Microstomum (Platyhelminthes, Macrostomorpha, Microstomidae) from the Swedish west coast: two new species and a population description

Two new species of marine Platyhelminthes, Microstomum laurae sp. nov. and Microstomum edmondi sp. nov. (Macrostomida: Microstomidae) are described from the west coast of Sweden. Microstomum laurae sp. nov. is distinguished by the following combination of characters: rounded anterior and posterior ends; presence of approximately 20 adhesive papillae on the posterior rim; paired lateral red eyespots located level with the brain; preoral gut extending anterior to brain and very small sensory pits. Microstomum edmondi sp. nov. is a protandrous hermaphrodite with a single ovary, single testis and male copulatory organ with stylet. It is characterized by a conical pointed anterior end, a blunt posterior end with numerous adhesive papillae along the rim, and large ciliary pits. The stylet is shaped as a narrow funnel with a short, arched tip. In addition, the fi rst records of fully mature specimens of Microstomum rubromaculatum von Graff, 1882 from Fiskebäckskil and a phylogenetic analysis of Microstomum Schmidt, 1848 based on the mitochondrial cytochrome oxidase I (COI) gene are presented.


Introduction
Macrostomorpha Doe, 1986 is a group of free-living fl atworms that may be found in aquatic and semiaquatic habitats all over the world (Rieger 2001). Their relatively small size, typically only 1-2 mm (although animals as large as 5 mm have been recorded) and paucity of distinguishing morphological characters makes taxonomic identifi cation within the group diffi cult (Janssen et al. 2015).
Species identifi cation of Macrostomorpha is largely dependent on the morphology of the reproductive system, and identifi cation of species that predominately reproduce asexually, such as most species of Microstomum Schmidt, 1848, is particularly challenging. It is unsurprising, then, that while limited
Species of Microstomum primarily reproduce through asexual fi ssion, but their life cycle may also include short periods of sexual reproduction (Bauchhenss 1971;Heitkamp 1982). During that time, individual zooids will develop both male and female sexual structures, including for the male complex: single or bilateral testes, vas deferentia, a male copulatory apparatus with a seminal vesicle, stylet and antrum masculinum, and a male gonopore; and for the female complex: a single ovary, female antrum and female gonopore. Microstomum sexual maturity may be present for as little as two weeks in a year (Bauchhenss 1971;Faubel 1974) and therefore descriptions of the reproductive organs exist for only 15 of the 31 currently accepted nominal species.
Several members of Microstomum were encountered while investigating macrostomorph diversity in western Sweden. Herein, two new species of marine Microstomum are described, and a population of Microstomum rubromaculatum von Graff, 1882 from Fiskebäckskil is redescribed based upon the fi rst records of fully sexual specimens. Additionally, we present a hypothesis regarding the phylogenetic position of the three species based on the mitochondrial cytochrome oxidase I (COI) gene and make COI barcode sequences available.

Material and methods
Sediments and aquatic vegetation were collected by hand from sites around the Sven Lovén Centre Kristineberg in Fiskebäckskil, Sweden on 17-20 Aug. 2015 and the Sven Lovén Centre Tjärnö in Strömstad, Sweden on 17-19 Jun. 2016. Exact dates and coordinates are listed in Table 1. Samples were transported back to the laboratory and marine fl atworms were extracted within 48 hours following a standard MgCl 2 anesthetization-decantation technique (Martens 1984). Animals were manually isolated under a Nikon SMZ 1500 stereo microscope, transferred to a glass slide and identifi ed using a Nikon Eclipse 80i compound microscope equipped with DIC (differential interference contrast). Light micrographs and digital videos were captured with a Cannon EOS 5D Mark III digital camera. Measurements were taken with an ocular micrometer. Following documentation, individual specimens were fi xed in 95% ethanol and transported to the Naturhistoriska riksmuseet in Stockholm for DNA extraction and analysis.
DNA was extracted from whole animals using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Amplifi cation was performed via PCR using 0.2 ml PuReTaq Ready-To-Go PCR beads (GE Healthcare). A ~700 base-pair region of the COI gene was targeted using Mac_COIF+Mac_COIR (Janssen et al. 2015) or new (Mic_COIF 5'-GTT TGA GGA GGT TTG ATA GGC-3'; Mic_COIR 5'-ATC ACC CCC CTC CGT AGG AT-3') PCR and sequencing primers, and amplifi ed with the following program: 5 minutes hotstart at 94°C; followed by 40 cycles of 30 seconds at 94°C, 90 seconds at 50°C, and 60 seconds at 72°C; and a fi nal extension time of 10 minutes at 72°C. Products were viewed on a 0.8% agarose gel, purifi ed using ExoSAP-IT enzymes (Exonuclease and Shrimp Alkaline Phosphatase, GE Healthcare), and sent to Macrogen (Macrogen Europe, Netherlands) for commercial sequencing.
Thirty-fi ve COI sequences of Microstomum and seven outgroup sequences were used in the phylogenetic analysis (Table 1). The dataset included all COI sequences of Microstomum publically available in GenBank along with new sequences of the three species presented here. Outgroups were selected based on their position in the phylogenetic hypothesis of Janssen et al. (2015).
Sequence assembly was performed in MEGA v. 6.06 (Darriba et al. 2012) and trace fi les were manually edited. Sequences were aligned as amino acids using the standard fl atworm mitochondrial genetic code European Journal of Taxonomy 398: 1-18 (2018) and then back-translated to nucleotides. The general time-reversible model with gamma distribution and proportion invariant sites was determined to be the best model of sequence evolution with three substitution schemes by jModelTest2 (Tamura et al. 2011) based upon the Akaike information criterion (Akaike 1974). Maximum Likelihood Analysis (ML) was performed in RaxmlGUI v. 1.5b1 (Silvestro & Michalak 2012) with 1000 fast bootstrap replicates.
Patristic distances were calculated using the TN93-model (Tamura & Nei 1993) with rate variation among sites and gamma distribution. Alignment gaps and ambiguous sites were not considered.

Etymology
This species is dedicated to Laura R. Atherton, mother of the fi rst author.

Description
Microstomum with a total body length of 760 μm and two vegetative zooids. Body strap-shaped with very slight constrictions between zooids and at the level of the ciliary pits. Ratio of body width:length 1:5 in slightly compressed animal. Anterior and posterior ends rounded.
Ciliary pits very small and shallow (Fig. 1A), below eyespots, 185 μm from anterior. Paired red eyespots approximately 43 μm long and present in the lateral margins of the body level with brain ( Fig. 1A-B).
Many small (max. diameter 10 μm) orange lipid droplets derived from food scattered across body, heavily concentrated around pharynx and anterior end ( Fig. 1A-B). Body otherwise colorless or refl ective of intestine.
Mouth slit-like and 55 μm long. Pharynx encompassing the second fi fth of the body. Preoral gut extending well anterior to brain. Intestine evenly fi lled with orange droplets.
Reproductive system unknown.

Remarks
Eight of the currently recognized Microstomum species (M. bioculatum Faubel, 1984, M. gabriellae Marcus, 1950, M. giganteum Hallez, 1878, M. groenlandicum Levinsen, 1879, M. lineare Ørsted, 1843, M. melanophthalmum Steinbock, 1933, M. paràdii Graff, 1913and M. spiriferum Westblad, 1953 possess pigmented eyespots. The eyespots of M. melanophthalmum and M. paràdii are black with distinct lenses, while the eyespots of M. laurae sp. nov. are red. The eyespots in M. bioculatum, M. gabriellae, M. giganteum, M. lineare and M. melanophthalmum are situated far in the front of the animal, whereas they are level with the brain in M. laurae sp. nov. Finally, the eyespot of M. groenlandicum is unpaired, red and located medially above the brain. Microstomum laurae sp. nov. is most similar to M. spiriferum, being alike in general body size and shape as well as their small ciliary pits and large bundles of rhabdites. However, M. spiriferum can be differentiated based on the dorsal richly yellowish pigment cells and absence of adhesive papillae (Westblad 1953). M. laurae sp. nov., on the other hand, is colorless or sometimes colored by its gut contents; there are no pigment cells apart from the eyespots, and it possesses a distinct line of adhesive tubes along the posterior rim. Though both species are found on the Swedish west coast, M. spiriferum was described from sublittoral habitats between 15 and 60 m depth, while M. laurae sp. nov. was collected from eulittoral sand at around 10 cm depth.

Diagnosis
Microstomum with animal/zooid body length of 1700/750 μm. Conical pointed anterior end; blunt posterior end with numerous adhesive papillae along rim. Ciliary pits large, bottle shaped. Pigmented eyes absent. Dense fi eld of cilia clearly covering epidermis. Preoral gut extending anteriorly to brain. Mouth distinctly encircled by glands. Protandrous hermaphrodite. Male reproductive system with single large testis. Vesicula seminalis circular to elliptical, 98 μm long, and containing the ends of numerous prostate glands in the distal part. Stylet approximately 67 μm long; shaped as an elongate, narrow funnel, slightly curved in one plane with a short, arched tip. Female reproductive system with single ovary and gonopore. Eggs develop caudally. GenBank accession number for partial COI sequences MF185700-11.

Etymology
This species is dedicated to Edmond T. Atherton, father of the fi rst author.
Protandrous hermaphrodite. Male reproductive system with single large testis connected by short vas deferens to male copulatory apparatus (Fig. 3A). Vesicula seminalis circular to elliptical, 98 μm long, and containing the ends of numerous prostate glands in the distal part (Fig. 2D). Stylet 67 μm long, shaped as a tube very slightly curved in one plane and narrowing to a short, arched tip; width at base approximately 17 μm and terminal opening 4 μm (Figs 2D, 3C). Male pore not seen.
Female reproductive system including single mediolateral ovary and ventral female gonopore (Figs 2E, 3B). Eggs develop caudally. Very small testis posterior to ovary present in some animals (Fig. 2E).

Remarks
Almost all sexually mature specimens of Microstomum edmondi sp. nov. displayed only male or female sexual organs. Only one individual (Fig. 2E) contained both an ovary with a single egg and what appeared to be a small testis, roughly a quarter of the size of the testes of the other male specimens. This individual otherwise lacked any discernible male copulatory apparatus (vesicula seminalis or stylet). Furthermore, animals with male reproductive anatomy were generally composed of two zooids with the sexual organs in the posterior zooid only, and animals with female anatomy were always solitary. Previous life cycle studies on M. papillosum Graff, 1882 andM. spiculifer Faubel, 1974 found that male genital organs fi rst occur in asexually produced zooids that are otherwise well-developed, and sexual development then fi nishes in solitary individuals (Faubel 1974(Faubel , 1976Hellwig 1987 (Bauchhenss 1971;Faubel 1974Faubel , 1976Heitkamp 1982;Hellwig 1987).  Marcus, 1950) have distinctly and continuously curved or crescent shaped stylets. Furthermore, the stylet of M. edmondi sp. nov. clearly differs from that of M. melanophthalmum Steinböck, 1933 by its size (67 vs 30 μm, respectively), as well as the lack of very wide, almost fl at proximal rims and mid-way 90° bend.
Of the species of Microstomum for which sexual anatomy is known, M. edmondi sp. nov. is most similar to M. hamatum Westblad, 1953. The male reproductive system for both includes a large seminal vesicle and a 60-70-μm-long stylet with similar shape. Both species additionally include individuals with only a single large testis, although animals with smaller, paired testes were also found in M. hamatum (Westblad 1953). The stylet of M. edmondi sp. nov., however, can be distinguished by the narrower base, more gradual distal tapering and a small arched tip. Microstomum hamatum has a much broader funnel-shaped stylet ending in an 180° curve that forms a large hook. Other morphological differences include the pointed anterior end, large ciliary pits and lack of dark gray pigmentation in M. edmondi sp. nov. Finally, M. edmondi sp. nov. was collected from shallow, fairly clean marine sediments instead of deeper black mud.
Of the species of Microstomum for which the sexual organs remain undocumented, only eight inhabit marine waters (M. bioculatum, M. breviceps Marcus, 1951, M. davenporti von Graff, 1911 Graff, 1905, M. rhabdotum Marcus, 1951, M. rubromaculatum von Graff, 1882, M. ulum Marcus, 1950. Microstomum edmondi sp. nov. is clearly most similar to M. ulum in that both have conically pointed anterior ends, large ciliary pits and large rhabdite bundles, and both lack eyespot pigmentation. Morphological differences occur in the shape of the posterior end, where a clear constriction sets apart a rounded adhesive tail plate in M. ulum while the posterior of M. edmondi sp. nov. is more paddle-like and blunt, and perhaps in the density of the locomotory cilia. Both species may be found in shallow marine sediments but are described from very distant locales, M. ulum being from the southwest Atlantic near the Island of São Sebastião, Brazil (Marcus 1950) while M. edmondi sp. nov. was described from the Swedish west coast.

Type locality
ITALY: Gulf of Naples, Tyrrhenian Sea. Deposition not recorded.

Population description
Microstomum with fi eld of bright red pigmentation spots on each well-developed zooid; length of pigmentation stretches from just below the anterior tip to halfway to the brain, width of pigmentation somewhat variable: either predominately at the lateral margins and thinning toward the middle or, most frequently, a band that encircles the entire body (Fig. 4A). Other small orange-red droplets may be scattered within the parenchyma of some specimens, particularly around the anterior and pharynx (Fig. 4B-C). Body otherwise colorless, clear and refl ective of intestine.
Mouth slit-like at rest, but able to distend to encompass very large food. Pharynx spherical to elliptical, encompassing up to the length of the second quarter of the zooid. Preoral gut extending to brain or slightly anterior. Intestine yellow-brown or tinged with red or pink; may contain ingested prey.
Female reproductive system typical for the genus (Figs 4D, 5). Single ovary situated mid-body, ventral to intestine, leading to ciliated female antrum. Female gonopore separate. Eggs develop caudally.

Remarks
Collected specimens generally appeared morphologically similar to the type description (von Graff 1882) and to previous accounts of M. rubromaculatum from Fiskebäckskil. Westblad (1953) recorded M. rubromaculatum from Fiskebäckskil with vegetative chains up to four zooids long, yet all currently collected specimens except one were composed of either two weakly developed zooids separated by a faint fi ssion plane or one or two well-developed zooids only. This follows other patterns found in species of Microstomum in which slender chains of multiple, short zooids dominate during the asexual reproductive phase of the lifecycle while larger single or double zooid animals dominate during periods of sexual reproduction (Bauchhenss 1971).
The amount of eyespot pigmentation in M. rubromaculatum can greatly vary between individuals. Specimens from Fiskebäckskil generally agreed with the original description of Graff (1882): paired, lateral eyespots composed of an accumulation of red pigmentation that extends medially to form a ring around the anterior end. However, pigmentation spots in four of the observed specimens remained clearly distinct, a phenomenon that has been recorded in other populations of M. rubromaculatum (von Graff 1913;Steinböck 1931). COI sequences were identical between specimens with two distinct eyespots and those with a circular band, which indicates amount of pigmentation is not necessarily a systematically important character. Rather, accumulation of eyespot pigmentation may be more "correlated with light intensity", as in, e.g., Microstomum lineare (Bauchhenss 1971). Steinböck (1938) reported a single specimen of M. rubromaculatum from Iceland with a large central pigment spot that thinned toward the body margins. However, such a pattern was not observed in any of our specimens, nor otherwise recorded in any other population.
Red-orange droplets ( Fig. 4B-C), that have not been previously documented in M. rubromaculatum, were observed in 18 of the 20 live specimens. The droplets ranged in size from a diameter of ~2-10 μm and were most often located anteriorly, especially around the pharynx. The droplets were most likely lipid deposits whose presence and coloration stems from ingested food. While such deposits have not European Journal of Taxonomy 398: 1-18 (2018) been recorded before in Microstomum, colored lipid droplets are known to occur in other species of Macrostomorpha (Rieger et al. 1991).
The distribution of Microstomum rubromaculatum is wide, with populations reported from the Mediterranean, the North Sea and the Baltic. Although such patterns do occur for other macrostomorphs (e.g., Macrostomum pusillum, M. rubrocinctum, Paramalostomum dubium -see Ax 1956;Karling 1974;Armonies 1988), including other species of Microstomum (e.g., M. lineare, M. papillosum -see  Steinböck 1931;Karling 1974), the distribution may still be considered surprising giving the large geographic distances and differences in salinity and temperature (Boyer & Levitus 1994). Evidence has increasingly shown that widespread taxa previously thought to represent a single species are in fact morphologically indistinct complexes. However, all our specimens were collected from a single location in west Sweden, and thus different populations of M. rubromaculatum could not be compared at this time. Sexually mature specimens of M. rubromaculatum have not been recorded from any other populations, including those inhabiting the type locality, and therefore further research may be necessary to confi rm the identity of M. rubromaculatum from Fiskebäckskil, Sweden before sexual anatomy can be included in the description of the species as a whole.

Phylogeny
The maximum likelihood analysis found four moderately or highly supported clades of Microstomum (Fig. 6). M. rubromaculatum was sister to M. edmondi sp. nov. with moderate support and further formed Fig. 6. Phylogenetic relationships of Microstomum Schmidt, 1848 inferred from ML analysis of partial COI gene. Outgroups were selected based on the phylogenetic hypothesis presented in Janssen et al. (2015). Numbers at nodes represent bootstrap support. Genbank accession numbers are listed after each taxon name.
European Journal of Taxonomy 398: 1-18 (2018) a clade with an unidentifi ed species of Microstomum (species "D" in Janssen et al. 2015; see Table 1) and M. laurae sp. nov. The other three species of Microstomum represented in the analysis (species "B" in Janssen et al. 2015, M. lineare, M. papillosum) individually comprised the remaining three clades. Patristic distances are presented in Table 2.
A true understanding of the evolutionary relationships within Microstomum would require multiple nuclear and mitochondrial gene sequences as well as a much greater species representation (Maddison 1997). However, the results of the ML analysis and patristic distances presented here clearly separate specimens of M. edmondi sp. nov., M. laurae sp. nov. and M. rubromaculatum into three distinct lineages representing the three species.

Discussion
This study extends the number of species of Microstomum known to occur within Sweden to nine. Eight species (M. hamatum, M. jenseni, M. papillosum, M. rubromaculatum, M. septentrionale, M. spiriferum, Table 2. Patristic distances between DNA sequences used in this study. Distances were calculated using the TN93-model (Tamura & Nei 1993) with rate variation among sites and gamma distribution. Alignment gaps and ambiguous sites were not considered. Accession numbers are given after species name. Specimens with identical sequences are listed together. and now M. edmondi sp. nov. and M. laurae sp. nov.) are marine and from the Swedish west coast. The ninth species, M. lineare, occurs primarily in fresh or brackish waters with salinity up to 6-8‰ (Karling 1974) and is, perhaps, the most widespread species of Microstomum, with populations reported from waters throughout Europe, Asia and North America (Karling 1974;Kolasa et al. 1987; but see Janssen et al. 2015). Additionally, the occurrence of sexually mature specimens of M. rubromaculatum from Fiskebäckskil, Sweden brings the number of species of Microstomum with known sexual anatomy to seventeen.