Setaphyes algarvensis sp. nov., the first description of an allomalorhagid mud dragon (Kinorhyncha: Allomalorhagida) from Portugal (Eastern Atlantic Ocean)

. The kinorhynch fauna from Portugal has been explored, yielding a new species of the genus Setaphyes (Kinorhyncha: Allomalorhagida). This is the first description of an allomalorhagid species from Portugal. Specimens of the new species were collected at a subtidal muddy beach in Alvor, a village located in the southernmost region of Portugal. Setaphyes algarvensis sp. nov. may be distinguished from its congeners by a unique arrangement of the setae: paired paradorsal setae on segments 2–7 and 9, paradorsal seta on segment 8 unpaired, laterodorsal setae on segments 2–3 and 6–9 in males and 2–9 in females, paralateral setae on segment 1, lateroventral setae on segments 2–10 (two pairs on segment 5), ventrolateral setae on segment 1 in males and 1–3 in females, and ventromedial setae on segments 3–9 in males and 4–9 in females. The diagnostic features of Setaphyes algarvensis sp. nov. are discussed from a comparative perspective with the congener species. Additionally, morphometric analyses of selected features, namely the total trunk length and the relation between the total trunk length and the length of lateral terminal spines, turned out to be useful to distinguish between the new species and Setaphyes kielensis (its most similar congener).


Introduction
The kinorhynch fauna of the Iberian Peninsula has been extensively sampled since the late 1990s.The first ever report of a mud dragon species in this area occurred in 1998, when two new species of the genus Echinoderes Claparède, 1863 were described from the Cantabrian coast (Pardos et al. 1998): Echinoderes cantabricus Pardos et al., 1998 andE. hispanicus Pardos et al., 1998.Ten years later, three new species of the same genus were also discovered from this northern coastline (GªOrdóñez et al. 2008): E. isabelae GªOrdóñez et al., 2008;E. neospinosus GªOrdóñez et al., 2008 andE. parrai GªOrdóñez et al., 2008.The first Iberian allomalorhagid kinorhynch, Paracentrophyes quadridentatus (Zelinka, 1928), was also found a few years later in the Cantabrian Sea (Sørensen et al. 2010).The first descriptions of new allomalorhagid species were made by Sánchez et al. (2011), namely Pycnophyes aulacodes Sánchez et al., 2011 andHigginsium dolichurum (Sánchez et al., 2011), both species collected from the northwestern coast of Spain.Subsequently, E. gerardi Claperède, 1863 and a new cyclorhagid genus and species, Meristoderes macracanthus Herranz et al., 2012, were found along the Mediterranean coasts of Spain (Sánchez-Tocino et al. 2011;Herranz et al. 2012;Sørensen et al. 2020).Moreover, the first Iberian dracoderid kinorhynch, Dracoderes gallaicus Sørensen et al., 2012, was described from the north-and southwestern coasts of Spain (Sørensen et al. 2012).However, it was not until 2012 that the first comprehensive campaign to assess the diversity of Kinorhyncha Reinhard, 1885 in the Iberian Peninsula was accomplished (Sánchez et al. 2012).The inspection of 81 sampling stations yielded approximately 2000 specimens, whose examination by light and scanning electron microscopy eventually led to the identification of 11 genera and 29 kinorhynch species.Interestingly, only 11 of the 29 species (i.e., less than 40%) had previously been recorded from Iberian waters.Since then, five additional species of kinorhynchs have been described, namely Pycnophyes almansae Sánchez et al., 2014, Setaphyes cimarensis Sánchez et al., 2018, Leiocanthus lageria (Sánchez et al., 2014), Echinoderes lusitanicus Neves et al., 2016and E. reicherti Neves et al., 2016(Sánchez et al. 2014, 2018;Neves et al. 2016).The latter two species actually are the only kinorhynch species described from the Portuguese coast (Neves et al. 2016).As compared to the Spanish coastline, the knowledge on the Portuguese kinorhynch fauna is as yet extremely limited.
The aim of the present paper is thus to increase the knowledge on the Portuguese kinorhynch fauna and, hence, to provide more comprehensive data on the biogeography of Kinorhyncha in the Iberian Peninsula.In the present study, we describe a new intertidal species of Setaphyes Sánchez et al., 2016 from Portuguese waters, one the most common and abundant genera of Pycnophyidae Zelinka, 1896 in Europe.Five out of the seven species belonging to this genus were described from or reported in European waters; namely S. cimarensis Sánchez et al., 2018, S. dentatus (Reinhard, 1881), S. elenae Cepeda et al., 2020, S. flaveolatus (Zelinka, 1908) and S. kielensis (Zelinka, 1928) (see Reinhard 1881; Zelinka 1908Zelinka , 1928;;Neuhaus 2013;Dal Zotto & Todaro 2016;Sánchez et al. 2018;Cepeda et al. 2020).This is the first description of an allomalorhagid kinorhynch for the southwestern region of the Iberian Peninsula.

Sampling and study of specimens
Kinorhynch specimens were collected in December 2012 from a muddy beach at Alvor (37°07.714′N; 08°36.329′W), a small fishing village located in the southernmost region of Portugal (Fig. 1).Samples GONZÁLEZ-CASARRUBIOS A. et al., New kinorhynch from the Iberian Peninsula were taken by hand from an intertidal muddy area containing Zostera sp.Animals were extracted from the sediment following the bubble-and-blot method (Higgins & Thiel 1988;Sørensen & Pardos 2020), and subsequently preserved in 70% ethanol.
Kinorhynchs were sorted under a stereo microscope (ZEISS Stemi SV 6).For light microscopy (LM), specimens were dehydrated through a graded series of ethanol, transferred to 100% glycerine for 24 h, and then mounted on glass slides with Fluoromount G ® .Mounted specimens were studied and photographed with an Olympus © BX51-P microscope with differential interference contrast (DIC) optics equipped with an Olympus © DP-70 camera.
For scanning electron microscopy (SEM), specimens were dehydrated through a graded series of ethanol and then transferred to acetone through a graded ethanol/acetone series, critical point dried, mounted on aluminum stubs and sputter-coated with a gold/palladium mixture for examination with a JEOL ® JSM-6335F field emission scanning electron microscope at the ICTS Centro Nacional de Microscopía Electrónica (Universidad Complutense de Madrid, Spain).Line drawings and image plate compositions were made using Illustrator CC-2014 and Adobe ® Photoshop software, respectively.The type material of the new species is deposited at the Natural History Museum of Denmark (NHMD).

Morphometric statistics
Differences in selected morphometric measures (i.e., total trunk length and relative proportion between total trunk length and length of lateral terminal spines) of the new species and the most similar species of Setaphyes were tested.For this, we selected 30 specimens of S. kielensis (15 ♂♂, 15 ♀♀: ZMB-12294 to 12313, ZMB-12359 to 12361, ZMB-12363, ZMB-12367 and ZMB-12394 to 12398) collected at Sylt (Germany), and stored at the Museum für Naturkunde Berlin (Germany), as well as 19 specimens (18 ♂♂ and 1 ♀) of the new species described here.Saphiro-Wilk and Barlett tests were used to check the normality and the homoscedasticity of the variables, respectively.In addition, visual methods of density and quantile-quantile (Q-Q) plots were applied for the same purpose.To assess differences, a one-way analysis of variance (ANOVA) was used, except when the variable was heteroscedastic, in which case a Welch's ANOVA was applied.These statistical analyses were performed in R ver.1.1.453(R Core Team 2021) using the stats, ggplot2 and car packages (Fox et al. 2019).

Etymology
The species name, algarvensis, refers to the Algarve, the southern region of Portugal where the new species was found.
Additional material PORTUGAL • 8 adult ♂♂, five of them mounted as the holotype and three mounted for SEM; same collection data as for holotype; UCM Meiofauna Collection.

Description
See Table 1 for measurements and dimensions and Table 2 for a summary of the middorsal cuticular specialization, seta, tube, nephridiopore and sensory spot locations.
Head.With retractable mouth cone and introvert.Although two of the examined specimens had the head everted, their structures tend to collapse when mounted for LM; hence, only some details on the GONZÁLEZ-CASARRUBIOS A. et al., New kinorhynch from the Iberian Peninsula morphology of oral styles and scalids can be provided.Internal part of mouth cone with several rings of inner oral styles; exact number, arrangement and morphology not determined.External part of mouth cone with single ring of nine equally-sized outer oral styles, arranged as one anterior to each introvert sector, except for middorsal sector 6 where style is missing.Each outer oral style composed of single, flexible unit, wider at base, bearing fringed sheath, progressively tapering toward distally pointed tip.Introvert with six transverse rings of scalids and 10 longitudinal sectors defined by arrangement of primary spinoscalids.Primary spinoscalids larger than remaining scalids, each one composed of basal, rectangular, wide sheath and distal, elongated, distally pointed end-piece.Scalids from remaining rings regular-sized, similar in morphology to primary spinoscalids but smaller, also composed of a longer distal end-piece and a shorter basal sheath.Exact number, arrangement and detailed morphology of scalids not determined.
Statistical analysis (Fig. 5, Table 1) The comparison of the total trunk length (TL) between Setaphyes algarvensis sp.nov.and S. kielensis revealed statistically significant differences (p < 0.01; Fig. 5A).In contrast, significant differences were not found in the relationship between the total trunk length and the length of lateral terminal spines (LTS/TL) of both species (p = 0.86; Fig. 5B).However, due to the broad range observed in the LTS/ TL of S. kielensis (Fig. 5B), the ratio LTS/TL was compared between males and females in order to find sexually dimorphic differences.The statistical analysis revealed highly significant gender-based European Journal of Taxonomy 821: 150-165 (2022) differences within this species (p < 0.01; Fig. 5C).The LTS/TL seems to present sexual dimorphism in S. algarvensis sp.nov.as well (Fig. 5B, the arrow points out the single female value), but this fact could not be statistically verified due to the fact that only a single female of this species was collected.Therefore, the LTS/TL ratio was only tested between males of both species, which revealed significant differences (p < 0.01; Fig. 5D).

Taxonomical remarks
The new species described here clearly belongs to Setaphyes according to the diagnostic features of the genus provided in Sánchez et al. (2016).Setaphyes algarvensis sp.nov.possesses a combination of morphological features present in Setaphyes only, e.g., the absence of ventrolateral setae on segment 5, the presence of paradorsal setae on segments 2-9 and lateroventral setae on segments 2-10, as well as the presence of scattered, dot-shaped muscular scars (likely outlets of glandular cells) on both tergal and sternal plates.
The new species can be unequivocally distinguished from its congeners by the unique arrangement of dorsal setae and the absence of cuticular ornamentations.(Zelinka 1908;Sánchez et al. 2016Sánchez et al. , 2018;;Cepeda et al. 2020); Setaphyes australensis, S. iniorhaptus and S. kielensis lack cuticular ornamentations, but they can easily be distinguished from the new species by the number of paired lateroventral setae on segment 5, as also occurs for the remaining congeners (two pairs in S. algarvensis sp.nov.vs one pair in the remaining species of Setaphyes) (Higgins 1983;Lemburg 2002;Sánchez et al. 2016Sánchez et al. , 2018)).
A species that can potentially be problematic to compare with its congeners is S. iniorhaptus.Indeed, the presence of a dorsal series of setae in this species is difficult to verify due to the badly preserved condition of the type material, which prevents accurate observation of these morphological features (Sánchez et al. 2016).Nevertheless, S. iniorhaptus is easily distinguished from the new species by the presence of a single pair of lateroventral setae on segment 5, and furthermore by the absence of setae on the episternal plates of segment 1 (present ventromedially in S. algarvensis sp.nov.).
Regarding the setal arrangement, S. algarvensis sp.nov. is most similar to S. kielensis, as both species share the same paradorsal setal arrangement (Zelinka 1928;Sánchez et al. 2016), but again the presence of two pairs of lateroventral setae on segment 5 and a single pair of paralateral and ventromedial setae on segment 1 in the new species allows its differentiation from S. kielensis.

Morphometrical remarks
The morphometry of selected morphological measures in Setaphyes has turned out to be useful to distinguish between congeners (Sánchez et al. 2018;Cepeda et al. 2020).Specifically, in the present study, these analyses help to find more differences between S. algarviensis sp.nov.and S. kielensis, the most similar congener in terms of setal arrangement.The total trunk length could be used to differentiate GONZÁLEZ-CASARRUBIOS A. et al., New kinorhynch from the Iberian Peninsula both species, as S. algarviensis sp.nov. is substantially longer than S. kielensis.The ratio between the total trunk length and the length of the lateral terminal spines is also useful when comparing males, being significantly higher in S. kielensis.We expect the LTS/TL ratio to be different in females as well; however, this cannot be confirmed with the investigation of only one female and further analyses with more female specimens are needed.

Biological remarks
Knowledge about kinorhynch sex ratios is largely scarce for all ocean bathymetries.Only a few studies limited to the littoral zone have addressed this topic, showing a 1:1 ratio of females to males in Cristaphyes carinatus (Zelinka, 1928), Echinoderes coulli Higgins, 1977, and Pycnophyes communis Zelinka, 1908(Zelinka 1908, 1928;Higgins 1977).More recently, Hoffman et al. (2021) found similar proportions for several shallow water species of Echinoderes and Leiocanthus along the Alabama coast (Hoffman et al. 2021).By contrast, the ratio was biased toward females in the shallow water species Echinoderes dujardinii Claparède, 1863 (female:male 3:1) (Zelinka 1928;Higgins & Fleeger 1980), and in six deep-sea echinoderid species found in the Clarion-Clipperton Zone (Sánchez et al. 2019, in press).This bias may be explained as a reproductive strategy to enable minimization of energetic investment, as hypothesized for other meiofaunal groups (Hicks & Coull 1983;Shimanaga et al. 2009).
Our results show, for the first time, the opposite pattern, with a much higher proportion of males (female:male 1:20).Although most ecological and biological aspects for kinorhynchs are still poorly known, including sex ratio, mating system, and factors driving their spatial-temporal variations (Neuhaus 2013), the observed bias may be related to sex differential mortality after mating or disparities in the ability to move, triggered by external factors, e.g., females burrow more actively into sediment than males under dehydration/insulation conditions at low tide.However, the total number of collected specimens is too low to reach a clear conclusion, and more sampling along the studied area, throughout several years, and controlling time exposition to low tide would be needed in order to know whether our observations are merely fortuitous or define a consistent pattern for this species.

Fig. 1 .
Fig. 1. A. Map showing the sampling area of Alvor (Faro, Portugal).B. Close-up of the sampling area.Green point marks the sampling site.

Fig. 5 .
Fig. 5. Boxplots represent the ranges of different body measurements of Setaphyes algarvensis sp.nov., and S. kielensis(Zelinka, 1928).A. Total trunk length.B. Relationship between total trunk length and lateral terminal spine length; arrow points out the female of S. algarvensis sp.nov.C. Relationship between total trunk length and lateral terminal spine length among males and females of S. kielensis.D. Relationship between total trunk length and lateral terminal spine length among the males of both species.

Table 2 .
Summary of nature and arrangement of cuticular elevations, processes, spines, tubes, setae, sensory spots and nephridiopores in Setaphyes algarvensis sp.nov.
(Sánchez et al. 2018)cteristic cuticular ornamentation on segments 1 or 10 in S. algarvensis sp.nov. is remarkable, as most of the remaining congeners have a certain kind of ornamentation: S. dentatus and S. cimarensis possess longitudinal, parallel, folded cuticular thickenings in laterodorsal and ventrolateral positions on segment 10 (Sánchez et al. 2018); S. cimarensis additionally has a net-like structure forming rounded, isolated depressions near the anterior margin of the tergal plate of segment 1, whereas S. dentatus has a continuous, reticular ornamentation covering the entire anterior surface of the tergal plate of segment 1(Sánchez et al. 2018); both S. elenae and S. flaveolatus have the ornamentation present solely in the middle anterior region of the tergal plate segment 1