Zoosyst. Evol. 96 (1) 2020, 217-236 | DOI 10.3897/zse.96.50821 Gp husrum ror BERLIN Small is beautiful: the first phylogenetic analysis of Bryodelphax Thulin, 1928 (Heterotardigrada, Echiniscidae) Piotr Gasiorek!, Katarzyna Vonéina!, Peter Degma*, Lukasz Michalezyk! 1 Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387, Krakéw, Poland 2 Department of Zoology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska dolina, Ilkovicova 6/B1, 84215, Bratislava, Slovakia http://zoobank.org/0B80F F 1 B-5ED7-430B-A 471-ASDE02E8E6D3 Corresponding author: Piotr Gasiorek (piotr.lukas.gasiorek@gmail.com) Academic editor: Martin Husemann @ Received 10 February 2020 Accepted 17 April 2020 Published 27 May 2020 Abstract The phyletic relationships both between and within many of tardigrade genera have been barely studied and they remain obscure. Amongst them is the cosmopolitan Bryodelphax, one of the smallest in terms of body size echiniscid genera. The analysis of new- ly-found populations and species from the Mediterranean region and from South-East Asia gave us an opportunity to present the first phylogeny of this genus, which showed that phenotypic traits used in classical Bryodelphax taxonomy do not correlate with their phyletic relationships. In contrast, geographic distribution of the analysed species suggests their limited dispersal abilities and seems to be a reliable predictor of phylogenetic affinities within the genus. Moreover, we describe three new species of the genus. Bryodelphax australasiaticus sp. nov., by having the ventral plate configuration VII:4-4-2-4-2-2-1, is a new member of the weg/arskae group with a wide geographic range extending from the Malay Peninsula through the Malay Archipelago to Australia. Bryodelphax decoratus sp. nov. from Central Sulawesi (Celebes) also belongs to the weg/arskae group (poorly visible ventral plates VII:4-2-2-4-2-2-1) and is closely related to the recently described Bryodelphax arenosus Gasiorek, 2018, but is differentiated from the latter by well-developed epicuticular granules on the dorsum. Finally, a new dioecious species, Bryodelphax nigripunctatus sp. nov., is described from Mallorca and, by the reduced ventral armature (II/III:2-2-(1)), it resembles Bryodelphax maculatus Gasiorek et al., 2017. The latter species, known so far only from northern Africa, is recorded from Europe for the first time. A taxonomic key to the genus members is also presented. Key Words cradle hypothesis, Everything is Everywhere hypothesis, geographic distribution, miniaturisation, phylogeny, ventral plates Introduction Tardigrades are regarded as miniaturised panarthropods (Gross et al. 2019). The average body size of a limno- terrestrial tardigrade varies between 200 and 500 um, with some notable exceptions, such as milnesiids (Morek et al. 2016) or richtersiids (Guidetti et al. 2016), reaching body lengths up to 1200 um (Nelson et al. 2015). Marine heterotardigrades are the smallest representatives of the phylum, being usually below 200 um in body length (Jorgensen et al. 2014; Fontoura et al. 2017), but speciose heterotardigrade echiniscids fit to the aforementioned range for limno-terrestrial water bears (200-500 um), with single exceptions, such as Acanthechiniscus islandicus (Richters, 1904) (Maucci 1996) and some members of the genus Cornechiniscus (Maucci 1979; Kristensen 1987), which can grow up to 800 um. The opposite trend, Le. a potential reduction of the already small body size in the course of evolution, can be seen in five indirectly related genera: Antechiniscus, Bryodelphax, Parechiniscus, Pseudechiniscus and Stellariscus (Kristensen 1987; Claxton 2001; Gasiorek 2018; Gasiorek et al. 2018a). Of these genera exhibiting small body size, all but Bryodelphax and Parechiniscus share black crystalline eyes and sexual reproduction (Kristensen 1987). Furthermore, Bryodelphax is unique amongst Echiniscidae as it exhibits some peculiar apomorphies (e.g. ten peribuccal papulae) and plesiomorphies (e.g. ancestral type of the buccal Copyright Piotr Gasiorek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 218 apparatus). These characteristics make Bryodelphax a good example of mosaic evolution in tardigrades (Kristensen et al. 2010; Gasiorek 2018). The aim of this study was to elucidate the phyloge- ny of Bryodelphax in relation to morphological traits used in its taxonomy, with application of the integrative approach, 1.e. DNA barcoding and both qualitative and quantitative morphology, based on three new species that are described and illustrated herein. Our analyses reveal no congruence between the topology of the phylogenetic tree and the traditional taxonomic divisions of the genus (based on the presence of ventral armature), the repro- ductive mode or the development of dark, contrasting epicuticular granules on the dorsal plates. On the other hand, we show that phylogeny is tightly correlated with geography. In addition, an amended and updated key to the genus Bryodelphax is provided. Materials and methods Sample collection and processing, comparative material Specimens of the genus Bryodelphax were extracted from various moss and lichen samples collected in numerous European and Asian locales (details in Table 1). The an- imals were divided into three groups used in different analyses: (I) qualitative and quantitative morphology investigated in phase contrast microscopy (PCM) and Nomarski differential interference contrast microscopy Gasiorek, P. et al.: Phylogeny of Bryodelphax (NCM), collectively termed as light contrast microscopy (LCM); (II) qualitative morphology in scanning electron microscopy (SEM); and (III) DNA sequencing (details in Table 2). For morphological comparisons, the type se- ries of B. aaseae Kristensen et al., 2010, B. amphoterus (Durante Pasa & Maucci, 1975), B. asiaticus Kaczmarek & Michalczyk, 2004, B. brevidentatus Kaczmarek et al., 2005, B. iohannis Bertolani et al., 1996, B. meronensis Pilato et al., 2010, B. parvuspolaris Kaczmarek et al., 2012, B. sinensis (Pilato, 1974) and B. weglarskae (P1- lato, 1972) deposited in the Natural History of Denmark, University of Modena and Reggio Emilia, Museum of Natural History of Verona, Jagiellonian University and University of Catania, were studied. Microscopy, imaging and morphometrics Permanent microscope slides were made using Hoyer’s medium and examined under a Nikon Eclipse 501 PCM associated with a Nikon Digital Sight DS-L2 digital camera and Olympus BX51 PCM and DIC associated with a digital camera CCD ColorView III FW. Speci- mens for imaging in the SEM were prepared according to Stec et al. (2015) and examined in Versa 3D Dual- Beam SEM at the ATOMIN facility of the Jagiellonian University. All figures were assembled in Corel Pho- to-Paint X6, ver. 16.4.1.1281 or in Adobe Photoshop CS3 Extended, ver. 10.0. All measurements are given in micrometres (um) and were performed under PCM. Structures were measured only when not broken, de- Table 1. Collection data for the newly-sequenced species used in morphological and phylogenetic analyses. Species Sample | Coordinates, Locality Environment Sample type, Collector code altitude substrate Bryodelphax MY.240 | 5°27'05'"N, Malaysia, Pulau Pinang, Pantai beach dominated by | moss, tree branch Piotr Gasiorek & auStralasiaticus Sp. nov. 100°11'00"E, Keracut Casuarina equisetifolia Artur Oczkowski 4m asl MY.241 | 5°27'05'"N, Malaysia, Pulau Pinang, Pantai beach dominated by moss, tree branch Piotr Gasiorek & 100°11'00"E, Keracut Casuarina equisetifolia Artur Oczkowski 4m asl MY.242 | 5°27'13'N, Malaysia, Pulau Pinang, Panta lowland rainforest moss, rock Piotr Gasiorek & 100°11'08'E, Keracut Artur Oczkowski 53 m asl Bryodelphax decoratus ID.546 1°50:33'S, Indonesia, Central Sulawesi, Lore | cacao tree plantation moss, tree Piotr Gasiorek & Sp. nov. 120°16'34"E, Lindu, Bada Lembah Artur Oczkowski 800 m asl ID.548 1°50'33'S, Indonesia, Central Sulawesi, Lore | cacao tree plantation | moss+lichen, tree Piotr Gasiorek & 120°16'34'E, Lindu, Bada Lembah Artur Oczkowski 801 mas! Bryodelphax maculatus* | GR.O50 | 35°23'23'N, Greece, Crete ,Vilatos olive tree plantation moss, olive tree Peter Degma (=780) | 23°39'36'E, 374 m asl Bryodelphax ES.264 |} 39°57'00'N, Spain, The Balearic Islands, sea shore moss, rock Peter Degma nigripunctatus sp. nov.**| (=716) | 3°10'50"E, Mallorca, Cap de Formentor, Cala 160 m asl Figuera beach, near the road above Bryodelphax parvulus IT.O10 | 45°42'12'N, Italy, Trieste, Grignano Miramare urban park moss, wall Alicja Witwicka 3A OSE, 1 mas Bryodelphax sp. nov. ID.464 1°52'48'S Indonesia, Central Sulawesi, Lore | cacao tree plantation moss, tree Piotr Gasiorek & 120°15'48'E, Lindu, Bada Lembah Artur Oczkowski 778 m asl Bryodelphax sp. nov. ID.846 | 3°10'52'S, Indonesia, Ambon, pass between mountain rainforest lichen, palm tree Piotr Gasiorek & 129°02'58'E, Triana and Jerili/Sawai, Seram tukasz Krzywanski 295 m asl Tengah * First record for Greece, ** First record of a limno-terrestrial tardigrade for Balearic Islands (see Guil 2002). zse.pensoft.net Zoosyst. Evol. 96 (1) 2020, 217-236 Table 2. Processing data for populations of Bryodelphax inves- tigated in this study. Types of analyses: LCM — imaging and morphometry in PCM/NCM, SEM -— imaging in SEM, DNA — genotyping. Numbers indicate how many specimens were uti- lised in a given analysis. Species Sample code Analyses LCM SEM DNA Bryodelphax australasiaticus sp. nov. MY.240 14 10 4 MY.241 1 - - MY.242 2 - - Bryodelphax decoratus sp. nov. ID.546 9 - 5 ID.548 3 - - Bryodelphax maculatus GR.O50 9 - 6 Bryodelphax nigripunctatus sp. nov. ES.264 55 30 8 Bryodelphax parvulus IT.010 8 - 4 Bryodelphax sp. nov. ID.464 8 - 4 Bryodelphax sp. nov. ID.846 13 - 4 formed or twisted and their orientations were suitable. Body length was measured from the anterior to the posterior end of the body, excluding the hind legs. The sp ratio is the ratio of the length of a given structure to the length of the scapular plate expressed as a per- centage (Dastych 1999). The bs ratio is the proportion between the maximal body width and the body length in dorsoventrally orientated specimens (Gasiorek et al. 2018a). Morphometric data were handled using the Echiniscoidea ver. 1.2 template available from the Tar- digrada Register, www.tardigrada.net (Michalczyk and Kaczmarek 2013). Ventral plate configuration is given according to Kaczmarek et al. (2012). Genotyping and phylogenetics DNA was extracted from individual animals (all exam- ined under a 400x magnification PCM prior to DNA extraction) following a Chelex 100 resin (Bio-Rad) ex- traction method (Casquet et al. 2012; Stec et al. 2015). Three DNA fragments were sequenced: the small ri- bosome subunit 18S rRNA (primers 18S_Tar_Ffl and 18S_Tar_Rr2 from Stec et al. 2017 and Gasiorek et al. 2017b, PCR programme from Zeller 2010), the large ri- bosome subunit 28S rRNA (primers 28S_Eutar_F and 28SRO0990 from Gasiorek et al. 2018b and Mironov et al. 2012 and the PCR programme from Mironov et al. 2012) and the internal transcribed spacer ITS-1 (prim- ers ITS] Echi_F and ITS1_ Echi_R from Gasiorek et al. 2019a, PCR programme from Welnicz et al. 2011). Some of the less conservative markers, such as ITS-2 and COI, are often difficult to amplify for Brvodelphax. All fragments were amplified and sequenced according to the protocols described in Stec et al. (2015). Sequenc- es of 18S rRNA and 28S rRNA were aligned using the default settings of MAFFT version 7 (Katoh et al. 2002; Katoh and Toh 2008), with Echiniscus lineatus Pilato et al., 2008 and Echiniscus testudo (Doyere, 1840) used as the outgroup. The obtained alignments were edited and checked manually in BioEdit v7.2.6.1 (Hall 1999) and then trimmed to 967 bp (18S rRNA) and 754 bp (28S 219 rRNA), respectively. The aligned sequences were con- catenated using SequenceMatrix (Vaidya et al. 2011). PartitionFinder version 2.1.1 (Lanfear et al. 2016) with applied Bayesian Information Criterion (BIC) and greedy algorithm (Lanfear et al. 2012) were used to test for the best scheme of partitioning and substitution models for posterior phylogenetic analysis. The analysis was performed solely for MrBayes purposes. The pre- ferred evolution model was GTR+G for both markers (Nei and Kumar 2000), which was finally chosen for further analyses. ModelFinder (Kalyaanamoorthy et al. 2017) under the Akaike Information Criterion (AIC) and corrected AIC (AICc) was used to find the best substitution models for two predefined partitions (Chernomor et al. 2016). The programme indicated the following models: TVMet+G4 (18S rRNA) and TIM3e+G4 (28S rRNA). Maxi- mum-likelihood (ML) topologies were constructed using IQ-TREE (Nguyen et al. 2015; Trifinopoulos et al. 2016). Strength of support for internal nodes of ML construc- tion was measured using 1000 ultrafast bootstrap repli- cates (Hoang et al. 2018). Bootstrap (BS) support values > 85% on the final tree were regarded as well support- ed and those > 70% as moderately supported. Bayesian inference (BI) marginal posterior probabilities were cal- culated using MrBayes v3.2 (Ronquist and Huelsenbeck 2003). Random starting trees were used and the analysis was run for ten million generations, sampling the Markov chain every 1000 generations. An average standard devi- ation of split frequencies of < 0.01 was used as a guide to ensure the two independent analyses had converged. The programme Tracer v1.7 (Rambaut et al. 2018) was then used to ensure Markov chains had reached station- arity and to determine the correct ‘burn-in’ for the analy- sis which was the first 10% of generations. The Effective Sample Size values were greater than 200 and consensus tree was obtained after summarising the resulting topolo- gies and discarding the burn-in. In the BI consensus tree, clades recovered with posterior probability (PP) between 0.95 and | were considered well supported, those with PP between 0.90 and 0.94 were considered moderately sup- ported and those with lower PP were considered unsup- ported. All final consensus trees were viewed and visual- ised by FigTree v.1.4.3 (http://tree.bio.ed.ac.uk/software/ figtree). MEGA7.0.26 (Kumar et al. 2016) was used for calculation of uncorrected pairwise distances (Srivathsan and Meier 2012). Data deposition Raw morphometric data underlying the description of the new species are deposited in the Tardigrada Regis- ter under www.tardigrada.net/register/0064.htm (B. aus- tralasiaticus sp. nov.), Www.tardigrada.net/register/0065. htm (B. decoratus sp. nov.), www.tardigrada.net/regis- ter/0066.htm (B. nigripunctatus sp. nov.). Type DNA se- quences are deposited in GenBank. zse.pensoft.net 220 Results Molecular phylogeny Bayesian Inference and Maximum Likelihood trees shared identical topology (Fig. 1). Two lineages, each represented by four species, were recovered: the Orien- tal clade (B. arenosus Gasiorek, 2018, B. australasiaticus sp. nov., B. decoratus sp. nov. and Bryodelphax sp. nov.) and the Western Palaearctic clade (B. instabilis Gasiorek & Degma, 2018, B. maculatus Gasiorek et al., 2017, B. nigripunctatus sp. nov. and B. parvulus Thulin, 1928). Descriptions of new species Systematic account Phylum: Tardigrada Doyeére, 1840 Class: Heterotardigrada Marcus, 1927 Order: Echiniscoidea Richters, 1926 Family: Echiniscidae Thulin, 1928 Genus: Bryodelphax Thulin, 1928 Bryodelphax australasiaticus Gasiorek, Von¢ina, Degma & Michalczyk, sp. nov. http://zoobank.org/BE521B67-6769-4EF5-B3C9-56EB43BF2DFE Figures 2-4, 12, Table 3 B. australis sp. n. in Claxton (2004) Locus typicus and type’ material. 5°27'05"N, 100°11'00"E, 4 m a.s.l.; Pantai Keracut, Pulau Penang, Malaysia. Holotype (adult female; slide MY.240.01) and seven paratypes (5 females, 2 juveniles; slides MY.240.01—04) deposited in the Institute of Zoology and Biomedical Research, Jagiellonian University; two para- types (2 females; slide MY.240.05) deposited in the De- partment of Zoology, Comenius University in Bratislava; one paratype (1 female; slide MY.240.06) deposited in the Natural History Museum of Denmark, University of Copenhagen; two paratypes (2 females; slide MY.240.07) deposited in the Department of Animal Biology, Univer- sity of Catania; three paratypes (2 females, one larva; slides MY.241.02, MY.242.02) deposited in the Raffles Museum of Biodiversity Research, National University of Singapore. Etymology. The name refers to the currently identified geographic range of the new species that encompasses Asia and Australia. Adjective in the nominative singular. Adults. Body pink, pearly opalescent; eyes absent or not visible after mounting in Hoyer’s medium. Primary and secondary clavae small and conical. Cirri interni and ex- terni with poorly-developed cirrophores. Cirri A of typical length for Bryodelphax, i.e. reaching around 25% of the total body length. All dorsal plates with barely-discern- zse.pensoft.net Gasiorek, P. et al.: Phylogeny of Bryodelphax ible intra-cuticular pillars (better visible under a 1000~x magnification), the centro-posterior portion of the caudal (terminal) plate has evident, largest pillars (Fig. 2A). Dark epicuticular granules absent (Figs 3; 4A, B), but lateral margins of all dorsal plates and internal margins of facets constituting the scapular plate distinctly thicker and, con- sequently, darker (Fig. 2A). Pores large and easily detect- able (Figs 2A, 3A, B, 4A, B). Pores distributed uneven- ly, with the largest number present on the antero-central portion of the scapular plate (17-40 pores/100 twm?, x = 29, N = 16, Fig. 4A) and the central portion of the caudal plate (7-43 pores/100 um’, x =31, N= 16, Fig. 4B); other plates more variable in terms of pore density, which is always lower than in the aforementioned elements of the armour (0-26 pores/100 um’, x = 14, N = 16, Fig. 2A). Scapular plate gently faceted by transverse cuticular ex- tensions (Figs 3A, 4A), with deep sutures separating lat- eral portions from the central faceted part, extending from the base of cirrophore A to the posterior margin of the plate (Fig. 2A). Paired plates divided into two roughly equal anterior and posterior parts by a transverse stripe (Figs 2A, 3A, B). Caudal (terminal) plate with poorly developed sutures, not visible under PCM (Fig. 2A), but present and visible under SEM (Figs 3A, B, 4B). Medi- an plate 1 subdivided into anterior narrow portion with dark posterior edge and posterior pentagonal portion with transverse suture (Figs 2A, 3A). Median plate 2 is the largest amongst median plates, with well-developed an- terior pentagonal portion and poorly sclerotised posterior portion (Figs 2A, 3A). Median plate 3 with only the an- terior portion fully developed, the posterior portion trian- gular and rounded (Figs 2A, 3A). Supplementary lateral platelets present at the level of median plates (three pairs of platelets on each body side: a pair between the scapular plate and the first pair of the segmental plates, a pair be- tween the paired plates and a pair between the second pair of segmental plates and the caudal plate; Fig. 2A). Venter with seven rows of faint, greyish plates (VII:4- 4-2-4-2-2-1), of which two plates of the first, subce- phalic row are located in a more ventrolateral position (Figs 2B, 3C, D, 10). Under SEM, only the central sub- cephalic and genital plates are visible as true cuticular thickenings, whereas other plates are visible only as darker areas on the cuticular surface (Fig. 3C, D). Leg papillae undetectable under LCM (Fig. 2), but papillae IV visible under SEM (Fig. 3B, C). Both pulvini and pedal plates present, the former developed as thin rect- angular stripes in the proximal leg portions (Figs 2A, 3C) and the latter — as large swellings in the central leg portions (Figs 2A, 3C). Pedal plate I'V toothless, but with a distinct dark margin (Fig. 2A). External claws spur- less, but internal ones with minute spurs positioned close to the claw bases (Figs 2B [insert], 4C). Juveniles. Body 73—101 um long in the two found juve- niles. Dorsal and ventral plates developed similarly to adults. Scapular plate 12-16 um long. Claws 3.7-4.8 pm long. Zoosyst. Evol. 96 (1) 2020, 217-236 abi Echiniscus lineatus 100 — Echiniscus testudo 1.00 97 0.98 79 Bryodelphax decoratus sp. nov. CELEBES he Bryodelphax decoratus sp. nov. CELEBES 1.00 — Bryodelphax arenosus BORNEO 100 Bryodelphax arenosus BORNEO Bryodelphax sp. nov. SERAM 1.00 100 91 86 1.00 Bryodelphax australasiaticus sp. nov, PENANG Bryodelphax australasiaticus sp. nov. PENANG Bryodelphax sp. nov. CELEBES Bryodelphax sp. nov. CELEBES 221 oe 2 | | he 6 aoe 2 eee Pe Bi} UBL UL Figure 1. BI and ML concatenated (18S rRNA + 28S rRNA) phylogenetic tree of Bryvodelphax Thulin, 1928; Echiniscus spp. were used as outgroup taxa. Bayesian posterior probability values (> 0.90) are given above tree branches, whereas ML bootstrap support values (= 70) are below branches. The scale bar represents 0.01 substitutions/site in the Bayesian tree. Trait mapping (blue square — presence, empty square — absence): | — ventral plates; 2 — granules on dorsal plates; 3 — males in population. Figure 2. Habitus of adult female of Bryodelphax australasiaticus sp. nov. (holotype, PCM): A — dorsolateral view; B — ventrolateral view (insert with the claws III, arrowhead indicates spur on internal claw). Roman numerals signify ventral plate rows. Scale bars: 50 um. Larvae. Body 80 um long in a single found two-clawed specimen. Dorsal and ventral plates developed similar- ly to adults. Scapular plate 12.7 um long. Claws 4.0— 4.4 um long. Eggs. Up to one egg in exuvia was found. DNA_ sequences. Single 18S rRNA haplotype (MT333468), two 28S rRNA haplotypes (MT333460-1) and single ITS-1 haplotype (MT333477). Phenotypic differential diagnosis. Within the weg/arskae group, only B. decoratus sp. nov., B. sinensis and B. insta- bilis have seven plate rows, but B. olszanowskii Kaczmarek et al., 2018 exhibits peculiar ventrolateral plates in the sub- cephalic row and that is why this taxon is also taken into consideration in the differential diagnosis. Adult females of B. australasiaticus sp. nov. are differentiated from: ¢ B. decoratus sp. nov., by the ventral plate formula (VII:4-4-2-4-2-2-1 in the new species vs. VII:4-2- zse.pensoft.net 222 Gasiorek, P. et al.: Phylogeny of Bryodelphax Figure 3. Habitus of adult females of Bryodelphax australasiaticus sp. nov. (paratypes, SEM): A — dorsal view; B — lateral view; C — ventrolateral view; D — ventral view. Roman numerals signify ventral plate rows and arrowheads indicate papillae on fourth pair of legs. Scale bars: 50 um. Figure 4. Detailed morphology of Bryodelphax australasiaticus sp. nov. (paratypes, SEM): A — frontal part of the body, faceting of the scapular plate; B — caudal part of the body; C — claws III in close-up. Scale bars: 10 um. 2-4-2-2-1 in B. decoratus sp. nov.) and by dorsal ¢ B. instabilis, currently considered endemic to the plate sculpturing (merged epicuticular ridges sur- rounding the borders of all dorsal plates in the new species vs. large, dark epicuticular granules in B. decoratus sp. nov.); B. sinensis, known from Caucasus and China (the record from Spitsbergen (Dastych 1985) represents B. parvuspolaris), by the ventral plate formula (VII:4-4-2-4-2-2-1 in the new species vs. VII:2-2- 2-2-2-2-1 in B. sinensis) and the caudal (terminal) plate faceting (invisible under LCM in the new species vs. four facets formed by the raised plate areas between two longitudinal and one transversal sutures in B. sinensis), zse.pensoft.net Tatras and northern Slovakia, by the ventral plate formula (VII:4-4-2-4-2-2-1 in the new species vs. VII/TX:(2)-(1)-2/4-2-2/4-2-2-2-1 in B. instabilis), the presence of dentate collar IV (absent in the new species vs. present in B. instabilis), the detectabil- ity of papilla TV under LCM (undetectable in the new species vs. detectable in B. instabilis) and by the reproductive mode (parthenogenesis in the new species vs. dioecy in B. instabilis), ¢ B. olszanowskii, reported from the Antarctic, by the ventral plate formula (VII:4-4-2-4-2-2-1 in the new species vs. VIII:4-1-1-2-2-2-2-2 in B. olszanows- kii), the presence of dark epicuticular granules (ab- Zoosyst. Evol. 96 (1) 2020, 217-236 223 Table 3. Measurements [in um] of selected morphological structures of mature females of Bryodelphax australasiaticus sp. nov. mounted in Hoyer’s medium (N — number of specimens/structures measured, Range refers to the smallest and the largest structure amongst all measured specimens; SD — standard deviation; sp — the ratio of the length of a given structure to the length of the scap- ular plate expressed as a percentage). Character N Range Mean Holotype ym sp ym sp ym sp ym sp Body length 16 91 - 119 584 - 737 108 667 7 Al 111 666 Scapular plate length 16 L'5s2 - 17.9 - 16.2 - 0.8 - 16.6 - Head appendages lengths Cirrus internus 15 4.7 - 6.6 30.2 - 42.4 5? 35.4 0.6 4.0 5.6 33.9 Cephalic papilla 12 2.3 - 3.3 13.9 - 21.1 2.9 18.0 0.3 2.3 2.9 17.6 Cirrus externus 14 7A - 9.4 47.7 - 58.6 8.4 52.6 0.5 3.5 9.0 54.3 Clava 11 1.8 - Sul 10.7 - 20.4 2.3 14.0 0.4 2.8 1:9 11.2 Cirrus A 17 23.2 - 28.7 144.0 - 183.6 25.8 160.0 1.6 11.6 246 147.7 Cirrus A/Body length ratio 15 21% - 26% - 24% - 1% - 22% - Claw heights Claw | 13 45 - 5.7 26.2 - 33.9 opi! 31.2 0.4 2.2 bli 30.4 Claw II 14 4.3 - 5:5 26.7 - 33.3 5.0 30.4 0.3 2.3 5.5 33.3 Claw III 15 4.2 - 5e5 26.5 - 34.2 4.8 29.6 0.3 2.1 5.0 30.2 Claw IV 13 49 - 6.2 30.7 - 38.4 5.4 33.3 0.4 2./ 5.4 32.2 sent in the new species vs. present and accumulated on ventral plates in B. o/szanowskii) and the detect- ability of papilla [V under LM (absent in the new species vs. present in B. olszanowskii). Genotypic differential diagnosis: p-distances between the new species and the remaining Bryodelphax spp., for which DNA sequences are available, were as follows: ¢ 18S rRNA: from 0.3% (B. decoratus sp. nov., MT333469-70) to 3.4% (B. cf. parvulus, HM193371); ¢ 28S rRNA from 0.5% (Bryodelphax sp. nov. from Celebes, MT333467) to 9.3% (B. cf. parvulus, MT333466); ¢ ITS-1: from 2.6% (B. arenosus, MT346599-600) to 3.3% (B. decoratus sp. nov., MT333478). Remarks. Two ventrolateral plates were not drawn in Claxton (2004), which is an unpublished PhD dissertation, thus the species described therein are not valid. However, having ascertained that these structures exist in specimens from Australia, the compared populations from both con- tinents appeared identical in terms of morphology. Bryodelphax decoratus Gasiorek, Von¢tina, Degma & Michalczyk, sp. nov. http://zoobank.org/B009F420-45BF-4B38-B752-E9CE5S78AB5A5 Figures 5, 6, 12, Table 4 Locus typicus and _ type’ material. 1°50'33"S, 120°16'34"E, 800 m a.s.l.; Bada Lembah, Lore Lindu, Celebes (Sulawesi), Indonesia. Holotype (adult female, Slide ID.546.15) and three paratypes (females; slide ID.546.12) deposited in the Institute of Zoology and Biomedical Research, Jagiellonian University; three paratypes (females; slide ID.546.13) deposited in the De- partment of Zoology, Comenius University in Bratislava; three paratypes (females; slide [D.548.11) deposited in the Natural History Museum of Denmark, University of Copenhagen; one paratype (female; slide ID.546.14) de- posited in the Department of Animal Biology, University of Catania; one paratype (female; slide ID.546.16) depos- ited in the Raffles Museum of Biodiversity Research, Na- tional University of Singapore. Etymology. From Latin decoratus = beautified, embel- lished. The name highlights the intricate and beautiful pattern of the dark epicuticular granules. Adjective in the nominative singular. Adults. Body translucent; eyes absent or not visible af- ter mounting in Hoyer’s medium. Primary and secondary clavae minute and conical. Cirri interni and externi with poorly developed cirrophores. Cirri A of typical length for Bryodelphax, i.e. reaching around 25% of the total body length. All dorsal plates with well-visible intra-cuticular pillars, the largest pillars present on the scapular, posteri- or portions of paired segmental and the caudal (terminal) plates (Fig. 5). Dark epicuticular granules present on the dorsum (Figs 5, 6), forming visible transverse lines on the scapular plate, distributed along the margins of all plates, lateral scapular sutures and caudal sutures; additionally, two lines of granules parallel to the lateral margins of paired segmental plates are visible (Figs 5, 6). Pores large and easily detectable (Fig. 5), but their number varies considerably between both specimens and different ele- ments of the armour, with the largest numbers present on the antero-central portion of the scapular plate and median plate 2 (23-48 pores/100 um’, x = 32 and 23-47 pores/100 um’, x = 33, respectively; N = 12) and lower numbers on the central portions of the caudal plate and paired seg- mental plate II (1-38 pores/100 um’, x = 19 and 10—27 pores/100 pm’, x = 17, respectively; N = 12). Scapular plate with lighter rectangles (pseudofacets) between three or four transverse rows of merged dark epicuticular gran- zse.pensoft.net Gasiorek, P. et al.: Phylogeny of Bryodelphax Figure 5. Habitus of adult females of Bryvodelphax decoratus sp. nov. (PCM): A — holotype, dorsal view (insert with the claws I, arrowhead indicates spur on internal claw); B — paratype, lateral view; C — frontal part of the body; D — central part of the body, sculpture of median and paired plates. Scale bars in um. ules (Figs 5C, 6). Paired plates divided into two equal an- terior and posterior parts by a transverse stripe (Fig. 5D). Caudal (terminal) plate with poorly developed sutures (Fig. 5A). Median plate 1 subdivided into the narrow an- terior portion with dark epicuticular granules accumulated at the posterior edge and the posterior, unsculptured pen- tagonal portion with transverse suture (Fig. 5A, D). Me- dian plate 2 is notably the largest amongst median plates, with well-developed anterior pentagonal portion and weakly-sclerotised posterior portion (Fig. 5A, D). Median plate 3 with the anterior portion fully developed, triangu- lar and a smaller rounded posterior portion (Fig. 5A, D). Supplementary lateral platelets present and detectable at lateral-most margins of the segmental plates (Fig. 5B). Venter with extremely weakly delineated plates (VII:4- 2-2-4-2-2-1), only slightly darker than the surrounding ventral cuticle and without clear, sclerotised margins. Dark epicuticular granules and intra-cuticular pillars absent. Leg papillae undetectable under LCM. Both pulvini and pedal plates absent (Fig. 5B). Dentate collar IV absent. External claws spurless, but internal ones with minute spurs barely divergent from the claw branches (Fig. 5A, insert). Juveniles. Not found. Larvae. Not found. Eggs. Not found. zse.pensoft.net DNA sequences. Two 1 8S rRNA haplotypes (MT333469— 70) and two 28S rRNA haplotypes (MT333462-3) and single ITS-1 haplotype (MT333478). Phenotypic differential diagnosis. The new species be- longs to the weglarskae group and it must be compared with the three species (B. instabilis, B. olszanowskii and B. sinensis) with seven ventral plate rows or with ventrolateral plates in the first row present. Additionally, B. decoratus sp. nov. is compared with B. arenosus, as the new species can have very dim and barely discernible ventral plates and, in such cases, it resembles B. arenosus. Nevertheless, adult females of B. decoratus sp. nov. differ specifically from: ¢ B. arenosus, currently considered endemic to Borneo, by body length (99-120 um in the new species vs. 76-95 um in B. arenosus) and dorsal plate sculptur- ing (separate granules present on entire dorsum in the new species vs. continuous, thickened ridges present on the lateral portions of plates in B. arenosus); ¢ B. sinensis, by the ventral plate formula (VIH:4-2-2- 4-2-2-1 in the new species vs. VII:2-2-2-2-2-2-1 in B. sinensis) and the caudal (terminal) plate faceting (invisible under LCM in the new species vs. four fac- ets formed by the raised plate areas between two lon- gitudinal and one transversal sutures in B. sinensis); ¢ B. instabilis, by the ventral plate formula (VII:4- 2-2-4-2-2-1 in the new species vs. VII/IX:(2)-(1)- Zoosyst. Evol. 96 (1) 2020, 217-236 Figure 6. Semi-schematic drawing of the arrangement of epicutic- ular granules on dorsal armour of Bryodelphax decoratus sp. nov. 2/4-2-2/4-2-2-2-1 in B. instabilis), the absence of dentate collar IV (present in B. instabilis), the de- tectability of papilla TV under LCM (undetectable 225 in the new species vs. detectable in B. instabilis) and by the reproductive mode (parthenogenesis in the new species vs. dioecy in B. instabilis), ¢ B. olszanowskii, by the ventral plate formula (VIT:4-2-2-4-2-2-1 in the new species vs. VIII:4- ]-]-2-2-2-2-2 in B. olszanowskii), the presence of dark epicuticular granules on the dorsum (absent in B. olszanowskii) and by the detectability of papilla TV under LCM (undetectable in the new species vs. detectable in B. olszanowskii). Genotypic differential diagnosis: p-distances between the new species and the remaining Bryodelphax spp., for which DNA sequences are available, were as follows: ¢ 18S rRNA: from 0.3% (B. australasiaticus sp. nov., MT333468) to 3.1% (B. cf. parvulus, HM193371); ¢ 28S rRNA from 0.3% (B. australasiaticus sp. nov. and Bryodelphax sp. nov. from Celebes, MT333460, MT333461, and MT333467, respectively) to 9.5% (B. cf. parvulus, MT333466); ¢ ITS-1: from 3.1% (B. arenosus, MT346599) to 23.4% (B. maculatus, MT333479). Bryodelphax nigripunctatus Degma, Gasiorek, Von¢ina & Michalczyk, sp. nov. http://zoobank.org/48DA4500-2806-491 E-8A D8-D2C830A F39F4 Figures 7-12, Tables 5—6 Locus typicus and type material. 39°57'00"N, 3°10'50"E, 160 maz.s.l.; near the road above Cala Figuera beach, Cap de Formentor, NE Mallorca, the Balearic Islands, Spain. Holotype (adult female; together with one male paratype in slide 716/45), allotype (adult male; slide 716/9) and 30 paratypes (9 females, 14 males, 3 specimens of unknown sex, 2 juveniles and 2 larvae; slides 716/1—5, 10, 13, 17 —20, 25, 27-29, 32-34, 41, 45, 47-48, 50-51) deposit- ed in the Department of Zoology, Comenius University Table 4. Measurements [in um] of selected morphological structures of mature females of Bryodelphax decoratus sp. nov. mounted in Hoyer’s medium (N — number of specimens/structures measured, Range refers to the smallest and the largest structure amongst all measured specimens; SD — standard deviation; sp — the ratio of the length of a given structure to the length of the scapular plate expressed as a percentage). Character N Range Mean SD Holotype um sp ym sp ym sp ym sp Body length 13 99 - 120 595 - 694 107 633 6 32 104 640 Scapular plate length 3 16.1 - 18.2 - 16.9 - O27: - 16.2 - Head appendages lengths Cirrus internus ile 4.6 - 7.0 28.5 - 42.3 6.0 35.7 0.8 4.2 5.4 33.0 Cephalic papilla 11 2.5 - 3:5 15.4 - 20.5 3.1 18.4 0.4 1s7 328) 20.5 Cirrus externus 11 8.1 - 12.2 50.2 - 70.6 9.6 57.2 1.3 723 9.0 55.4 Clava 9 2.2 - 3.0 131 - 18.4 2.6 15.3 0.2 1:8 ? ? Cirrus A 12 24.1 - 2Ttf” “P3786: - L69:5% 25:9. = P5324 1.2 9.5 274 169.3 Cirrus A/Body length ratio 12 22% - 28% - 24% - 2% - 26% - Claw heights Claw | 11 5.0 - 6.2 29.8 - SOtl 5I5 325 0.4 1.9 ? ? Claw II 11 4.4 - 5.5 26.5 - 33.3 areal 30.2 0.4 2.0 4.8 29.8 Claw III 13 4.6 - 57, 28.1 - 34.1 52 30.8 0.4 EET, 4.9 30.1 Claw IV 13 BZ - 6.4 yar - 39.2 5.8 34.6 0.4 2.4 5.2 32.2 zse.pensoft.net 226 Gasiorek, P. et al.: Phylogeny of Bryodelphax Figure 7. Habitus of adults of Bryodelphax nigripunctatus sp. nov. (PCM): A— female (holotype, dorsolateral view); B — male (al- lotype, dorsal view); C — female (paratype, lateral view; Roman numerals signify epicuticular belts of granules on legs); D— male (allotype, ventral view, Roman numerals point out reduced ventral armature; insert with the claws I, arrowhead indicates spur on internal claw). Scale bars: 50 um. Figure 8. Habitus of adult male of Bryodelphax nigripunctatus sp. nov. (paratype, SEM): A — dorsal view; B — lateral view. Scale bars: 50 um. in Bratislava; 13 paratypes (6 females, 6 males and one specimen of unknown sex; slides 716/26, 38, 40, 42-44, 46, 49) deposited in the Institute of Zoology and Biomed- ical Research, Jagiellonian University; 9 paratypes (4 females and 5 males; slides 716/6—8, 11-12, 22—24, 35) deposited in the Natural History Museum of Denmark, University of Copenhagen; 9 paratypes (4 females and 5 males; slides 716/14—16, 21, 31, 36-37) deposited in the Department of Animal Biology, University of Catania. Single paratype (male) mounted on a SEM stub (14.19) deposited in the Institute of Zoology and Biomedical Re- zse.pensoft.net search, Jagiellonian University. Eight specimens used for DNA extraction. Bryodelphax nigripunctatus sp. nov. was not accompanied by other species in the sample. Etymology. From Latin niger = black + punctum = dot, spot. The name underlines evident epicuticular granules appearing dark in PCM and contrasting with other ele- ments of dorsal sculpture. Adjective in the nominative singular. Zoosyst. Evol. 96 (1) 2020, 217-236 224 Figure 9. Detailed morphology of Bryodelphax nigripunctatus sp. nov. (paratype, SEM): A — frontal part of the body; B — central part of the body; C — caudal part of the body. Scale bars: 10 um. Figure 10. Details of cuticular sculpturing of Bryodelphax nigripunctatus sp. nov.: A— microscope focused on the epicuticular gran- ules (allotype, NCM); B— microscope focused on the intracuticular pillars (allotype, NCM); C — frontal part of the body (allotype, PCM); D — frontal part of the body (holotype, PCM). Scale bars in um. Adults. Body translucent without distinct colour and usually stout in females and more slender in males (Figs 7, 8), bs = 43.0-49.2% (x = 45.6%, N = 8) in fe- males and 37.5—44.7% (xX = 39.8%, N = 10) in males. Eyes absent or not visible after mounting in Hoyer’s me- dium. Cephalic papillae and clavae elliptical with round- ed apex. Cephalic papilla is relatively broader in males than in females. Both cephalic papillae and primary cla- vae relatively longer in males than in females (Figs 7A, B, 10C, D; compare the sp of the cephalic papilla and clava in Tables 5, 6). Cirri interni always shorter than cirri externi and both with poorly-developed cirrophores. Cirri A reach around 1/5—1/4 of the total body length (Ta- bles 5, 6). Unappendaged. Cuticular sculpture consists of large epicuticular granules, true pores and intra-cuticular pillars (Fig. 10). In PCM, these structures appear, respec- tively, as conspicuous large dark spots, smaller bright spots and fine dark and dense punctuation (pseudogranu- lation). Epicuticular granules of irregular shape and size (up to ca. 1.6 um) are merged together (as visible in SEM, Figs 8-10) and arranged in rows along the margins of all plates, although they are least visible or absent in posteri- or median plate | and posterior median plate 2. Moreover, in the cervical (neck) plate, the row or a double row of granules is also present along its transverse axis. Gran- ules in rows appear as dark spots in PCM (Fig. 7A—C). Similar rows of granules cover also folds which create the faceting of the scapular and caudal plate: median and two lateral longitudinal folds (at the level of cirrophores A) together with 3-4 posterior transverse folds on the scap- ular plate and two longitudinal folds dividing the caudal plate into three parallel portions (Figs 7A, B, 8, 9A, C). Finally, short longitudinal rows of granules divide both paired segmental plates and posterior portions of anteri- or ml—2 plates into left and right portions (Figs 7A, B, 8, 9B). Scattered granules also irregularly cover the sur- face of the cephalic, paired, caudal, anterior m1l—2 and anterior parts of scapular plates (Figs 7A, B, 8, 9, 10A). Round, focusable pores (0.3—0.4 um in diameter) are un- equally distributed on dorsal plates in spaces between zse.pensoft.net 228 a Figure 11. Variability of the dentate collar IV in Bryodelph the scattered granules, on pedal plates IV and between transverse rows of granules in the scapular plate, but they are absent on the rows of granules (Figs 7C, 8, 9A-C, 10B). The density of pores varies between the sexes and elements of armour, with the largest pores present on the segmental plate II, anterior m2 and the scapular plate (21-40 pores/100 um’, x = 29, 14-28 pores/100 um?, X = 22 and 18—3lpores/100 pm’, x = 26, respectively, N = 15 in females and 7-35 pores/100 um?, x = 29, 0-34 pores/100 um’, x = 25 and 11-33 pores/100 m7, x = 25, respectively; N = 15 in males) and lowest density on cau- dal plate (16-27 pores/100 um’, x = 22 in females and 7-28 pores/100 um’, xX = 20 in males; N = 15). On the scapular plate (in the area delimited with lateral longitu- dinal rows of granules), lines of pores tend to alternate with transverse rows of granules (Figs 7A—C, 8A, 9A, 10A, B). Regularly distributed round intra-cuticular pil- lars (0.1—0.2 um in diameter) reinforce the entire cuticle (also under the granules), but they are well-visible only in the cephalic, scapular, both paired segmental, caudal, an- terior median | and anterior median 2 plates (Fig. 10B), as well as on pedal plates IV. On the remaining cuticle, they are weakly (venter) or scarcely (legs) detectable. Cephalic plate with an anterior chalice-like incision. Each segmental and median plate consists of the anterior and the posterior portion separated each from other with a transverse bright poreless stripe in PCM. Therefore, paired segmental plates are subdivided into the narrow- zse.pensoft.net Gasiorek, P. et al.: Phylogeny of Bryodelphax ax nigripunctatus sp. nov. (paratypes, PCM). Scale bars: 10 um. er anterior (ca. 1/3—2/5 of the plate length) and the wid- er posterior portions, trapezoidal anterior median plate 1 is subdivided just behind its anterior margin, pentagonal anterior m2 (the largest amongst the median plates) is di- vided at approximately equally-long portions, triangular anterior portion of median plate 3 is ca. two times as long (along median body axis) as the posterior one with round- ed posterior margin (dividing transverse line of anterior median plates 2—3 correspond with posterior corners of paired segmental plates). Pentagonal posterior median plates 1-2 subdivided at portions of about same lengths. On each body side, the first two pairs of supplementary lateral platelets are connected with anterior and posterior median plates 1—2 and the third pair is connected with the posterior portion of m3 and with the anterior edge of cau- dal plate. Anterior platelets of each pair have very distinct- ly-thickened lateral (lower) margins (Figs 7A, B, 8, 9). Venter with transverse rows of weakly-developed plates unevenly sculptured with epicuticular granules similar to those on the dorsal plates, but a bit smaller. There are three rows of ventral plates in females (the plate formula IIJ:2-2-1) and two rows in males (II:2- 2) (Fig. 7D). The outer surface of legs with a narrow well-visible proximal pulvinus and a wide weak dis- tal pedal plate placed in the central part of the leg. A single row of small epicuticular granules (similar to those on ventral plates) on the distal edge of pulvini in legs I-III (rarely, the second row also on their proximal Zoosyst. Evol. 96 (1) 2020, 217-236 229 B. aaseae B. kristenseni B. iohannis adult 2 adult 2 B. weglarskae adult adult 9 i@ Il pq B. instabilis B. olszanowskii adult 2 adult B. instabilis juvenile B. instabilis larva B. parvuspolaris B. sinensis adult 2 adult 2 B. australasiaticus B. decoratus adult 2 adult 2 B. nigripunctatus B. amphoterus adult 3 adult B. maculatus adult 2 B. nigripunctatus adult B. maculatus juvenile B. maculatus larva Figure 12. Schematic arrangement of the ventral plates in all members of the weg/arskae group (species are arranged in order of the increasing reduction of the ventral armature). Known cases of ontogenetic variability and sexual dimorphism are depicted. Follow- ing species taken from Kaczmarek et al. (2012): B. aaseae, B. iohannis, B. parvuspolaris, B. sinensis and B. weglarskae;, Gasiorek et al. (2017a): B. maculatus; Gasiorek & Degma (2018): B. instabilis. zse.pensoft.net 230 Gasiorek, P. et al.: Phylogeny of Bryodelphax Table 5. Measurements [in um] of selected morphological structures of mature females of Bryodelphax nigripunctatus sp. nov. mounted in Hoyer’s medium (N — number of specimens/structures measured, Range refers to the smallest and the largest structure amongst all measured specimens; SD — standard deviation; sp — the ratio of the length of a given structure to the length of the scap- ular plate expressed as a percentage). Character N Range Mean SD Holotype ym sp ym sp ym sp ym sp Body length 15 114 - 144 562 - 689 126 645 9 38 131 657 Scapular plate length 15 18.0 - 21.7 - 19.6 - Le - 19.9 - Head appendages lengths Cirrus internus 11 6.1 - 8.9 32.9 - 42.8 Vee} 37.4 0.8 3.3 75 37.4 Cephalic papilla 14 2.6 - 337 12.6 - 19.7 3.1 15.9 0.4 1.9 3.3 16.5 Cirrus externus 12 10.2 - 12.8 54.1 - 66.9 11.8 60.1 0.7 4.4 12.2 61.4 Clava 10 2.7 - Beil 12.3 - 16.9 2.9 14.8 0.1 1.3 3.0 15.0 Cirrus A 15 24.8 - 31.0 120.7 - 163.7 28.0 143.6 1.6 11.0 26.3 132.4 Cirrus A/Body length ratio 15 20% - 25% - 22% - 2% - 20% - Body appendages lengths Papilla on leg IV length 8 1.4 - 2.3 TL? - 12.5 1.8 975) 0.3 2.0 1.5 7.6 Number of teeth on the collar 10 2 - 5 - 383 - 0.9 - 2 - Claw heights Claw | 12 7.0 - 8.3 35.1 - 44.4 7.8 39.8 0.5 Sel 7.2 36.0 Claw II 9 7.0 - 8.8 36.1 - 44.3 7.6 39.6 0.5 2.8 7.7 38.8 Claw III 13 6.7 - 8.3 36.1 - 46.2 7.6 39.3 0.5 Sal Tale: 38.1 Claw IV 9 7.4 - 8.9 38.1 - 48.0 8.4 43.0 0.5 3:5 ? ? Table 6. Measurements [in um] of selected morphological structures of mature males of Bryodelphax nigripunctatus sp. nov. mounted in Hoyer’s medium (N — number of specimens/structures measured, Range refers to the smallest and the largest structure amongst all measured specimens; SD — standard deviation; sp — the ratio of the length of a given structure to the length of the scap- ular plate expressed as a percentage). Character N Range Mean SD Allotype ym sp ym sp ym sp ym sp Body length 15 106 - 134 618 - 129 123 685 8 30 130 704 Scapular plate length 15 16.1 - 19.5 - 17.9 - Tel - 18.5 - Head appendages lengths Cirrus internus 13 6.3 - 9.9 3528 - SeER/2 LA: 42.8 1.0 4.6 8.5 46.0 Cephalic papilla 14 3.1 - 5:6 19.0 - 32.0 4.6 25.5 0.8 339 5.2 28.1 Cirrus externus 113 12.3 - 15.9 70.6 - 85.8 14.2 78.6 1.0 4.3 aReigs) 75.4 Clava 11 2.8 - 4.7 17.6 - 24.2 3.8 21.2 0.5 1.9 4.1 21.9 Cirrus A 15 26.8 - 32.6 141.3 - 187.3 29.3 16358 1.9 11.4 30.2 163.4 Cirrus A/Body length ratio 15 21% - 26% - 24% - 2% - 23% - Body appendages lengths Papilla on leg IV length 8 IS, - 2.4 oy - 12.8 2.0 11.4 0.3 1.0 2.0 10.9 Number of teeth on the collar 12 3 - 5 - 329 - 1.0 - 5 - Claw heights Claw | 11 Jx3 - 8.7 37.6 - 50.3 8.1 45.1 0.5 3.4 8.3 44.8 Claw II 10 Tel - 8.8 41.2 - 48.5 8.0 44.7 0.5 2:5 8.8 47.9 Claw III 14 7.0 - 9.2 38.5 - 49.5 8.0 44.7 0.6 3:3 9.0 48.6 Claw IV 9 7.8 - 9.4 43.6 - 52.6 8.7 48.3 0.6 2.8 2 ? edge). Pedal plates I-III sculptured usually with three (sometimes with more) transverse rows of epicuticular granules, which can be either shortened or connected at their ends (Figs 7A—C, 8B). The pedal plate IV sculp- tured with distinct intra-cuticular pillars and scattered pores and distally hemmed with dentate collar. The col- lar with sharp teeth, always longer than the width of their bases and with the distance between teeth simi- lar to their basal widths, although some pairs of teeth can be merged (Fig. 11). Papilla or spine on legs I-III absent, papilla on legs IV well developed. Claws slen- der, claws IV always slightly longer than claws I-III. External claws smooth, internal ones with a small spur pointing downwards and placed very close to the claw bases (Figs 7A—C, 7D, insert). zse.pensoft.net Juveniles. In appearance as adults, but smaller (111- 112 um) and with ventral plates just marked with rows of granules. Selected measurements of a shorter specimen: cephalic papilla 3.1 um, scapular plate 14.9 um long, claws I-III 5.1—5.6 and claws IV 6.5 um long. Larvae. 83—85 um long. Dorsal plates (especially medi- an ones) mostly with poorly-delineated margins, supple- mentary lateral platelets absent. Epicuticular granules less numerous than in adults, concentrated mainly on posterior margins of the cephalic, scapular, both paired and caudal plates. Cuticular pores less numerous than in adults, but intracuticular pillars, stripes of granules on the outer surface of legs, papilla on legs IV and den- tate collar IV well developed. Ventral plates not visible Zoosyst. Evol. 96 (1) 2020, 217-236 in laterally orientated larvae. Claws with spurs formed as in adults. Some measurements of shorter specimen: cephalic papilla 2.5 um, claws II-III 4.6—5.3 and claws IV 6.4 um long. Eggs. Not found. DNA sequences. Two 18S rRNA haplotypes (MT333472- 3), single 28S rRNA haplotype (MT333465). Phenotypic differential diagnosis. Having ventral plates, Bryodelphax nigripunctatus sp. nov. belongs to the weglarskae group. Within the group, only B. ampho- terus and B. maculatus have a reduced number of ventral plate rows to two or three, as in the new species. Consec- utively, B. nigripunctatus sp. nov. differs from: ¢ B. amphoterus, known from Croatia (Istria) and Greece (Crete) (McInnes 1994), by: the mode of reproduction (dioecy in the new species vs. par- thenogenesis in B. amphoterus), the presence of lateral supplementary platelets (absent in B. am- photerus), the presence of epicuticular granules on dorsal and ventral plates (absent in B. ampho- terus), a different number of ventral plate rows in females (ventral plate formula HI:2-2-1 in females of the new species vs. II:2-2 in B. amphoterus) and by the lack of spurs on external claws (extremely small spur very difficult to observe just near the base in B. amphoterus); ¢ B. maculatus, known from Tunisia and Greece, by: the mode of reproduction (dioecy in the new spe- cies vs. parthenogenesis in B. maculatus), large contrasting granules on dorsal plates (granules not contrasting and clearly visible only in SEM in B. maculatus), the absence of patches or transverse stripes of epicuticular granules on ventral cuticle between legs (present in B. maculatus), a smaller maximal pore density (21—40 pores per 100 um? in segmental II plate in the new species females vs. 48-61 pores per 100 pum? in the same plate in B. maculatus), a relatively longer internal peribuccal cirrus (sp is 33-43% in females of the new spe- cies vs. 21-30% in B. maculatus) and by relatively longer claws I-IV (sp for claws II is 36-44%, for claws III 36-46%, for claws IV 38-48% in females of the new species vs. 29-36%, 30-34% and 32— 38%, respectively in B. maculatus). Genotypic differential diagnosis: p-distances between the new species and the remaining Bryodelphax spp., for which DNA sequences are available, were as follows: ¢ 18S rRNA: from 0.4% (B. maculatus, KY 609137 and M1T333471) to 2.9% (B. australasiaticus sp. nov., MT333468); ¢ 28S rRNA from 4.2% (B. instabilis, MH414965) to 8.1% (B. decoratus sp. nov., MT333462, MT333463). 231 Discussion Phylogeny and evolution of traits in Bryodelphax Inter-generic tardigrade relationships are constant- ly being unravelled (Bertolani et al. 2014; Fujimoto et al. 2016; Gasiorek et al. 2019a, b). Recently, Guil et al. (2019) proposed a new classification of Echiniscidae, with Bryodelphax included within Echiniscinae and having its own tribe Bryodelphaxini. Not only is such a proposal unjustified morphologically, as Bryodelphax is more similar to the Pseudechiniscus-like genera than to the Echiniscus lineage (Gasiorek et al. 2018a), but, im- portantly, the current phylogenetic evidence 1s also not conclusive (different positions of the genus on echiniscid phylogenetic trees in Guil et al. 2019). In fact, the trait used to delimit putative Bryodelphaxini from Echinisci- ni, 1.e. the presence of peribuccal cirrophores, is biased and unreliable — Bryodelphax has weakly outlined cirro- phores due to the miniaturised body, but, essentially, the anatomy of cephalic cirri within both dubious tribes is identical. Therefore, the systematic distinction between Bryodelphaxini and Echiniscini is controversial and their status should be further verified. In terms of morphology, the genus should be currently recognised as a separate lineage of Echiniscidae, with the unsolved, long-stand- ing problem of Bryochoerus (Kristensen 1987; Lisi et al. 2017; Gasiorek 2018). Regarding the phyletic relationships within the genus Bryodelphax, some intriguing conclusions can be drawn from the mapping of various phenotypic traits onto the phylogenetic tree (Fig. 1). Firstly, the division of the ge- nus, based on the presence (weg/arskae group) or absence (parvulus group) of ventral armature, has only practical significance for taxonomic purposes (see below), as the members of both groups are phylogenetically intermin- gled. This suggests that this trait is not conservative and its appearance should be regarded as convergent. Ventral plates are strongly sclerotised and evident in B. amphoter- us, formerly affiliated within the parvulus group, which corroborates the supposition by Gasiorek et al. (2017a) that these structures have been previously overlooked. Peculiarly, the reduction of ventral armature to plesio- morphic subcephalic and genital plate rows 1s known in Bryodelphax only in three Mediterranean species (B. am- photerus, B. maculatus and B. nigripunctatus sp. nov.). Secondly, since the description of B. maculatus, dark epicuticular granules have received attention of taxono- mists (Lisi et al. 2017; Kaczmarek et al. 2018). To date, these structures were recognised in eight Bryodelphax spp. during examination of comparative material (B. aaseae, B. atlantis Fontoura et al., 2008, B. decoratus sp. nov., B. instabilis, B. kristenseni Lisi et al., 2017, B. maculatus, B. nigripunctatus sp. nov. and B. olszanows- kii). Although this trait was not described in many pre- vious descriptions, our analysis indicated no granules in ten spp. (B. amphoterus, B. arenosus, B. asiaticus, B. zse.pensoft.net 232 australasiaticus sp. nov., B. brevidentatus, B. mateusi (Fontoura, 1982), B. meronensis, B. parvulus, B. tatren- sis (Weglarska, 1959) and B. weglarskae). Similarly to the traits described above, a glance at the distribution of species with dark epicuticular granules on the tree (Fig. 1) implies that this taxonomically-useful criterion bears no phylogenetic signal. Thirdly, species exhibiting different modes of repro- duction are scattered on the tree. Two of the three known dioecious Bryodelphax spp., B. instabilis and B. nigri- punctatus sp. nov., are not directly related (Fig. 1). This pattern, that is parthenogenetic and dioecious taxa mixed on the tree, 1s consistent with recent data for Paramac- robiotus and Milnesium (Guidetti et al. 2019; Morek and Michalczyk 2020). However, most of the other echiniscid genera are more consistent in terms of the mode of repro- duction (Kristensen 1987). Last but not least, in contrast to phenotypic traits, geographic distribution of the analysed species suggests their limited dispersal abilities and seems to be a reliable predictor of phylogenetic affinities within the genus. This intriguing pattern has been recently shown in the genus Milnesium Doyere, 1840 by Morek and Michalczyk (2020). Considering the remote phyletic relationship and contrasting body sizes in the two groups (Milnesium comprises largest tardigrades), these results suggest that tardigrade species, in general, may have much more re- stricted geographic distributions than the "Everything is everywhere" hypothesis predicts (Beijerinck 1913). It ought to be noted that the lengths of the tree branches in the case of the Oriental clade are consid- erably shorter than those for the Western Palaearctic clade (Fig. 1), whereas the taxa of both lineages are well-separated from each other (with the exception of B. arenosus and Bryodelphax sp. nov. from Seram, which requires more data to solve its phyletic relation- ship with other congeners). Deeper nodes in the West- ern Palaearctic clade may result from: (a) longer diver- gence time needed for cladogenesis in this region (e.g. Ricklefs 2004), (b) higher extinction rate in the tropics (e.g. Jablonski et al. 2006) or (c) from under-sampling of lineages in the Palaearctic (e.g. Chown and Gaston Gasiorek, P. et al.: Phylogeny of Bryodelphax 2000). If the last possibility 1s excluded, then the ob- served pattern may mean that speciation could be more rapid in tropical tardigrades, as postulated by the trop- ical cradle biodiversity hypothesis, which assumes that young evolutionary lineages are prevalent in the tropics (Stebbins 1974; Stenseth 1984; Jablonski et al. 2006; Moreau and Bell 2013). A similar scenario was recently demonstrated for oribatid mites (Pachl et al. 2017), but a much greater sampling effort 1s required for Bryodel- phax spp. in order to test this hypothesis. Taxonomic key to the genus Bryodelphax Since the last key by Fontoura et al. (2008), the num- ber of described Bryodelphax species almost doubled (from 15 spp. considered valid in 2008 to 25 spp. gath- ered in the present contribution). Moreover, the 2008 key has several inadequacies: (1) three members of the weglarskae group (B. iohannis, B. sinensis and B. we- glarskae) had an under-estimated number of ventral plates (nine instead of ten, six instead of seven and eight instead of nine, respectively); (II) B. asiaticus was de- limited from B. parvulus by the absence of supplemen- tary lateral platelets, but these structures are present in both species (Gasiorek 2018). Our analysis of B. am- photerus paratypes revealed the presence of reduced, but evident ventral armature (formula II:2-2), thus the species belongs to the weglarskae group (Fig. 12). All these facts inclined us to present a new key allowing for the delimitation of females of the genus members at the adult life stage. The distinction between members of the weglarskae group (12 spp.) is rather straightforward, but the identification of species within the parvulus group (13 spp.) may pose a problem for beginner taxonomists. Consequently, we advise the greatest caution when iden- tifying its members. Importantly, B. /ijiangensis Yang, 2002 is designated as nomen dubium due to the insuffi- cient description and the general habitus not conforming to the characteristics of the genus (trunk cirri in all later- al positions suggest its affinity to Echiniscus) and hence it is omitted from our key. Key 1 VEnthalnplate Stone SOWMUClIas Wella Shae LOU yc uk o8. ...a0lece ama teeta analaa bans eet Pere as dies peace Mialnehnelcesad aaa anette Matte 2 - META PISteS ABSeM EEE OSIVAIUS BLOW erie) eee oA shan oec las cnessellsces de Reestaa ones elite dasade stheedes ceesttAlhe sn