First records of limnoterrestrial tardigrades (Tardigrada) from Haida Gwaii, British Columbia, Canada

Abstract Moss samples were collected from trees and rocks in Haida Gwaii, British Columbia, Canada, and examined for the presence of tardigrades. Specimens from 24 taxa were found in 17 out of the 22 examined samples. New species records for British Columbia are provided and undescribed Grevenius and Crenubiotus species were found; a division in four morphogroups of Grevenius, based on number and presence of placoids, is provided to aid in the future identification. In addition, three specimens of a new species belonging to a potential new undescribed Diploechiniscus species were identified. The finding of Macrobiotus occidentalis occidentalis also provides the occasion to transfer the latter one to the genus Diaforobiotus, for which a new dichotomous key for the identification of its species is given and to redefine the family Richtersiusidae. The DNA sequences of selected taxa are also provided. The high number of tardigrade species collected from a relatively low number of samples highlight how still unexplored is tardigrade diversity, particularly in still-largely insular island systems like Haida Gwaii.

At the same time, the Canadian Pacific coast region includes essentially isolated island systems like the Haida Gwaii archipelago, about 80 km offshore of northwestern British Columbia, which comprises several hundred islands and islets over an area of 1 million hectares.It is likely that Haida Gwaii flora and fauna had been isolated from the mainland since the inundation of the area of the present-day Hecate Strait by about 10,700 years ago (Fedje et al. 2021).Despite extensive development in many areas, significant protected areas, including Naikoon Provincial Park and associated reserves, still support high numbers of endemic, disjunct, and rare species (Golumbia 2000).
In this paper, we provide the first account on tardigrades from Haida Gwaii, with (when possible) DNA sequences to corroborate some of the identification and to allow future comparisons with the recovered taxa.In addition, we provide a division of the genus Grevenius in four morphogroups, based on number and presence of placoids, to aid in the future identification of species in this genus, an amended diagnosis for the family Richtersiusidae, and an updated dichotomous key for the genus Diaforobiotus amending the one given by Stec et al. (2022).

Sampling and tardigrades extraction
Moss samples (22 in total) were collected by H. Choong in several locations in Haida Gwaii as part of a biodiversity survey in collaboration with the BC Parks (i.e., an agency of the British Columbia Ministry of Environment and Climate Change Strategy) and colleagues from the Haida Nation (Table I; Figure 1; map prepared with the R package "ggOceanMaps"; Vihtakari 2022) from 1st to the 5th of June 2022.Moss samples were air dried and preserved in paper bags.To extract tardigrades from their substrates, fragments of all samples (1-3 g of dry sample) were placed in distilled water for ca.30 minutes.After soaking, samples were sieved (sieves meshes: 500 and 32 μm) to separate tardigrades and eggs from the substrate; animals and eggs were then isolated using a needle and a glass pipette under a stereomicroscope.
The remaining fragments of the samples are vouchered in the Invertebrate Zoology Collections, Royal BC Museum, Victoria, BC, Canada (RBCM) and coded following Table I.

Microscopy and imaging
Specimens were mounted at Department of Biological and Environmental Sciences, University of Jyväskylä, Jyväskylä, Finland (JYU) on slides in Hoyer's medium, dried at 60°C for 1 week and then sealed with transparent nail polish.Observations with Light Microscope (LM) and measurements were carried out under both phase contrast (PhC) and differential interference contrast (DIC) up to the maximum magnification (100× oil objective) with a Leica DM RB microscope equipped with a AmScope MU1803 digital camera, at the Department of Life Sciences, University of Modena and Reggio Emilia (Unimore).For structures that could not be satisfactorily focused on a single light microscope photograph, stacks of images were taken and assembled into a single deep-focus image with the AmScope Camera software.To ensure the presence or absence of minute structures of selected specimens, stacks of images were acquired taking advantage of the autofluorescence (light length excitation 488 nm and emission signal collected 509-614 nm) with the Confocal Laser Scanning Microscope [CLSM] Leica SP8 equipped with white laser available at "Centro Interdipartimentale Grandi Strumenti" at the Unimore and 3D reconstructions were performed with the software Leica LAS X (Guidetti et al. 2019;Massa et al. 2021Massa et al. , 2023)).Photographs were assembled into figures with FigureJ (Mutterer & Zinck 2013).
Specimens are deposited in the Bertolani Collection (BC) at the Department of Life Sciences, University of Modena and Reggio Emilia, Italy, and in the Invertebrate Zoology Collections of the Royal BC Museum (RBCM).Microscope slides are reported in the taxonomic account with the sample code (Table I) and slide number both given at JYU (S#_SL#) and BC (C#s#) or RBCM (RBCM#).

Genotyping
The DNA was extracted at JYU from individual animals following a Chelex® 100 resin (BioRad) extraction method by Casquet et al. (2012) with modifications described in detail in Stec et al. (2020a).When possible, carcasses were recovered and mounted on slides as voucher specimens.Voucher specimens are deposited in BC.Slides with mounted voucher specimens are reported in the taxonomic account with the sample code and slide number both given at JYU (S#.Name.V#) and BC (C#v#).Amplification and sequencing of fragments of the 18S ribosomal RNA gene and of the COI mitochondrial gene were amplified and sequencing according to Stec et al. (2020a).Sequencing products were read with the ABI 3130×l sequencer at the Department of Biological and Environmental Science of JYU.Sequences 2 E. Massa et al. were processed in MEGA7 (Kumar et al. 2016) and submitted to NCBI GenBank (Accession numbers provided in Table II).To aid in the taxa identification to species level, the produced sequences were searched against all the NCBI nr database assigned to tardigrades (NCBI: txid42241) with the blastn algorithm (Madden 2002).Matches of taxa from this study and sequences in Blast are discussed in the respective taxonomic accounts.The blastn results are provided in SM.01.

Results
Tardigrades and eggs (149 and 85 respectively) belonging to 24 taxa were found in 17 over 22 samples.The taxa found in each sample are presented in Table III.By the presence of only cirri A, and two pair of lateral projections, a dorsal plates sculpture composed by large multangular granules with massive striae without intracuticular layer of pillars (Gąsiorek et al. 2023), the male individual can be confidently attributed to C. mauccii.One individual with a characteristic plate's ornamentation of the Claxtonia wendti morphotype (Gąsiorek et al. 2023) (large epicuticular granules, which at a high magnification show intracuticular layer composed of numerous pillars).Due to the unsuitable orientation of the individual, and the presence of multiple cryptic species in Claxtonia (Gąsiorek et al. 2023), it is not possible to confirm the identification of this species.
-3 individuals (Slide S2013_SL1 -C5087s1) A possible new species of Diploechiniscus (by having dorsolateral spicules).The description of this species will be the subject of future investigations.

Echiniscus merokensis merokensis Richters, 1904
-1 individual (Slide 2015_SL1 -RBCM 023-00006-004) Table II.Sequences produced in this study.For each sequenced individual the specific name, the sample code as listed at University of Jyvaskyla (Finland; JYU), the individual code as listed at JYU, the slide containing the voucher individual coded as listed at JYU (S#) and as reposited at   First tardigrades record from Haida Gwaii (Canada) By having lateral cirri in A, C, D, E, a spine in Cd and plates, ornamentation composed by irregularshaped granules randomly distributed, the specimens can be confidently assigned to E. merokensis merokensis.

Roszkowska, Grobys, Bartylak & Kaczmarek, 2020
-2 individuals (Slide 2010_SL1 -C5084s1) Despite this species (Figure 2(c-d)) being described from the maritime Antarctic (Roszkowska et al. 2020) and the absence of DNA data on the newly found population, we confirm this record due to the striking morphological match with the original description.

Testechiniscus laterculus (Schuster et al. 1980)
-1 individual (Slide S2016_SL1 -RBCM 023-00007-003) -1 individual sequenced (Voucher slide S2016.Tes. V03 -C5090v1) The individuals found match the original description by Schuster et al. (1980) and the amended diagnosis by Gąsiorek et al. (2018).These are characterized by a peculiar ornamentation of dorsal plates (Figure 2(e)), the presence of evident spines on legs I (Figure 2(f)), and the presence of small upward spurs on the external claws of legs IV (Figure 2(g)).This species has been already recorded from British Columbia by Kathman and Dastych (1990).Due to the very few animals found, we refrain to describe this species; however, we present a detailed morphological account and a differential diagnosis compared to the other species in the genus.

Class
In the buccal apparatus, lamellae absent and AISM present.Ventral lamina absent.Three grainlike macroplacoids and a microplacoid present (Figure 3(a)).The first and the second macroplacoid are almost fused and the third macroplacoid is spaced from the first two with a distance similar to the size of the microplacoid in length.Claws of the same leg are similar in size (Figure 3 Cuticle sculptured from the II claws to the posterior end of the body, with small tubercles forming a faint reticulum (Figure 3(d)).
The individuals found belongs to an undescribed species in the lineatus morphogroup that differs from G. lineatus by the sculptured cuticle in form of tubercles creating a reticulum (sculpture in form of lines in G. lineatus).The morphology of the individuals fits the original description of Hypsibius convergens (Urbanowicz, 1925).The individuals are characterised by the presence of faint septula in the pharynx, together with II macroplacoid long two-thirds of the I macroplacoid which present a median constriction, and robust claws with short common tract.Hypsibius convergens was previously reported as cosmopolitan  First tardigrades record from Haida Gwaii (Canada) absent lunulae under each claw.Primary branch of the external (or posterior in the hind legs) claws slender.Internal (or anterior in the hind legs) claws stumpy.On the main branches of each claw accessory points are present, those are thick on internal (or anterior in the hind legs) and faint on external (or posterior in the hind legs).Eggs unknown.

Ramazzottius gr. oberhaeuseri
-1 egg (Slide S1996_SL1 -C5070s1) Egg of the Ramazzottius oberhaeuseri complex (Stec et al. 2018) without evident cap-like structures on the hemispherical process and with wrinkled surface over the egg chorion and over the processes.The processes are surrounded by dots.The wall of each process is formed by an internal and an external wall.The internal is hemispherical ad slightly flatted on the apex, the width is slightly higher than the height.The external presents in few processes an elongated tip with variable eight.The space between the two walls has the same optical density of the walls.This egg could possibly belong to Ramazzottius sp. 1. considering its proximity to the two samples; however, due to the absence of an embryo, it is not possible to confidently assign it.The morphology is consistent with the redescription and the neotypic material by Ramazzotti and Maucci (1983).A very evident thickening of the buccal tube wall is present after the stylet supports insertions (Figure 5 The individuals and eggs fit with the species redescription by Bertolani & Rebecchi (1993).The identification was also confirmed by the DNA sequencing (Table II).The COI sequences have >99% identity with sequences found in GenBank (SM.01) from the neotype population (HQ876584-7) of M. hufelandi, confirming the morphological identification.Murray, 1910 -4 individuals (Slide 2011_SL1 -C5085s1) In the alive individuals, a space between the cuticle and the epidermis is present.This space is filled First tardigrades record from Haida Gwaii (Canada) with liquid in which crystal-like structures (refractive at PhC) float (https://youtu.be/Wsic3w_j0AY).

Macrobiotus occidentalis occidentalis
The specimens resemble Macrobiotus occidentalis occidentalis Murray, 1910 described from Victoria Harbour (Vancouver Island, British Columbia), including the very peculiar floating crystal-like structures between the cuticle and the epidermis.

Mesobiotus sp.
-3 individuals (Slide S2000_SL1 -C5074s1) -2 individuals (Slide S2011_SL1 -C5085s1) -5 individuals (Slide S2012_SL1 -C5086s1) -1 individual (Slide S2015_SL1 -RBCM 023-00 006-001) -1 individual (Slide S2016_SL2 -C5090s1) Mesobiotus with oral cavity armature with anterior row of teeth not visible or very faint, posterior row composed by two line of large teeth, and transversal ridges divided into a ventral line composed by two ventral bars and a median mucrone and a dorsal line composed by three large bar-like transversal teeth.Faint granulation on IV legs not exceeding between legs.Lunule IV finely dentate.Accessory points lifted from the primary branch of each claw.Eggs unknown.12 E. Massa et al.

Paramacrobiotus klymenki
The morphology of the individuals and eggs fits the original description.

Discussion
Despite its relatively sparse research history with few, limited, discontinuous sampling efforts, British Columbia has yielded an astonishing tardigrade species diversity, indicating that an incredible hidden biodiversity that only awaits to be unveiled.As shown in other tardigrade studies, an increase in sampling effort increases the number of species found (Nelson & Bartels 2013;Vuori et al. 2020;Massa et al. 2021), and it is similarly expected that additional efforts in characterizing the tardigrade fauna of Haida Gwaii will provide a large increase in the number of identified species.At the same time, due to climate change, anthropogenic effects, and resulting loss of habitats, species are becoming extinct even before we have the chance to find and describe them (Lees & Pimm 2015), and microscopic invertebrate like tardigrades are no exception to this trend (Vicente 2010;Zawierucha et al. 2018).Our results underscore the importance of continuing to sample and to document tardigrade diversity in this region, particularly in islands off the British Columbia coast such as Haida Gwaii and Vancouver Island.We provided the first account on tardigrades diversity from Haida Gwaii, showing tardigrade diversity and reporting of two Macrobiotus species (M.hufelandi and M. occidentalis occidentalis) which provides the occasion to briefly discuss biogeography and taxonomic issues.First tardigrades record from Haida Gwaii (Canada) Historically, Macrobiotus hufelandi was the first formally described tardigrade species (C.A.S. Schultze 1834) and was initially thought to be cosmopolitan.Subsequent studies involving fine morphology and DNA barcoding suggested instead the presence of multiple species with similar morphology (Bertolani & Rebecchi 1993;Cesari et al. 2009;Bertolani et al. 2011;Kaczmarek & Michalczyk 2017).Based on this, the cosmopolitan nature of M. hufelandi was questioned.In this study, we showed unequivocally (through DNA sequencing) that other than its type of locality in Germany, this species is present also in Canada.In addition, molecular data supporting the presence of this species in USA (Colorado; Vecchi et al. 2022) and Russia (Tumanov et al. 2022) expand the distribution of this species to most of the Boreal hemisphere and assigning this specie to the Holarctic bioregion.Future integrative studies focusing on M. hufelandi specimens in the Austral hemisphere are needed to clarify whether the species is truly cosmopolitan or not and will be an important contribution in the discussion on the cosmopolitan vs. localized distribution of tardigrade species (see for example Jørgensen et al. 2007;Gąsiorek et al. 2019;Kaczmarek et al. 2020;Morek et al. 2021;Kayastha et al. 2023).
Macrobiotus occidentalis occidentalis is an enigmatic species of which many worldwide records have been published (McInnes 1994).In its original description, based on different characters, Murray (1910) pointed out an affinity between M. o. occidentalis and many species now belonging to different genera (Richtersius coronifer (Richters, 1903), Adorybiotus granulatus (Richters, 1903), Crenubiotus crenulatus (Richters, 1904), Mesobiotus harmsworthi (Murray, 1907), and Diaforobiotus islandicus (Richters, 1904)) but was recognized to be most similar to D. islandicus.Despite this ambiguity in the morphological affiliation of M. o. occidentalis, its taxonomic position has never been challenged in extenso, and it is still nowadays included in the genus Macrobiotus (Guidetti & Bertolani, 2005;Degma & Guidetti. 2007, 2019-2023) despite the revolutions in tardigrades taxonomy that happened in the last decades after the introduction of the molecular phylogenesis in tardigrade taxonomy (e.g., Cesari et al. 2009;Guidetti et al. 2009;Bertolani et al. 2014).Numerous reports of M. o. occidentalis started accumulating quickly after its description from different localities, for example, Sweden (Thulin 1911) and France (Cuénot 1932).By the time of the publication of Marcus' monograph on tardigrades (Marcus 1936), M. o. occidentalis had also been recorded from Ireland, Scotland, Peru, and Brasil. When McInnes (1994) published a list of tardigrade records of terrestrial and freshwater habitats in various countries, M. o. occidentalis was reported from all continents except Antarctica.One subspecies was described from North Korea (Macrobiotus occidentalis striata Dastych, 1974).It should be noted that, in Murray (1910), the description of M. o. occidentalis from Australia (p 139, indicated as "Macrobiotus occidentalis, Murray?") and indicated as "Macrobiotus occidentalis(?)") comes before than the proper description of the species from Canadian material (p.169-175, indicated with "Macrobiotus occidentalis sp.n".).This may be the reason that led Dastych (1988) to erroneously indicate its locus typicus in Australia instead of in Victoria, Vancouver Island, Canada.Given the morphological characters reported for the individuals to Macrobiotus o. occidentalis found in our survey (including the 18S similarity with D. islandicus SM.01) and the speculations on its affinities with Diaforobiotus islandicus already presented by Murray (1910), we propose Diaforobiotus occidentalis (Murray, 1910) comb.nov.stat.nov.The presence of a very small thickening within the pharynx that we found to be a microplacoid, confirming it with both LM and CLSM (SM.02), is not in contrast with the original Murray (1910) description as due to the small size of this structure, that could have been easily overlooked at the time.A similar structure has also been reported for Paramacrobiotus areolatus (Stec et al., 2020c) and in Stec et al. (2020b) Figure 4(e) a similar structure can be glimpsed for Richtersius coronifer.
The genus Diaforobiotus was first described by Guidetti et al. (2016) as belonging to the family Richtersiidae, considered invalid name in Guidetti et al. (2021) that thus proposed the name Richtersiusidae.Since its proposal by Guidetti et al. (2016) based on molecular and morphological data, the family Richtersiusidae has undergone a first amendment by Lisi et al. ( 2020), followed by two redefinitions by Guidetti et al. (2021) and Stec et al. (2020d).Stec et al. (2020d), among the other features, added in the amended diagnoses the absence of the microplacoid as diagnostic, but the presence of a microplacoid within the pharynx in the D. occidentalis comb.nov.stat.nov.and its possible presence in R. coronifer imposes a further diagnostic amendment of the family Richtersiusidae.
According to Stec (2022), which showed hidden cryptic diversity within the genus Diaforobiotus, and the morphological characters more affine to Macrobiotus described by Dastych (1974) 1977), and among the remaining material conserved in the Royal Scottish Museum, there is no trace of D. occidentalis comb.nov.stat.nov.type specimens (Morgan 1977).As the type material of D. occidentalis comb.nov.stat.nov.can be considered lost, an integrative redescription of this species and a neotype assignation are needed.However, given the distance of the population found in this study to the type locality of D. occidentalis comb.nov.stat.nov.(700 km), we temporarily refrain from assigning a neotype and from providing an amended diagnosis of the species.However, an extensive descriptive account of D. occidentalis comb.nov.stat.nov. is given in the results.
Moss samples for tardigrade extraction in this study were collected at or about sea-level (Table I).Most of the moss samples were collected from "standard" terrestrial habitats (rocks, living trees, and tree stumps).Previous studies in British Columbia (e.g., Kathman & Nelson 1989) have focused on tardigrade diversity from elevations as high as 760 m.However, Hypsibius cf.convergens (S2002), Echiniscus cf.robertsi, Pseudechiniscus (Meridioniscus) dastychi, Diphascon cf.claxtonae (S2010), and Claxtonia maucci (S2011) (Table III) came from moss collected from beached trees in several localities in the intertidal zone (Figure 8).These beached trees, which are primarily western hemlock (Tsuga heterophylla (Raf.)Sarg.), are large and remain relatively stationary, having been in their approximate positions for at least 30 years (Chris Ashurst pers.comm).While partially submerged during high tide, the upper-most level of the beached trees remains exposed and have accumulated enough sediment to support a permanent assemblage of terrestrial vegetation, including mosses.The diversity and peculiarity of tardigrade taxa found in these "non-standard" habitats shows how keeping exploring new habitat types is important in obtaining a complete view and understanding of tardigrade diversity.Interestingly, there is no overlap between the taxa found at all beached tree locations.Past studies remain divided as to the extent to which ecological factors affect tardigrade distribution in the Americas (Meyer 2013), but the results indicate that further data are needed to determine whether a consistent pattern can be elucidated.
The exploration of Haida Gwaii conducted in this study also increases the knowledge on American tardigrades fauna adding seven taxa (Table I) to the 274 taxa already known for North America based on the last available checklist for this area (Kaczmarek et al. 2016) and nine taxa (Table I) to the 129 found in Canada (Kaczmarek et al. 2016;Kayastha et al. 2021;Vecchi et al. 2022b).

Family Richtersiusidae amended diagnosis
Double claws Y-shaped, with the two branches forming an evident common tract of a variable length with system of internal septa.Teeth present or absent on lunulae I-III and always present on lunulae IV.Buccal tube with ventral lamina  III).
First tardigrades record from Haida Gwaii (Canada) exhibiting ventral thickening in its anterior portion (sometimes hardly visible under light microscope) and a cuticular thickening on the anterior, dorsal wall of the buccal tube (which can form a large apophysis).Transverse crests in the buccal armature absent.Two macroplacoids in the pharynx.Microplacoid present or absent (in most taxa).Cuticular pores (at least in a phase of the life cycle).Eggs laid freely with conical (usually spiky) processes and without areolation on their surface.Body and leg granulation absent in all currently recognized species.
1. -Teeth present on lunulae of legs I-III (2) -Teeth absent on lunulae of legs I-III (3) 2. -The claw common tract longer than the half of the entire claw height, egg surface between processes with evenly distributed dark dots (seen in PCM) -Diaforobiotus islandicus (Richters, 1904) -The claw common track constitutes one-third of the entire claw length, egg surface between processes without dark dots (seen in PCM) -Diaforobiotus caelicola (Kathman, 1990) 3. -The claw common tract shorter than the half of the entire claw length, the dorsal portion of the third band of teeth in the oral cavity armature (OCA) -where the transversal ridge is generally present -comprises only one tooth -Diaforobiotus hyperonyx (Maucci, 1982) -The claw common tract longer than the half of the entire claw height, dorsal portion of the third band of teeth in the OCA -where the transversal ridge is generally present -comprises three teeth (4) 4. -Microplacoid absent, chorion surface with evenly distributed, minute, faintly visible light refracting dots in the egg surface between processes (visible in PCM), egg process base/height ratio between 56% and 83% -Diaforobiotus svalbardicus Stec, 2022 -Microplacoid present (even if small and not always clearly visible), chorion surface with irregularly distributed dark dots in the egg surface between processes (visible in PCM), egg process base/height ratio between 23% and 50% -Diaforobiotus occidentalis (Murray, 1910) cf. robertsi -4 individuals (Slide S2010_SL1 -C5084s1) -2 individuals sequenced (Voucher slides S2010.Ech.V03-6 -C5084v1-2) By having only appendages in positions A and D lateral (Figure 2(a)) and a polygonal sculpture of the dorsal plate (Figure 2(b)), the individuals resemble Echiniscus robertsi Schuster & Grigarick, 1965 (a rare species recorded only twice by Schuster & Grigarick 1965 and Biserov 1998).Schuster and Grigarick (1965) described the presence of spines in position D lateral, while in the animals analyzed in this study, longer filaments were present in D lateral.

Figure 1 .
Figure 1.Map of Haida Gwaii (British Columbia, Canada) showing the sampling localities.
(b-c)).Pseudolunulae under each claw present (Figure 3(b)), larger under the external (or posterior in the hind legs) claws.Two cuticular bars present on each leg I-III: one intern to the internal claws and the other extern to the external claws.The cuticular bars are well visible only in one of the two specimens.Cuticular bar present claws IV.
Diphascon cf.claxtonae -1 individual (Slide S2010_SL1 -C5084s1)The species identification follows the key byFontoura and Pilato (2007) for the species of the D. pingue group, to which Diphascon claxtonae Pilato & Binda, 1998 belongs, and it is characterized by having: smooth cuticle; pharyngeal bulb with apophyses, three macroplacoids, microplacoid, and septulum; lunules and other cuticular thickenings on the legs absent; claw bases without indentations(Fontoura et al. 2010).However, being the species of the D. pingue group differing only for few morphometric characters and our sample composed by one individual only, we prefer to be prudent and name this taxon D. cf.claxtonae.Hypsibius cf.convergens -3 individuals (Slide S2002_SL1 -C5076s1)
https://youtu.be/Wsic3w_j0AY).Oral cavity armature with an anterior band of teeth at the base of the buccal lamellae absent or not visible with LM; a posterior band of teeth covering the internal side of the buccal ring composed of irregular rows of granular teeth, increasing in size from the front to the back (well visible with LM; Figure6(a-b)); a band of teeth present at beginning of the buccal tube is divided into the dorsal and the ventral portion.The ventral portion is composed of small faint teeth (in LM faintly visible as granular; Figure6(a)).The dorsal portion is composed of three evident granular teeth (Figure6(b)).An additional tooth caudal and median to the dorsal portion (at the level of the buccal crown) in shape of a mucrone is also present (Figure6(b)).Two macroplacoids without evident constrictions are present (Figure 6(c)), and a rugose-margined microplacoid is present, although not well visible in all the specimens (Figure 6(c), SM.02).Claws of the Richtersiusidae type (Figure 6(d)), as described by Lisi et al. (2020).Lunulae under claws I-III smooth (Figure 6(d)), whereas lunulae under claws IV weakly dentate (Figure 6(e)).The eggs are light orange, spherical with slender conical processes, and a dotted surface (Figure 6(f)).Measurements of animals and eggs are provided in SM.03.

Figure 8 .
Figure 8. Picture of beached tree (western hemlock -Tsuga heterophylla) in the intertidal zone, showing growth of moss and other terrestrial plants, on which sample of mosses used for this study were collected (see sample code in TableIII).

Table I .
List of moss samples used in this study with relative codes, localities, and coordinates expressed in decimal degrees (WGS84), and substrate on which moss samples were collected.Beached trees are western hemlock (Tsuga heterophylla).The other tree species mentioned are western redcedar (Thuja plicata) and Sitka spruce (Picea sitchensis).