it is still under the infl uence of other regions. As such, our results confi rm the hypotheses of Spalding et al. (2007) and Freitas et al. (2019) who propose Cape Verde as an independent archipelago outside of Macaronesia with its own ecological and biological history and as a specifi c hotspot (Freitas et al. 2019).
Most of the studied species of Cape Verde belong to the island of São Vicente (Laidlaw 1906) and only 9 species were collected in Boa Vista, Santo Antão and Santiago. In the present study, samples from the island of São Vicente were examined, but only two species coincide with those recorded by Laidlaw (1906).
The second best studied archipelago is the Canary Islands (Cuadrado et al. 2017). In the present study, Discocelis tigrina and Gnesioceros sargassicola are new records added to the biodiversity of the archipelago and the distribution of Pseudoceros mororum, to date limited to the island of Gran Canaria, has been extended to Tenerife.
While the focus of this study doesn't lie in the analysis of the Macaronesian species from a molecular point of view, such analyses were carried out to verify the previous morphological determination and to add another source of proof to the delimitation of new species. (2019) are of particular relevance. As the last authors discuss the previous studies, we will mainly compare our results with their analyses. In general, the phylogenetic trees (both ML and BI) obtained in the present study show an almost identical topology at the family level to those presented by Litvaitis et al. (2019).
Regarding the newly sequenced and analysed material (Bordeaux in Fig. 8), Latocestus plehni, within Acotylea, shows the same close relationship with Leptostylochus gracilis Kato, 1934 as mentioned by Litvaitis et al. (2019), therefore we include the genus Leptostylochus together with Latocestus in the family Latocestidae.
The genus Notoplana certainly presents a problem in our analysis. While some species from Brazil (Notoplana sp.) appear within the family Notoplanidae, others from Japan are closely related to the family Stylochoplanidae Faubel, 1983. Finally, Notoplana australis (Laidlaw, 1904) is included within the family Notocomplanidae Litvaitis, Bolaños & Quiroga, 2019. This shows the variety of the genus Notoplana, at least with respect to the 28S gene. With new morphological analyses and the use of more molecular markers, perhaps we will obtain more satisfactory results to resolve the relationship of the genus Notoplana.
On the other hand, and within Leptoplanoidea, Pseudostylochus and Koinostylochus are considered synonymous and belong to the family Pseudostylochidae (Oya & Kajihara 2020). Planocera pellucida, regardless of the origin location (Canarias or Cape Verde) seems to represent the same species despite differences in populations and well framed within the family Planoceridae. The same happens with Discocelis tigrina, although there are differences between the populations of the Atlantic (Canary Islands) and the Mediterranean (Catalonia).
Within Cotylea, Cestoplana rubrocincta is included together with Cestoplana salar Marcus, 1949 andCestoplana techa Du Bois-Reymond Marcus, 1957 within the family Cestoplanidae. Next, the family Diposthidae shows a clear relationship between the genera Pericelis and Marcusia, but in both analyses (ML and BI) the two genera are clearly differentiated and confi rmed as valid genera.
Anonimus ruber within Anonimidae, presents the same condition as Planocera pellucida (Acotylea), where no differences appear between the populations of the Canary Islands and Cape Verde.
Prostheceraeus roseus and Enchiridium magec, both species captured in the Canary Islands, are well defi ned and included within their respective families, Euryleptidae and Prosthiostomidae. On the other hand, Eurylepta cornuta var. melobesiarum shows clear differences with Eurylepta sp., an immature specimen from Martinique, possibly E. cornuta but could not be confi rmed.
Within the family Pseudocerotidae the relationships are more complex. In general, the species of the genus Pseudoceros are well framed within a clade, while the rest of the genera appear scattered. Pseudobiceros Faubel, 1984, Thysanozoon Grube, 1840, Monobiceros, Phrikoceros Newman & Cannon, 1996 and Yungia Lang, 1884 appear closely related and well separated from the genus Pseudoceros. The fi rst three genera share a double male copulatory organ while in Phrikoceros and Yungia it is single. Monobiceros and Phrikoceros are grouped in a single clade in our analysis, but they are morphologically different.
Close relationships among diverse morphotypes are already mentioned by Lang (1884), which includes clearly different morphotypes under the species Pseudoceros maximum. According to Lang (1884) this species could present externally three coloration patterns: brown with dark striations (Form A; Lang 1884: pl. 9, fi g 1), dark with white splatters (Form B; Lang 1884: pl. 9, fi g. 2) or beige with lighter, rounded spots (Form C; Lang 1884: pl. 9, fi g. 3). Internally, Pseudoceros maximus could present one (type a) or two (type b) male copulatory organs, both types with a single gonopore. Faubel (1984) separates both morphotypes into Pseudoceros maximus, with a male copulatory organ type a, and in Monobiceros langi with a type b, but no reference to the coloration pattern of these two species is made.
As such, species with one of the different color patterns described for P. maximus by Lang (1884) are: Phrikoceros mopsus (Marcus, 1952) (form C, type a) and Pseudobiceros caribbensis Bolaños, Quiroga & Litvaitis, 2007 (form A; type b, but with 2 gonopores) from the Caribbean. In our analysis, Phrikoceros mopsus and Monobiceros langi (form B, type b) appear together, while Pseudobiceros caribbensis appears grouped in a single isolated branch. These results indicate the close relationship between P. mopsus and M. langi, which seem to support the different morphotypes of Lang's P. maximus. Unfortunately, without sequences of Pseudoceros maximus (form A, type a) the resolution of the complex of species (or morphotypes) 'P. maximus' remains for a future study.
The species whose sequences were obtained from GenBank (NCBI) appear in the same positions described in other works (Bahia et al. 2017;Litvaitis et al. 2019) but some showcase new relationships that should be discussed.
Leptostylochus gracilis, which appears as closely related to Latocestus and Idioplana in Litvaitis et al. (2019), appears as a sister group to Latocestus in our analyses, a relationship that indicates a clear belonging to the family Latocestidae and not family Stylochidae.
The Hawaiian Paraplanocera oligoglena (Schmarda, 1859) is found within Stylochidae and clearly related to Stylochus and Imogine, genera which present little differences from one another. These results indicate that Stylochus and Imogine are possibly one genus (Dittman et al. 2019), to which the Hawaiian species belongs.
Amemiyaia pacifi ca does not seem to belong to the Cryptocelidae Laidlaw, 1903 as it appears in the results obtained by Litvaitis et al. (2019), but it is closely related to this family. Adenoplana evelinae, in comparison, appears in both analyses (ML and BI) within Cryptocelidae and not within Discocelidae, a fact that looks to corroborate the results of Bahia et al. (2017).