A taxonomic review and revisions of Microstomidae (Platyhelminthes: Macrostomorpha)

Microstomidae (Platyhelminthes: Macrostomorpha) diversity has been almost entirely ignored within recent years, likely due to inconsistent and often old taxonomic literature and a general rarity of sexually mature collected specimens. Herein, we reconstruct the phylogenetic relationships of the group using both previously published and new 18S and CO1 gene sequences. We present some taxonomic revisions of Microstomidae and further describe 8 new species of Microstomum based on both molecular and morphological evidence. Finally, we briefly review the morphological taxonomy of each species and provide a key to aid in future research and identification that is not dependent on reproductive morphology. Our goal is to clarify the taxonomy and facilitate future research into an otherwise very understudied group of tiny (but important) flatworms.


Introduction
Macrostomorpha DOE, 1986 is a group of free-living Platyhelminthes found worldwide in aquatic and semi-aquatic habitats [1]. They are very small in size, typically only 1-2 mm, but are nevertheless important and often abundant members of their communities, feeding on diatoms and other microorganisms or even other small invertebrates [2][3][4]. While the deep splits within Macrostomorpha remain ambiguous, current phylogenetic hypotheses [5][6][7], place Microstomidae as sister to Dolichomacrostomidae within the group Dolichomicrostomida.
Microstomidae LUTHER 1907 are characterised by ciliated sensory pits at the frontal end, an intestine extending anteriorly past the mouth, and the ability to reproduce asexually through fissioning. Currently, Microstomidae is unevenly divided between three genera: Myozonella BEKLEMISHEV, 1955, with only a single species Myozonella microstomoides BEKLEMISHEV, 1955 collected from a village in western Russia [8] and characterized by a muscular ring surrounding the anterior intestine; Alaurina BUSCH 1851, with three currently accepted species all with an elongated and unciliated (apart from sensory cilia) anterior proboscis; and the highly diverse and speciose genus Microstomum ØRSTED 1843.
Species of Microstomidae primarily reproduce through asexual fission, but their life cycle may also include short periods of sexual reproduction occurring primarily in the autumn [10][11]. During this time, individual zooids will develop male and female sexual structures, including for the male complex: single or bilateral testes, vasa deferentia, and a male copulatory apparatus with vesicula seminalis, stylet and antrum masculinum; and for the female complex: a single ovary and a female antrum. Species of Microstomidae may be sexually mature for as little as two weeks a year or less [10,19] and thus full or partial sexual anatomy is currently only known for roughly half of the nominal species. Since species identification of Macrostomorpha has often been based heavily or, in some cases, entirely on the morphology of the reproductive organs (e.g. [22][23][24]), some authors have argued that any species with unknown sexual anatomy should be instantly considered species dubiae [20].
For Microstomidae with limited morphological data available, analysis of nucleotide sequence data will be an indispensable tool when solving taxonomic problems. DNA taxonomy is not without its own pitfalls (e.g. sensitivity to sampling, overlap between intra and interspecific variation [25][26][27]), but has nevertheless been demonstrated as a reliable and reproducible method to identify lineages without depending solely on morphological characters that may be sensitive to environmental conditions or on access to sexually mature specimens [28][29]. A number of different approaches to sequence-based species discovery, or DNA-taxonomy, have been developed, including haplotype networks based on statistical parsimony [30], Bayesian species delineation [31][32][33], and maximum likelihood approaches with the General Mixed Yule-Coalescent model [34].
DNA taxonomy may be more effective than traditional morphological methods in discerning species, as re-examination of morphological taxa often reveals hidden biodiversity in morphologically inseparable, i.e. cryptic species [35]. Successful DNA-taxonomy requires use of a molecular marker with an appropriate level of variation, or, preferably, more than one such marker [36][37]. Recent molecular investigations into the widespread taxon Microstomum lineare revealed an assemblage of four genetically divergent but morphologically similar species, some of which were found in the same sample [4]. The presence of multiple distinct but cryptic lineages in single locations potentially could have much larger implications for research on Microstomidae, especially since studies in the past have often been performed on specimens collected from environmental samples (e.g. [10][11][38][39]).
In this paper, we reconstruct the phylogenetic relationships of Microstomum using both previously published and new 18S and CO1 nucleotide sequences. Our dataset comprises sequences from 336 specimens representing 15 previously described nominal species and eight new species. The new taxa are diagnosed on the basis of DNA-taxonomy as well as morphological characters that distinguish them from all other known Microstomum species. We also review the morphological taxonomy of the species within Microstomidae and provide a key to aid in future research and identification. Our goals are to simplify the taxonomy, demonstrate that asexual Microstomum specimens can often be identified, and to facilitate future research into an otherwise very understudied group of microscopic flatworms that are highly abundant in both limnic and marine habitats. determined using the ModelFinder algorithm [55] implemented in IQTree. We performed 1000 ultrafast bootstrap replicates [56] in IQTree, applying edge-proportional partitions with best-fit models independently selected for each gene in the concatenated datasets.
Species of Microstomidae were inferred using the Bayesian implementation of the Poisson Tree Processor (bPTP) in the web-based interface (http://species.h-its.org/) with the default parameters [32]. Input trees were generated from ML analyses of concatenated datasets that included Microstomidae sequences and two sequences each of Myozonaria bistylifera, Myozonaria fissipara and otherwise unidentified Myozonariinae downloaded from GenBank as outgroups (S1 Table). All bPTP tests were re-run up to three times with the input tree pruned so that each prospective species included a maximum of five randomly selected sequences in order to ensure that results were not influenced by an imbalance in the number of specimens in any given clade. Following recommended guidelines, outgroups were not included in any bPTP analysis. Since bPTP does not account for possible divergent rates of intraspecific variation that may occur from, e.g., selection or population bottlenecks, we additionally implemented the multi-rate Poisson Tree Processor (mPTP) via its web-based interface (https:// mptp.h-its.org). mPTP is an improved model and implementation of Poisson tree processor that alleviates previous shortcomings by fitting the branching events of each delimited species to a distinct exponential distribution [33]. The alignment used for the input tree as well as the ML input tree are given in S2 File and S3 File respectively. All DNA sequences are deposited in Genbank and their accession numbers are given in S1 Table. Alignments and trees are deposited in Dryad and may be accessed via https://doi.org/10.5061/dryad.jn95jm1.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub: 17D56064-22E3-4DA8-908D-A12E256A639F. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Results
The full-concatenated alignment had a total of 336 specimens and total length of 2348 bp. The K3Pu+F+G4 substitution model was selected as the best fit for the CO1 gene sets and the TIM2e+I substitution model was selected for the 18S gene sets. Tree topologies were consistent across all individual gene and concatenated analyses. Fig 1 summarizes the results of the entire concatenated phylogeny (see also S1 File). Results were generally consistent with Janssen et al. [7], with maximum (1.00) support for the two major subgroupings Macrostomidae and Dolichomicrostomida. Results found further high (>0.90) support for: a monophyletic Macrostomidae consisting of Macrostomum and Psammomacrostomum; Myozonariinae as sister to Dolichomacrostominae, including the genera Paramalostomum, Austromacrostomum, Cylidromacrostomum and Dolichomacrostomum; and a monophyletic Microstomidae, which in this analysis included Alaurina composita nested within Microstomum.
bPTP and mPTP results were consistent across analyses, and a total of twenty species were identified within the Microstomum clade (Fig 2). Twelve of these were previously described: We obtained high support for four main clades within Microstomum (Figs 1 and 2 Unfortunately, nucleotide sequences from two species of Alaurina and one species of Myozonella were unavailable due to lack of specimens; however, results from our phylogenetic analyses found all specimens of Alaurina composita nested within the Microstomum clade (Figs 1 and 2), and thus we herein propose to synonymise Alaurina with Microstomum and transfer its three species to Microstomum.

Nomenclature acts
Synonymization of Alaurina and amended diagnosis of Microstomum Schmidt 1848.

Gen. Microstomum SCHMIDT 1848
Microstomidae with ciliary pits at frontal end and preoral intestine. Two excretion openings in the anterior body. Rhabdites and ingested nematocysts present in some species. Asexual reproduction through fissioning as well as sexual reproduction. Sexual anatomy including one or two testes beside or behind the intestine; one or two ovaries consisting of one or more lobes and containing a central oocyte and a peripheral layer of nutrient cells. Body size of solitary individual 0.6-4mm, with chains comprising up to 18 zooids and 15 mm.
Type species: Microstomum lineare (Müller, 1773). 43 species: 33 marine, 9 limnic, and 1 inhabiting both brackish (up to 7‰) and freshwaters. (Fuhrmann, 1896). M. lucidum and M. hamatum are morphological identical accepting the ambiguously-defined "dark greyish ± pigment spots" in M. hamatum [57]. Both species have bodies colored yellow due to the intestine, with identical body shapes and sizes and adhesive papillae on the posterior ends. Both have rhabdites sparsely present over the entire body, concentrated at the posterior end and a preoral intestine extending to the brain. Moreover, both species inhabit the Northern Atlantic in overlapping habitats, in mud on opposite sides of the English Channel: Baie de la Forêt near Concarneau for M. lucidum, and the harbor near Plymouth for M. hamatum. Unfortunately, Fuhrmann [58] provided no images of M. lucidum apart from a drawing of a single rhabdite bundle and was unable to collect specimens with sexual anatomy. Nevertheless, the similarities in morphology and ecology suggest it is highly likely that these represent the same species if-as we have interpreted it-the "±" of Westblad's [57] description means that some individuals may not have dark grey spots. Regardless, body pigmentation is known to be highly variable in this genus [8,59], and Westblad [57]  Diagnosis: Marine Microstomum with an almost elliptical body shape; body length 0.8 mm, 2 zooids. Colorless and without pigmented eyespots. Anterior end smoothly rounded. Posterior end slightly more tapered but also with rounded tip. Ciliary pits small and shallow; may be difficult to discern in some specimens. Few adhesive papillae present on anterior and posterior ends of body. Rhabdites sparsely present throughout body. Nematocysts particularly concentrated at posterior end. Preoral intestine extending anterior to brain. Reproductive system unknown. GenBank accession number for partial CO1 sequence MK504554; for 18S MK504480.

Synonymization of Microstomum hamatum Westblad 1953 and Microstomum lucidum
Etymology: Named for Lars Fredrik Afzelius, the first director of the Tjärnö Marine Laboratory, in appreciation of his contributions to the marine scientific community.
Additional materials, asexual specimen, same as type (SMNH-Type-9152). Description: Microstomum with a total body length of 0.8 mm, 2 zooids. Body colorless and without pigmented eyespots. Body shape almost elliptical, with widest point occurring approximately at midpoint or slightly anterior, lightly decreasing in width to a rounded anterior end, and a slightly more pronounced tapering to the rounded posterior end. Ration of body width: length~1:2.5 in slightly compressed animal.
Ciliary pits weakly present, may be difficult to distinguish in some specimens. Small and shallow, located at level of posterior brain.
Mouth circular, 50 μm in diameter, surrounded by cilia. Pharynx also ciliated, 138 μm long, surrounded by relatively few pharyngeal glands. Preoral intestine extending anterior to brain. Intestine filled with lightly orange-brown food.
Reproductive system unknown. Notes: There are currently 23 species of Microstomum that have neither pigmented eyespots nor distinct body pigmentation. Of these, M. afzelii sp. nov. can generally be easily separated by its body shape, especially the rounded body ends, preoral intestine extending anterior to the brain and the presence of the adhesive papillae on the anterior end. The other species of Microstomum without very distinct and characteristic pigmentation can specifically be separated from M. afzelii sp. nov. in the following ways: M. album (ATTEMS 1896)-anterior proboscis present; more numerous posterior adhesive papillae, presence of adhesive papillae on midbody; more numerous rhabdites; short preoral intestine M. bispiralis STIREWALT 1937 -posterior end pointed; preoral intestine short; adhesive papillae and rhabdites absent; limnic habitat; also distinguished via bPTP and mPTP analyses (Fig 2).
M. mundum GRAFF 1905 -characteristic lateral bulges in intestine; preoral intestine extending only to level of brain; posterior end tail-like; anterior adhesive papillae absent M. papillosum (GRAFF 1882)-more numerous rhabdites at anterior end; adhesive papillae scattered across entire body; also distinguished via bPTP and mPTP analyses (Fig 2).
M. rhabdotum MARCUS 1950 -biparte pharynx; paired stripes of rhabdite bundles at ciliary pits; preoral intestine extending only to level of brain M. spiculifer FAUBEL 1974 -anterior adhesive papillae absent; more numerous posterior rhabdites; longer preoral intestine reaching almost to very tip of body M. trichotum MARCUS 1950 -long and tapered anterior end; more numerous rhabdites concentrated at anterior; anterior adhesive papillae absent; preoral intestine extending only to level of brain M. ulum MARCUS 1950 -preoral intestine short; deep ciliary pits; distinct spatulate tail plate; more numerous rhabdites concentrated at posterior end M. weberi sp. nov.-bottle-shaped ciliary pits; anterior crown of adhesive papillae; also distinguished via bPTP and mPTP analyses (Fig 2).
M. westbladi nom. nov.-more numerous rhabdites highly concentrated at anterior end; adhesive papillae scattered across entire body; characteristic shape of field of adhesive papillae at anterior end; also distinguished via bPTP and mPTP analyses (Fig 2).
Microstomum curinigalletti sp. nov. urn:lsid:zoobank.org:act:0385A782-B841-4DE0-BD6B-EA4B34141D0B Fig 4 Diagnosis: Marine Microstomum with a body length of 1.4 mm, 2 zooids. Anterior body end colored ochre to dark yellow-orange. Body otherwise colorless without pigmented eyespots. Body shape with a conically rounded anterior end and wide, blunt posterior end. Numerous adhesive papillae present at posterior end. Rhabdites present but few, scattered across the body. Ciliary pits large and bottle-shaped, located anterior to pharynx. Preoral intestine short. Reproductive system unknown. GenBank accession number for partial 18S sequence MK504534.
Etymology: This species is dedicated with many thanks to Dr. Marco Curini-Galletti, who found the first specimens of these animals.
Description: Microstomum with a total body length to 1.4 mm, 2 zooids. Anterior body end colored ochre to dark yellow-orange. Body otherwise colorless and reflecting intestines. Pigmented eyespots absent. Body shape with a conically rounded anterior end; posterior end blunt and almost as wide as rest of body. Ratio of width generally consistent across body, width:length is 1:4 in slightly compressed animal.
Ciliary pits large, clearly distinguishable anterior to pharynx; bottle shaped, extending at a 45˚angle posterior into the body~65 μm; opening 30 μm long.
Mouth small and circular surrounded by cilia. Pharynx surrounded by normal ring of pharyngeal glands. Preoral intestine very short, hardly extending past mouth.
Reproductive system unknown. Notes: In body shape and size and, especially, size, shape and location of ciliary pits ( Fig  4C), Microstomum curinigalletti sp. nov. is most similar to M. edmondi. The two species also share habitats and distributions and were collected in the same samples. M. curinigalletti sp. nov., however, can easily be distinguished from M. edmondi through the yellow pigmentation of the anterior end and the shorter preoral intestine. Results from the phylogenetic and bPTP analyses also clearly separated the two species (Figs 1 and 2 Description: Microstomum with a total body length to 0.85 mm, 2 zooids. Body colorless and without pigmented eyespots. Body shape with a conically pointed anterior end and a tapered posterior end with a blunt tip. Maximum body width just posterior to pharynx; ratio of maximum width:length is~1:3 in slightly compressed animal. Ciliary pits clearly distinguishable at level of anterior pharynx 170 μm from anterior body end; roundish to cup-shaped, extending at a slight angle into the body 27 μm; opening 20 μl long.
Epidermis uniformly covered with cilia. Very few nematocysts present, primarily at body ends. Rhabdites absent. Numerous adhesive papillae,~15 long, present at posterior end and along up to~⅕ of the posterior body; may also be present at zone of division in very well developed zooids.
Mouth elliptical, 45 μm long, surrounded by cilia. Pharynx 125 μm long, surrounded by normal ring of pharyngeal glands. Preoral intestine extending well anterior to brain, reaching almost to anterior body tip.
Reproductive system unknown.
Notes: Microstomum inexcultus sp. nov. can be distinguished from all other species of Microstomum through a combination of the following characters: absence of pigmentation or eyespots; preoral intestine very long and extending almost to anterior body tip ( Fig 5C); rhabdites absent. The absence of rhabdites in particular distinguished this species, since only 11 other species of Microstomum do not have rhabdites recorded; these can be distinguished from M. inexcultus sp. nov. by the following: M. artoisi ATHERTON & JONDELIUS 2018 -anterior red eyestripes present; preoral intestine short; posterior end tail-like; adhesive papillae absent; limnic habitat; also distinguished via bPTP and mPTP analyses (Fig 2).
Description: Microstomum with a total body length to 0.8 mm, 2 zooids. Body colorless and without pigmented eyespots. Anterior end conically pointed with row of adhesive papillae at the frontal end,~30 μm from anterior tip. Posterior end forming a distinct tail-plate separated by a constriction of the body width. Width to length ration is approximately 1:5.
Ciliary pits large, present posterior to brain; each forming a roundish indentation angled slightly anteriorly; extending inward 21 μm with 16 μm long openings.
Epidermis uniformly covered with cilia. Rhabdites bundles scattered throughout the body, 12-17 μm. In addition to those at the frontal end, numerous adhesive papillae, 10 μm long, are present at the tail-plate and less frequently at the margins to approximately halfway up the body.
Mouth circular and surrounded by cilia. Pharynx 220 μm long, surrounded by normal ring of pharyngeal glands. Preoral intestine reaching to level halfway between pharynx and brain.
Reproductive system unknown.  Ciliary pits present, cup shaped, located at level between anterior pharynx and brain. Preoral intestine short, reaching just anterior to pharynx. Rhabdites absent, but with many nematocysts intensely concentrated at posterior end. Male stylet a curved tube tapering distally to a slightly enlarged end. GenBank accession number for partial CO1 sequence KP730580; for 18S KP730505.
Description: Microstomum with a total body length to 0.65 mm, 2 zooids. Body colorless and without pigmented eyespots. Anterior end long, measuring 95 μm from tip to anterior brain, with a widely rounded tip. Body width slightly decreasing from frontal end to ciliary pits, slightly increasing toward the middle of the animal and then decreasing again to a blunt posterior end. Animal measures approximately 80:70:120:45 μm at frontal end: ciliary pits: body middle: posterior end.
Ciliary pits present posterior to brain; each forming a roundish indentation angled at approximately 45˚into the body; extending 10 μm with 10 μm long openings.
Epidermis uniformly covered with cilia. Rhabdites absent. Very many nematocysts present throughout the animal and especially intensely concentrated at the posterior end. Numerous adhesive papillae,~10 μm long, present along~⅕ of the posterior body and also sparsely occurring anteriorly.
Mouth surrounded by cilia. Pharynx 95 μm long, surrounded by normal ring of pharyngeal glands. Preoral intestine short, terminating just posterior to level of brain.
Male reproductive system with single developing testis connected by short vas deferens to a rounded vesicula granulorum. Male stylet, 63 μm long, a slightly tapering tube, 9 μm wide at base and~3 μm at narrowest part of distal end; curving a little less than 90˚, with a slight widening at the distal tip to~4.5 μm. Genital pores separate. Male pore 18 μm in diameter.
Notes: The stylet and reproductive system of Microstomum marisrubri sp. nov. (Fig 7E) is very similar to M. papillosum, although it is smaller (62 μm compared to 80 μm, respectively). Other differences between the two species can be found in the size of the preoral intestine, which for M. papillsum extends past the brain, and the presence of numerous rhabdites at the anterior end. As discussed above, few species of Microstomum lack rhabdites, and these can be distinguished: M. artoisi ATHERTON & JONDELIUS 2018 -anterior red eyestripes present; posterior end taillike; adhesive papillae absent; limnic habitat; also distinguished via bPTP and mPTP analyses (Fig 2).
Microstomum schultei sp. nov. urn:lsid:zoobank.org:act:589757DC-E737-4951-BB29-4ADB204C0FAD Fig 8 Diagnosis: Marine Microstomum with a body length of 1.5 mm, 1 zooid. Body appears light reddish-brown without pigmented eyespots. Anterior end rounded and set apart from the rest of the body by a slight constriction at the ciliary pits. Posterior end conically blunted. Adhesive papillae and rhabdites concentrated at anterior and posterior ends. Ciliary pits present, cup shaped. Preoral intestine short. Reproductive system with curved male stylet, tapering from a wider base to a narrow tube with a beveled tip. GenBank accession number for 18S KP730494.
Etymology: This species is named in honor of Gregor Schulte, who was a field assistant during the 2010 BIOSAND workshop and aided in sample collection.
Description: Microstomum with a total body length to 1.5 mm, 1 zooid. Body light reddishbrown, without pigmented eyespots. Anterior end rounded and set apart from the rest of the body by a slight constriction at the ciliary pits. Posterior end conically blunted. Ratio of maximum width:length is~1:5 in slightly compressed animal.
Ciliary pits present at level just posterior to brain, 170 μm from anterior body end; roundish to cup-shaped, extending into the body 30 μm with 15 μm long openings.
Epidermis uniformly covered with cilia. Rhabdites present throughout the body, but especially concentrated at the anterior and posterior ends. Numerous short adhesive papillae, 10 μm long, present, heavily concentrated at posterior end along~⅙ of the body and also anterior to brain.
Reproductive system with single medial ovary and caudally developing eggs. Male system with vesicula granulorum connected to male stylet, 60 μm long, curved and tapering from a wider base to form a narrow tube with a beveled tip. Stylet base 20 μm wide, distal tube~4 μm wide, opening~5 μm long. Sperm~70 μm long, with a rounded head and a single flagellum with lateral setae.
Notes: There are a few species of Microstomum with a similarly shaped male stylet (Fig 8D). M. schultei sp. nov. can be separated from each of these through: M. crildensis FAUBEL, 1984 -stylet much smaller (32 μm instead of 60 μm), with a more narrow base; rhabdites absent; anterior adhesive papillae absent; body colorless; also distinguished via bPTP and mPTP analyses (Fig 2).
M. gabriellae MARCUS, 1950 -stylet even more curved, tracing an entire half circle 180˚; terminal end not beveled; preoral intestine large, extending past brain; red eyespot present M. septentrionale (SABUSSOW 1900)-stylet without beveled terminal end; preoral intestine reaching to level of anterior brain; body color with dorsal yellow pigmentation; also distinguished via bPTP and mPTP analyses (Fig 2).
In addition, M. schultei sp. nov can generally be distinguished from the other species of Microstomum through a combination of the presence of adhesive papillae at the anterior and posterior ends and a short preoral intestine (defined here as a preoral intestine that does not extend to at least the level of posterior brain). M. schultei sp. nov. can be separated from the other species of Microstomum with these characters by the following ways: M. album (ATTEMS 1896)-body colorless or white; anterior proboscis present; ciliary pits small M. crildensis FAUBEL, 1984 -see above M. curinigalletti sp. nov.-frontal end colored ochre; ciliary pits large and bottle shaped; anterior adhesive papillae absent; fewer rhabdites; also distinguished via bPTP and mPTP analyses (Fig 2).
M. punctatum DORNER 1902 -body brown-yellow with black spots; pointed anterior and tail-like posterior ends; ciliary pits small and shallow; rhabdites absent; limnic habitat M. rubromaculatum (GRAFF, 1882)-red pigmentation stripe at anterior end present; widely blunt posterior end; also distinguished via bPTP and mPTP analyses (Fig 2). Description: Body length 0.65 mm, 2 zooids. Body colorless and without pigmented eyespots. Anterior end conically rounded. Body width smaller at anterior end and increases perceptively just after ciliary pits. Posterior tapering to a wide blunt end.
Ciliary pits bottle-shaped, present at level of anterior pharynx; extending 13 μm into the body at slight posteriorly-directed angle; with 12 μm long openings.
Epidermis uniformly covered with cilia. Large rhabdites bundles, to 20 μm, present only at anterior and posterior ends. A row of adhesive papillae at the frontal end, situated ventrally 25 μm from anterior tip. Additional adhesive papillae,~7-10 μm long, also present at posterior end, but are otherwise absent from the body margins.
Mouth eliptical and surrounded by cilia. Pharynx 105 μm long, surrounded by normal ring of pharyngeal glands. Preoral intestine reaching just past anterior brain.
Reproductive system unknown.
Notes: This is only the second species of Microstomum with a ventral row of adhesive papillae on the frontal end (Fig 9C), the other being M. lotti sp. nov. M. weberi sp. nov. can be distinguished by general body shape and size, through the bottle-shaped ciliary pits (Fig 9C), the longer preoral intestine and by the large rhabdite bundles present only near the body ends. Results from the phylogenetic and bPTP analyses also clearly separated the two species (Figs 1  and 2).
[57]-Norway, Herdla, Kvaernepoll current; Sweden, Gullmarfjord This paper: Sweden, Fiskebäckskil, 58˚15'05"N 11˚27'52" E Diagnosis: Marine Microstomum with strap shaped body, up to 2.0 mm long and 5 zooids. Colorless and without pigmented eyespots. Anterior end rounded. Posterior tapered to a blunt end. Ciliary pits small. Numerous rhabdites intensely concentrated at frontal end and present throughout body. Numerous adhesive papillae present at posterior end and more sparsely anterior. Characteristic grouping of adhesive papillae covering the anterior tip: field longest at both lateral sides and curving upwards toward the middle. Preoral intestine extending to brain. Reproductive system including male stylet, 60 μm long, and shaped as a smooth curve 180˚curve, with a maximum width~⅙ of the way to the distal end, decreasing proximally and also distally to a point. GenBank accession number for partial CO1 sequence MK504633; for 18S MK504553.
Etymology: This species is named in honor of Dr. Einar Westblad, who first described these animals.
Description: Microstomum with a total body length to 2.0 mm, 5 zooids. Body colorless and without pigmented eyespots. Body strap-shaped with a bluntly rounded anterior end, an almost equal width along entire body until a slight decreasing in width to a bluntly rounded posterior end.
Ciliary pits small and shallow cups, located at level of anterior pharynx. Epidermis uniformly covered with cilia. Nematocysts not seen or recorded. Rhabdites present,~15-30 μm long, throughout entire body, but intensely concentrated at anterior end. Numerous adhesive papillae,~12-15 μm long, present at posterior end and more sparsely anterior. Further adhesive papillae present at the anterior tip grouped in a very distinct pattern: the field covers the anterior tip, toupée like, i.e. longest at both lateral sides and curving upwards toward the middle.
Mouth elliptical surrounded by cilia. Pharynx 110 μm long, surrounded by normal ring of pharyngeal glands. Preoral intestine extending to brain.
Reproductive system mostly unknown. Following Westblad [57] stylet recorded as 60 μm long, shaped as a smooth curve 180˚curve, with the width first increasing along the most prox-imal~⅙ of the stylet and then smoothly decreasing in width to a pointed, terminal opening.
Notes: Microstomum westbladi nom. nov. can be readily distinguished from M. papillosum by the characteristic pattern of the anterior adhesive papillae (Fig 10B and 10C), the very high concentration of rhabdites in the anterior end (Fig 10D) and the shape of the stylet, which decreases in width toward the proximal end as well as distally. In addition, results of the phylogenetic and bPTP analyses clearly separate the two species (Figs 1 and 2).
M. westbladi nom. nov. is most similar to M. septentrionale, particularly since both species are of similar size and shape, have a very high concentration of rhabdites in the anterior end, and are known to have overlapping habitats. However, M. westbladi nom. nov. can be distinguished morphologically by its colorless body without dark or orange-yellow in the anterior and through the characteristic pattern of anterior adhesive papillae. Additionally, the stylet shape of M. westbladi nom. nov. differs by decreasing in width toward the proximal end. M. septentrionale and M. westbladi nom. nov. are closely related sister taxa (Figs 1 and 2).

Phylogenetic analyses and DNA taxonomy
The use of nucleotide sequences to delineate species and capture biodiversity is an important advancement in taxonomy of Microstomidae. As noted in the introduction, the animals may have relatively few morphological characters on which to base descriptions, and thus identification from morphology alone may require high amounts of effort, specimens and specialized taxonomic expertise. Further, many morphological features are highly variable depending on environmental conditions [10][11]59]. Laboratory studies of Microstomum lineare have demonstrated that body shape and size varies based on food availability and sexual maturity; body color ranges from colorless, white, yellow, grey, reddish or brownish based on light, food type and habitat; and even amount of pigmentation in the characteristic red eyespots can range from large bright red slashes at the frontal end to non-existence depending on light intensity [4,[9][10][11]. DNA taxonomy provides a robust, reproducible and relatively easy method of identification that can also account for the hidden biodiversity of cryptic species [28][29].
The results of the bPTP analyses on the concatenated 18S and CO1 datasets (Fig 2) were able to reliably separate the species of Microstomidae and supported the more traditional morphological delineation. Support for four species (M. crildensis, M. laurae, M. lotti, M. septentrionale) was consistent but low (0.49-0.68) and is unsurprising given the overall low intraspecific genetic diversity within each Microstomum species. Support was moderate to high (�0.79) for all other species.
Within the Microstomidae, our results found all limnic species (M. artoisi, M. bispiralis, M. lineare, M. tchaikovskyi, M. zicklerorum) grouped together in a single clade with high support (0.91), indicating a potential for a single instance of freshwater invasion from marine habitats. The transition of marine to freshwaters within a lineage is a common occurrence for many different animal taxa [60][61] and has occurred independently numerous times within the Platyhelminthes, including within Macrostomidae [44,62]. Currently, ten species of Microstomidae, including the sole species of Myozonella: Myozonella microstomoides, have been reported exclusively from freshwaters while one (M. lineare) inhabits both limnic and brackish waters to 7‰. Further broad sampling efforts in different parts of the world will be required to illuminate the origin and distribution patterns of freshwater Microstomidae, particularly as sampling efforts-and thereby our knowledge of biodiversity-have thus far been strongly biased towards temperate regions and shallow littoral waters.

Morphological taxonomy
Morphological identification of flatworms in general is often dependent on the sexual anatomy, and species identification of Macrostomorpha specifically is heavily or entirely based on the morphology of the reproductive organs (e.g. [22][23][24]). Some authors have argued that species of Microstomidae with unknown sexual anatomy are incompletely described and should not be considered valid [20]. Unfortunately, specimens of Microstomidae are most often immature when collected due to very short seasons of sexual reproduction, and thus scientists are often reluctant to describe new species (e.g. [7,[63][64]). Ignoring or invalidating immature Microstomidae leads to large parts of biodiversity left unexplored in a group of animals that are ubiquitous, abundant (e.g. [65][66]) and ecologically important [67] and prevents the discovered taxa from being included in future research or conservation efforts [68]. Further, invalidating asexually reproducing immature species assumes that all species must have sexual anatomy, an assumption that is not necessarily legitimate. Instances of obligate asexuality are widespread throughout the animal kingdom (reviewed in [69]), and animal taxa already possessing the ability to reproduce asexually can give rise to asexual lineages without sexual organs, as has been demonstrated in e.g. aphids [70], ostracods [71] as well as numerous other meio-and macrofaunal taxa [72].
In order to facilitate future research, we created a key to Microstomidae that is not dependent on the reproductive system so that species may be distinguished even when immature (Table 1). As the taxonomic literature is often old and may be difficult to acquire, we further summarize the morphological and molecular taxonomy of all currently accepted species within Microstomidae. Important distinguishing morphological characters include: shape of anterior end (blunt, conical, proboscis, rounded; Fig 11); shape of posterior end (blunt, pointed, rounded, spatulate, tail-like; Fig 12); size and shape of ciliary pits (rounded, bottle-shaped; Fig 13); length of preoral intestine (defined as long if extending anterior to the brain, medium if reaching to level of brain, and short if terminating posterior to level of brain; Fig 14); body pigmentation; presence, location and color of pigmented eyespots; presence and location of rhabdites; presence, pattern and location of adhesive papillae; as well as other characters specific to individual species (e.g. the lateral intestinal bulges of M. mundum; the biparte pharynx of M. breviceps; Fig 15). S3 Table summarizes the morphological characters for each species.

Fam. Microstomidae LUTHER, 1907
Macrostomida with intestine extending anterior to the mouth (preoral intestine). Ciliated sensory pits present at frontal end, typically around level of brain. Frontal glands present surrounding pharynx. Asexual reproduction through fissioning occurring. Marine, brackish and freshwater habitats; two genera.
Type: Microstomum SCHMIDT 1848 Gen. Microstomum SCHMIDT 1848 Microstomidae with ciliary pits at frontal end and preoral intestine. Two excretion openings in the anterior body. Rhabdites and nematocysts present in some species. Asexual Pigmented Eyes Absent 13.
Pigmented eyes dark, not red 9.
3. Adhesive papillae present at the posterior end of the animal 4.
Posterior adhesive papillae not present 7.
4. Adhesive papillae present at the anterior end of the animal 5.
Anterior adhesive papillae not present 6.
5. Eyespots marginal dots close to anterior tip; body color reddish; preoral intestine to level of brain

M. gabriellae
Field of red pigmentation forming broad transverse band at anterior end; preoral intestine small, not reaching to brain M. rubromaculatum 6. Paired red eyespots as small dots located at lateral margins of the body at level of brain, posterior to small ciliary pits

M. laurae
Single medial eyespot, oval shaped, located anterior to the brain; intestine with lateral lobules and bulges Anterior adhesive papillae not present 28.
14. Posterior end forming a spatulate tail plate set off by a constriction in the body width 15.
Posterior end not so 17.
15. Ciliary pits small and shallow; preoral intestine reaching anterior to brain; pharynx biparte with longer anterior cilia and shorter posterior
16. Adhesive papillae on frontal end situated in a row~30 μm from the anterior tip of the animal

M. lotti
Rhabdites heavily concentrated at anterior end above pharynx M. ulum 17. Preoral intestine short, terminating posterior to level of brain 18.
Preoral intestine extending to brain or anterior 22.

Anterior end enlarged to form a proboscis, non ciliated but with sensory bristles M. album
Anterior end not so 20.
20. Anterior end bluntly rounded; field of red pigmentation forming broad transverse band at anterior end M. rubromaculatum Body length of solitary animal to 1.6 mm, chains to 2.5 mm, 4 zooids. Body colorless or white, displaying yellow-orange intestine. Pigmented eyes absent. Anterior end smoothly tapering to conically pointed proboscis, non-ciliated but with tactile bristles and a ring of papillae. Posterior end rounded. Small ciliary pits present slightly anterior to mouth. Rhabdites present, numerous over entire body. Preoral intestine short. Adhesive papillae sparsely present along entire mid to posterior body and highly concentrated at posterior end.
Reproductive system with single testis and ovary. Stylet arched projecting into antrum masculinum. True prostatic vesicle present in addition to seminal vesicle present. Female system with ovary and short duct. Gonopores separate.
Reproductive system unknown.

Microstomum bioculatum FAUBEL, 1984
Type: Not deposited Type locality: North Sea, Sylt Island, off Keitum Habitat: Marine, eulittoral and superlittoral lentic upper mudflats and salt meadows. Body length to 1.4 mm, maximum 4 zooids. Body yellow-green with two very small red eyespots at frontal margins. Ends rounded; posterior with long, stiff cilia that are twice the length of the locomotory cilia. Rhabdites present. Ciliated pits, preoral intestine and adhesive papillae all apparently absent.
Reproductive system not seen. Notes: The absence of the preoral intestine and, especially, ciliated pits is extremely unusual, as both are defining characters of the genus. Additionally, there seems to be some confusion regarding the preoral intestine, since the original written description by Faubel [20] specifically precludes its presence ("Ein Kopfdarm und typische Einschnürungen des Körpers in Höhe des Gehirns fehlen"), but Faubel's original drawing of the species distinctly includes a preoral intestine that extends anterior to the brain.

Microstomum bispiralis STIREWALT, 1937; Figs 16E and 17
Type: Deposition not recorded Type locality: near University of Virginia, Virginia, USA Habitat: Freshwater pools This paper: Sweden, Finland GenBank Accession: 18S -MK504481 -MK504525, specimens from Sweden CO1 -MK504555 -MK504601, specimens from Sweden Maximum 8 zooids, 4 most common, with body length to 4 mm. Following laboratory cultures, individuals can be transparent-grey to bright yellow. Pigmented eyespots absent. Anterior end short conically pointed. Posterior end tapered to a point. Ciliary pits present anterior to mouth, each shaped as a tube extending posteriorly at 45˚angle and ending in an enlarged spherical pocket. Pharynx ciliated, and occupies ⅓ to ½ length of anterior zooid. Preoral intestine short. Adhesive papillae and rhabdites absent. Nematocysts may be present in some specimens.
Nervous system and cerebral ganglion as typical for the genus; consisting of two anterior ganglia connected by a short and wide transverse commissure. A dorsolateral and a ventrolateral nerve extend posteriorly from each ganglion. Ventrolateral nerves are connected by a commissure ventral to the pharynx. One nerve extends laterally from each ganglion to the ciliary pit and another from each ganglion to branch profusely in the anterior end of the body.
Protanderous hermaphrodites, with male formation occurring in laboratory cultures during January and February. Male genital system consisting of two compact and spherical testes, one developing earlier than the other. Vasa deferentia extending posteriorly from each testis to empty dorsolaterally into the vesicula seminalis. Vesicula seminalis shaped as a small sphere, located ventral and posterior to the intestine; glandular and surrounded by a heavy muscular sheath of the copulatory organ. Copulatory stylet a smooth spiral sharply bent in two planes, 120 μm long, widened at the base and drawn out distally to a sharp point.
Female reproductive system includes a single ovary lying slightly anterior to the middle of the zooid, ventral to the intestine. Numerous single-celled glands open to the vagina, which opens to a female pore at the beginning of the posterior ⅓ of the zooid.
Somatic chromosome number 16 (n = 8) Notes: This is the first time that M. bispiralis is reported from Sweden or anywhere else outside of the original location. The specimens collected in Finland and Sweden matched the original descriptions, including the male stylet and other sexual anatomy that was found in three individuals (Fig 17C). The very great distance between the locations does suggest the possibility of cryptic species or much larger dispersal abilities than has previously been shown in any species of Microstomum [see 4], although true support for either scenario must be deferred until DNA sequence data can be attained from specimens collected at the type locality.
The absence of adhesive papillae and rhabdites combined with a short preoral intestine is a unique set of characters that separates this species from nearly all of the other currently accepted Maximum 3 zooids, 0.7 mm. Color and pigmented eyes unknown as worms were only described based on preserved state [76]. Anterior end rounded, posterior spatulate. Adhesive papillae present anteroventrally above level of the brain, at the posterior tip and in the zone of division in mature zooids. Ciliated pits small. Rhabdites occur throughout the body. Preoral intestine extends anterior to brain. Mouth circular. Pharynx divided into two parts by an annular fold, with longer cilia in the anterior part and shorter cilia in the posterior. Glands around the pharynx may be pale pink in color. Intestine unciliated.
Reproductive organs not seen. Notes: Microstomum breviceps is similar to M. davenporti in that both have similar patterns of adhesive papillae, a long preoral intestine and a characteristic divided pharynx with long and short cilia and with an annular fold. M. breviceps can be separated from M. davenporti by its body shape: the anterior end of M. breviceps is truly round instead of tapering blunt, and the posterior spatulate tail-plate is very distinct from the rest of the body. Other differences can be found in the high concentration of rhabdites surrounding the oval mouth in M. davenporti, and the very large distances separating their locations (São Sebastião, Brazil and Woods Hole Massachusetts, USA for M. breviceps and M. davenporti, respectively). �� A type specimen was not previously designated for M. breviceps. However, two original specimens were deposited at the Swedish Museum of Natural History, and thus, following the rules of the International Code of Zoological Nomenclature, we hereby designate one sectioned asexual specimen (SMNH-108136) as lectotype and one whole mount asexual specimen (SMNH-108137) as paralectotype for Microstomum breviceps MARCUS, 1951.
Reproductive system not observed. Notes: Ciliary pits were not described for this species, nor were they included in Schmarda's [77] drawing. However, the original description occurred at a very early time period, and positively identifying ciliary pits may be difficult even with modern equipment when they are small and/or shallow. The importance of the character for this genus (as first argued by [80][81]) suggests that the presence or absence of ciliary pits for this species should remain in question until they can be re-examined. Even without absolute knowledge of the adhesive papillae, ciliary pits, and reproductive system, the combination of blue body color, absence of pigmented eyespots, large pre-oral intestine, blunt body ends and freshwater habitat is enough to separate this species from all other currently described species of Microstomum.
Reproductive system unknown. Notes: Hofsten [83] and Meixner [84] postulated that M. canum is a synonym of M. lineare based on variability of the latter. While the wide morphological variations of M. lineare undoubtedly creates difficulty in absolutely ruling out synonymy, potential differences between the two species exist in the extension of the preoral intestine anterior to the brain, a more posterior position of the ciliary pits and, perhaps, a more pointed anterior end ( GenBank Sequences 18S -MK504476 -MK504479, specimens from Sweden CO1 -MK504602 -MK504607, specimens from Sweden Body length of solitary animal to 0.9 mm, chains to 3.0 mm. Maximum 10 zooids, 4 most common. Body color light yellow with greenish anterior (proboscis). Black pigmented eyespots present. Anterior end sharply decreasing in width to form distinct proboscis, with sensory cilia, papillae and numerous circular folds, and denoted by paired transverse tufts of cilia at base. Posterior tip with thin tail and terminal sensory cilia. Preoral intestine extending anterior to brain. Intestine non-ciliated, often with diatoms and protozoa. Rhabdites may be present in the anterior end.
Male reproductive system including a single testis, which may be bilobed in some specimens. Stylet weakly arched connecting to seminal vesicle.
Female system unknown. Notes: Brinkmann [74] (pg 66) argued that both Alaurina viridirostrum and A. claparedii were identical to this species, stating that the very few differences were due to errors derived from outdated methods of investigation and/or a lack of sufficient specimens. GenBank Accession: 18S -MK504526 -MK504529, specimens from Sweden CO1 -MK504608 -MK504611, specimens from Sweden Body to 1.5 mm, maximum 4 zooids. Solitary animal 0.9 mm. Body colorless; pigmented eyes absent. Ends rounded, posterior with adhesive papillae. Constriction at level of brain at ciliary pits. Preoral intestine short, not quite reaching level of brain. Rhabdites and nematocysts absent.
Single testis, located at the very caudal end of the animal. Vesicula granulorum connected to a 32 μm long stylet, shaped as a curved tube of approximately consistent width and with distal terminal opening. Single ovary present.
Notes: Three specimens identified as Microstomum crildensis were collected from Swedish Skagerrak coast. Unfortunately, none of these specimens displayed sexual organs, and so the reproductive system-most importantly the male stylet-could not be used to corroborate identification. However, in every other respect, these specimens matched the original description and drawings, including: overall body shape including a body constriction at the ciliary pits, presence adhesive tubes on the posterior end only, absence of rhabdites, and small preoral intestine. The habitat-eulittoral muddy sediments of the North Sea-was also consistent.
Currently, the only other species of Microstomum with posterior adhesive papillae that entirely lacks rhabdites are M. inexcultus sp. nov., M. marisrubri sp. nov. and M. punctatum. M. crildensis can be distinguished from each of these species through: M. inexcultus sp. nov.-long preoral intestine; constriction of the anterior body width near the ciliary pits; also distinguished via bPTP and mPTP analyses (Fig 2).
M. marisrubri. sp. nov.-shape and size of the male stylet; presence of anterior adhesive papillae and more numerous posterior adhesive papillae; also distinguished via bPTP and mPTP analyses (Fig 2).
Reproductive system unknown.

Microstomum davenporti GRAFF, 1911
Type: Deposition not recorded Type locality: Woods Hole, Massachusetts, USA and Stamford, Connecticut, USA Habitat: Marine, on Ulva sp. and Sargaassum sp. Body length to 1.5 mm; maximum 4 zooids. Body colorless or whiteish with a yellow-ocher intestine. Eyes absent. Anterior conically pointed; posterior end broad with numerous adhesive papillae. Ciliary pits present but very shallow. Rhabdites present, 12 μm, highly concentrated at the anterior end and around the oval mouth. Preoral intestine long. Pharynx divided into two parts by an annular fold, with longer cilia in the anterior part and shorter cilia in the posterior Reproductive system not seen.
[91]-Adriatic sea Small, body length to 0.32 mm in solitary animal. Anterior end short conical; posterior end tapering to a rounded tip. Body colorless. Marginal eyes present very near the anterior end. Ciliary pits small and indistinct, at level posterior to marginal eyes. Rhabdites concentrated at anterior and posterior ends of the body. Adhesive papillae not recorded. Preoral intestine not quite reaching level of brain. Pharynx with 5.4 μm long cilia. Intestine unciliated.
Paired testes, sausage-shaped, obliquely lateral and posterior to ovary. Vesicula granulorum 20 μm long, 13 μm wide, strongly muscled. Male stylet strait tube with distal toothed thickening; small~10-12 μm long (based on drawing). Antrum masculine small, spherical. Genital pores separate. 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.

Microstomum gabriellae MARCUS, 1950
Lectotype: Asexual, whole mount, SMNH-108140; E. Marcus leg. �� Type location: Brazil, São Sebastião Habitat: Marine, between algae, mainly Sargassum stenophyllum Body length to 3 mm; maximum 7 zooids. Color reddish, with anterior marginal paired red eyespots. Anterior end short and conically rounded; posterior end rounded. Ciliated pits are thin and deep, located at level of posterior brain. Adhesive papillae present at the posterior of each well-developed zooid and more sparsely along the body. Rhabdites present, scarce and throughout the body. Nematocysts not seen. Pharyngeal nerve ring present, connected to brain by two short nerves. Preoral intestine reaching to level of brain.
Reproductive system based on original drawings [92]. Male system with paired round testes connecting separately to vesicular seminalis by vas deferens. Stylet approximately 45-50 μm long, shaped as a tube with near consistent width throughout, broadly curved 180˚.

Microstomum giganteum
Male copulatory organ with an oblong seminal vesicle, 150-180 μm long. Stylet with three parts: 85 μm distal part inserted in a 30 μm mid part which is inserted in turn into a 40 μm long proximal part. Total length of the stylet to 180 μm, shaped as a spiral with 2.5 full turns. Caudal ovary with three follicles with triangular ovicels and 4-5 abortive eggs.
Notes: There was much discussion in the literature about whether Microstomum giganetum should be considered a "safe species" or a larger variant of M. lineare. The species name was reduced to a synonym by Luther [9], who considered it to represent a pure line of M. lineare that had developed over a long period of asexual reproduction. M. giganteum eventually regained its independence after Kostenko [105] carried out a comparative study of both forms and found several morphological differences, most importantly in the different structures of the reproductive system and male stylet, without intermediate or transitional forms.
M. giganteum differs from M. lineare through its larger size (8 zooid body length of �8.5 mm compared with 4-5 mm, respectively); lack of elongated tail, adhesive papillae and pigmented eyespots; pharyngeal glands restricted to oral cavity; and reproductive system including larger male stylet with three parts forming greater number of spiral turns (2.5 versus 1.5, respectively). A single medial red eyespot anterior to the brain, oval shaped. Anterior tip of the body conically pointed. Posterior end long, tail-like with many adhesive papillae. Rhabdites present throughout the body, concentrated at the anterior end. Small ciliary pits located at level between the brain and the mouth. Preoral intestine extending to the brain. Intestine with lateral lobules and bulges.

Microstomum groenlandicum
Testes not seen. Male stylet narrow, cylindrical and weakly spiraling, ending with a small, spoon-shaped thickening. Single medial ovary.
18 Pigmented eyespots present as paired red stripes at dorso-anterior end. Amount of pigmentation highly variable and may be absent in some individuals based on light intensity.
Sensory bristles present. Cilia covering body, shorter in mouth. Ciliary pits at level of brain. Intestine ciliated and often including food remnants, including Cladocera, chironomid larvae, Copepoda, hydroids, Nematoda and other small animals. Nematocysts occuring throughout body.
Following TEM an SEM observations by Reuter [187], groups of 6-8 adhesive papillae, each to 4 μm long, present in row at ventrolaterally at frontal end.
Nervous system includes brain, three pairs of nerve chords and pharyngeal ring system. Protonephridia with bilateral stems and pores in anterior body.
Male reproductive system with bilateral testes, circular or eliptical vesicular seminalis with prostate glands and separate gonopore. Male stylet spiraled, to 200 μm long. Female system with single ovary, ciliated oviduct and ventral pore. Proterandrous development.
Notes: Microstomum lineare is an extremely common and abundant species in brackish and freshwater habitats worldwide and can be found February through late November or early December [10][11]. Lifecycle includes 4 generations per year, with sexual reproduction occurring primarily in late summer and fall. M. lineare purportedly has a global distribution and extremely wide tolerance for differing environmental conditions, including salinity (freshwater to 7‰ [188]), temperature (to 47˚C [75]), calcium (to 50 mg/L [185]), oxygen and pH [11,138,141].
Positive identification of M. lineare should be cautiously done since several other species of Microstomum are either very similar or identical in appearance to M. lineare. In part this is due to the very wide morphological variability reported for the species itself. Three species, M. artoisi, M. tchaikovskyi, and M. zicklerorum, were separated from M. lineare primarily through DNA evidence based on the mitochondrial cytochrome c oxidase subunit I and nuclear internal transcribed spacers I and II and 5.8S rDNA gene loci, and thus have few or no morphologically distinguishing characters [4]. A fourth, M. giganteum, though separable based on morphological evidence, can easily be misidentified and has historically been challenged as a "safe species". However, M. giganteum is a larger species than M. lineare (8 zooid body length of �8.5 mm compared with 4-5 mm, respectively), without an elongated tail, adhesive papillae or pigmented eyespots, with a clearly visible esophagus, with pharyngeal glands restricted to the oral cavity only and with a three-part stylet [21,105] 18S -KP730505 CO1 -KP730580 Body length 0.65, 1 zooid. Body colorless, without pigmented eyespots. Body anterior to brain long, with rounded end. Posterior end blunt with numerous adhesive papillae. Adhesive papillae sparsely present over entire body. Ciliary pits present at level between anterior pharynx and brain. Preoral intestine short. Rhabdites absent. Nematocysts intensely concentrated at posterior end.
Male reproductive system including a single testis connected by short vas deferens to a rounded vesicula granulorum. Male stylet 63 μm long, shaped as a slightly tapering curved tube with a slight widening at the distal tip. Genital pores separate.

Microstomum melanophthalmum STEINBÖ CK 1933
Type: deposition not recorded Type location: Croatia, Rovinj, S. Andrea Habitat: Marine, sublittoral to 7 m depth [91,191]-Croatia, Isola Saint Andrea; Italy, Capo di Sorrento, Sicily, Porto Venere; France, Banyuls-sur-mer Body length to 2.0 mm, to two zooids. Clear with yellow intestine. Black or dark brown eyes with distinct crescent lens near the frontal end. Anterior tip blunt. Posterior end conically rounded with adhesive tubes. Ciliary pits present just anterior to brain. Preoral intestine large, extending to well above brain. Rhabdites present.
Male reproductive system with seminal vesicular attached to stylet, 30 μm long, shaped as a sharply decreasing flat funnel connected to a tube hooked at an angle of 90˚with a pointed end and terminal opening. Female system not seen.

Microstomum mortenseni RIEDEL 1932
Type: deposition not recorded Type location: Greenland, Disco Bay Habitat: Marine, sublittoral benthal to 300 m depth Body to 1.2 mm in solitary zooid. Based on Riedel's [192] original drawings, anterior end conically rounded and posterior end rounded and wide. Ciliary pits present at frontal end. Red color with yellow tinged rhammite glands. Pigmented eyespots absent. Rhabdites present at lateral parts of body. Preoral intestine long, anterior to brain. Intestine non-ciliated. Adhesive papillae not recorded.
Reproductive incompletely known. Ovary single with medial female gonopore. S shaped stylet.

Microstomum mundum GRAFF 1905
Type: Deposition not recorded. Type locality: Black Sea, Sevastopol Bay, St. Georges Monastery Habitat: Marine, benthic in the sand Body to 2 mm, 8 zooids. Colorless without pigmented eyespots. Anterior end conical. Adhesive papillae present in the posterior quarter of each well-developed zooid. Ciliary pits present at level of brain. Rhabdites present throughout the body, 16-20 μm long. Nematocysts present. Intestine, including preoral intestine, with characteristic lateral bulges and smaller lobules. Preoral intestine extending to a level halfway between the ciliary pits and anterior tip.
Reproductive system unknown.
-Microstomum ornatum ULJANIN 1870 Species dubiae by Westblad [57] based on its incomplete description, perfunctory drawings, the posterior positioning of the ovary, which is unique among species of Microstomum, and the anterior direction of egg development, which seems to conflict with the posterior position of the female duct.
Male genital system consisting of unpaired testis and vesicula seminalis. Stylet shaped as a curved tube, tapering to a thin distal terminal opening, 80 μm long. Female system including single ovary.
Notes: Microstomum papillosum has a somewhat similar stylet shape as M. gabriellae and M. crildensis, although its stylet is larger (80 μm compared to 50 and 32 μm, respectively) and tapers to a thinner tube distally. Other differences include, for M. crildensis, the size of the preoral intestine and the ciliary pits and the absence of rhabdites; and for M. gabriellae, the red body coloring and red eyespots.

Microstomum paradii (DUGÉS 1828)
Synonyms: Derostoma squalus- [118] Pigmented eyes present lateral and posterior to mouth. Anterior end with conically pointed proboscis, non-ciliated, with many circular folds and denoted at the base by a tuft of cilia on each side. Sensory bristles present evenly spaced along lateral margins of the body, twice as long as the cilia, and single even longer bristle present at posterior end. Adhesive papillae present along entire body.
Notes: Brinkmann [74] argued that this species is identical to Alaurina composita since any differences were either errors or derived from outdated methods of investigation. Graff [75] argued against this, saying the two species differ in the sensory cilia and the location of the pigmented eyespots. Single ovary. Male system unknown.

Microstomum rhabdotum MARCUS 1950
Type: Deposition not recorded Type location: Brazil, São Sebastião Habitat: Marine, between Sargassum stenophyllum Body length to 0.7 mm, 3 zooids. Rounded anterior and posterior ends. Colorless and without pigmented eyespots. Adhesive papillae present at anterior, mouth, dividing zones, posterior. Biparte pharynx separated by annular fold with larger anterior cilia than posterior. Paired stripes of rhabdite bundles on the dorsolateral sides, joining at level of ciliary pits; median dorsal and ventral areas free of rhabdites; Preoral intestine reaches to anterior border of brain.
Reproductive system not seen. 18S -MK504546 -MK504551, specimens from Sweden CO1 -MF185684 -MF185696, specimens from Sweden Body length to 2 mm; maximum of 8 zooids. Very large red eyespot pigmentation, merging dorsally to form a broad transverse band at the anterior of the animal. Otherwise colorless or slightly pinkish reflective of intestines. Body ends bluntly rounded. Adhesive papillae present and concentrated at anterior and posterior ends. Rhabdites present throughout animal. Ciliary pits present with long cilia. Preoral intestine small.
Reproductive system based on specimens collected from Fiskebäckskil, Sweden [18]. Male system with paired testes, copulatory apparatus, antrum masculinum and separate gonopore. Stylet 75-112 μm long, shaped as a single wide spiral bent around a 90˚angle, terminating with a fingerlike hook. Female system with a single ovary situated mid-body, antrum, and a separate gonopore. Eggs develop caudally.
Notes: The amounts of eyespot pigmentation may be greatly variable, with pigmentation ranging from a bright circular band around the anterior end to two clearly distinct separate spots. Steinböck [90] found one animal with a single large pigment spot in the middle that became thinner towards the margins of the body. 18S -KP730494 Body length 1.5 mm, 1 zooid. Body light reddish-brown without pigmented eyespots. Anterior end rounded, with a small constriction at the ciliary pits. Posterior end conically blunted. Adhesive papillae and rhabdites present, concentrated at body ends. Ciliary pits present posterior to level of brain. Preoral intestine short.
Reproductive system with curved male stylet, tapering from a wider base to a narrow tube with a beveled tip. 18S -MK504552, specimen from Sweden CO1 -MK504630 -MK504632, specimens from Sweden Maximum body length 1 mm, 2 zooids. Body translucent yellow with bright yellow intestines. Some specimens may have darker yellow or orange yellow in the anterior end. Pigmented eyespots absent. Anterior end rounded with a tapering rounded posterior end. Adhesive papillae sparsely scattered over the entire body. Preoral intestine reaching to level of anterior brain. Rhabdites present throughout the body, and especially concentrated in anterior end. Ciliary pits present at level of brain.

Microstomum septentrionale
Single ovary and bilobed testis. Copulatory organ a curved tube, with a wide base and tapering distally to a point. Stylet length to 68 μm. Antrum masculinum present just anterior to stylet, large and ciliated. Separate ventral male and female pores.

Microstomum spiculifer FAUBEL 1974
Type: Sagittal section series; Deposition numbers not listed Type location: North Sea, North Frisian Uslands, Sylt Island, List Habitat: Marine, eulittoral, semi-exposed sandy beach Body length to 1.5 mm long, maximum 4 zooids. Body color clear with reddish brown intestine. Eyes absent. Ends rounded, posterior with multiple adhesive papillae. Preoral intestine reaching to level of brain or slightly anterior. Ciliated pits present at brain. Rhabdites present across body, concentrated at anterior. Testis unpaired, located laterally and posterior to male copulatory apparatus. Masculine antrum elongated. Stylet strait, ca. to 55 μm long, decreasing from a width of 7-8 μm to a distal point. Ovary unpaired. Male and female pores separate.
With two female ducts and orifices, both connected to the ovary. The anterior acts as a ciliated oviduct and is separated from the ovary by a sphincter. The posterior acts as a female copulatory organ and may contain spermatozoids in the proximal end.
�� A type specimen was not previously designated for M. spiriferum. However, fourteen original specimens were deposited at the Swedish Museum of Natural History, and thus, following the rules of the International Code of Zoological Nomenclature, we hereby designate one whole mount hermaphroditic specimen (SMNH-94968) as lectotype for Microstomum spiriferum WESTBLAD 1953. The remaining thirteen specimens (SMNH-94966 to 94967, SMNH-94969 to 94973, SMNH-97839 to 94944) are thus designated as paralectotypes. Body length approximately 4 mm, 6 zooids. Body clear with darker intestines. Preoral intestine short. Anterior end abruptly tapering, posterior end tail-like without adhesive papillae. Sensory bristles present. Ciliary pits present at the level of anterior pharynx. Red eye pigmentation in two stripes, located dorsally at anterior end. May be weak in some individuals. Nematocysts few, scattered across the body.
Reproductive system not recorded. Notes: See notes of M. lineare.
Male reproductive system with single testis. Stylet shaped as a curved funnel, approximately 12 μm long. Female system not seen. sparsely along body entire length, with characteristic grouping present at anterior tip: field lon-S2 File. Alignment used to create the ML tree for the mPTP analysis. (FAS) S3 File. Tree used in the mPTP analysis. (TREEFILE) S1 Table. Collecting information for the specimens used in this study. Collecting location, specific coordinates and dates are given for each specimen, where available. In addition, the GenBank Accession Numbers for 18S, and CO1 sequences and references are listed. (DOCX) S2