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Research Article
Small is beautiful: the first phylogenetic analysis of Bryodelphax Thulin, 1928 (Heterotardigrada, Echiniscidae)
expand article infoPiotr Gąsiorek‡, Katarzyna Vončina‡, Peter Degma§, Łukasz Michalczyk‡
‡ Jagiellonian University, Kraków, Poland
§ Comenius University, Bratislava, Slovakia

Corresponding author: Piotr Gąsiorek ( piotr.lukas.gasiorek@gmail.com )

Academic editor: Martin Husemann
© 2020 Piotr Gąsiorek, Katarzyna Vončina, Peter Degma, Łukasz Michalczyk.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Gąsiorek P, Vončina K, Degma P, Michalczyk Ł (2020) Small is beautiful: the first phylogenetic analysis of Bryodelphax Thulin, 1928 (Heterotardigrada, Echiniscidae). Zoosystematics and Evolution 96(1): 217-236. https://doi.org/10.3897/zse.96.50821
ZooBank: urn:lsid:zoobank.org:pub:0B80FF1B-5ED7-430B-A471-A5DE02E8E6D3
Open Access

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 newly-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 weglarskae 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 weglarskae group (poorly visible ventral plates VII:4-2-2-4-2-2-1) and is closely related to the recently described Bryodelphax arenosus Gąsiorek, 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 Gąsiorek 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 µm, with some notable exceptions, such as milnesiids (Morek et al. 2016) or richtersiids (Guidetti et al. 2016), reaching body lengths up to 1200 µm (Nelson et al. 2015). Marine heterotardigrades are the smallest representatives of the phylum, being usually below 200 µm in body length (Jørgensen et al. 2014; Fontoura et al. 2017), but speciose heterotardigrade echiniscids fit to the aforementioned range for limno-terrestrial water bears (200–500 µm), 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 μm. The opposite trend, i.e. 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; Gąsiorek 2018; Gąsiorek 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 apparatus). These characteristics make Bryodelphax a good example of mosaic evolution in tardigrades (Kristensen et al. 2010; Gąsiorek 2018).

The aim of this study was to elucidate the phylogeny of Bryodelphax in relation to morphological traits used in its taxonomy, with application of the integrative approach, i.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 reproductive 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 animals 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 (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 series 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 (Pilato, 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.

Table 1.
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Collection data for the newly-sequenced species used in morphological and phylogenetic analyses.

Species Sample code Coordinates, altitude Locality Environment Sample type, substrate Collector
Bryodelphax australasiaticus sp. nov. MY.240 5°27'05"N, 100°11'00"E, 4 m asl Malaysia, Pulau Pinang, Pantai Keracut beach dominated by Casuarina equisetifolia moss, tree branch Piotr Gąsiorek & Artur Oczkowski
MY.241 5°27'05"N, 100°11'00"E, 4 m asl Malaysia, Pulau Pinang, Pantai Keracut beach dominated by Casuarina equisetifolia moss, tree branch Piotr Gąsiorek & Artur Oczkowski
MY.242 5°27'13"N, 100°11'08"E, 53 m asl Malaysia, Pulau Pinang, Pantai Keracut lowland rainforest moss, rock Piotr Gąsiorek & Artur Oczkowski
Bryodelphax decoratus sp. nov. ID.546 1°50'33"S, 120°16'34"E, 800 m asl Indonesia, Central Sulawesi, Lore Lindu, Bada Lembah cacao tree plantation moss, tree Piotr Gąsiorek & Artur Oczkowski
ID.548 1°50'33"S, 120°16'34"E, 801 m asl Indonesia, Central Sulawesi, Lore Lindu, Bada Lembah cacao tree plantation moss+lichen, tree Piotr Gąsiorek & Artur Oczkowski
Bryodelphax maculatus* GR.050 (=780) 35°23'23"N, 23°39'36"E, 374 m asl Greece, Crete ,Vlatos olive tree plantation moss, olive tree Peter Degma
Bryodelphax nigripunctatus sp. nov.** ES.264 (=716) 39°57'00"N, 3°10'50"E, 160 m asl Spain, The Balearic Islands, Mallorca, Cap de Formentor, Cala Figuera beach, near the road above sea shore moss, rock Peter Degma
Bryodelphax parvulus IT.010 45°42'12"N, 13°42'53"E, 1 m asl Italy, Trieste, Grignano Miramare urban park moss, wall Alicja Witwicka
Bryodelphax sp. nov. ID.464 1°52'48"S 120°15'48"E, 778 m asl Indonesia, Central Sulawesi, Lore Lindu, Bada Lembah cacao tree plantation moss, tree Piotr Gąsiorek & Artur Oczkowski
Bryodelphax sp. nov. ID.846 3°10'52"S, 129°02'58"E, 295 m asl Indonesia, Ambon, pass between Triana and Jerili/Sawai, Seram Tengah mountain rainforest lichen, palm tree Piotr Gąsiorek & Łukasz Krzywański

* First record for Greece, ** First record of a limno-terrestrial tardigrade for Balearic Islands (see Guil 2002).
Table 2.
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Processing data for populations of Bryodelphax investigated 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 utilised 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.050 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

Microscopy, imaging and morphometrics

Permanent microscope slides were made using Hoyer’s medium and examined under a Nikon Eclipse 50i 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. Specimens for imaging in the SEM were prepared according to Stec et al. (2015) and examined in Versa 3D DualBeam SEM at the ATOMIN facility of the Jagiellonian University. All figures were assembled in Corel Photo-Paint X6, ver. 16.4.1.1281 or in Adobe Photoshop CS3 Extended, ver. 10.0. All measurements are given in micrometres (μm) and were performed under PCM. Structures were measured only when not broken, deformed 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 percentage (Dastych 1999). The bs ratio is the proportion between the maximal body width and the body length in dorsoventrally orientated specimens (Gąsiorek et al. 2018a). Morphometric data were handled using the Echiniscoidea ver. 1.2 template available from the Tardigrada 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 examined under a 400× magnification PCM prior to DNA extraction) following a Chelex 100 resin (Bio-Rad) extraction method (Casquet et al. 2012; Stec et al. 2015). Three DNA fragments were sequenced: the small ribosome subunit 18S rRNA (primers 18S_Tar_Ff1 and 18S_Tar_Rr2 from Stec et al. 2017 and Gąsiorek et al. 2017b, PCR programme from Zeller 2010), the large ribosome subunit 28S rRNA (primers 28S_Eutar_F and 28SR0990 from Gąsiorek et al. 2018b and Mironov et al. 2012 and the PCR programme from Mironov et al. 2012) and the internal transcribed spacer ITS-1 (primers ITS1_Echi_F and ITS1_Echi_R from Gąsiorek et al. 2019a, PCR programme from Wełnicz et al. 2011). Some of the less conservative markers, such as ITS-2 and COI, are often difficult to amplify for Bryodelphax. All fragments were amplified and sequenced according to the protocols described in Stec et al. (2015). Sequences 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 (Doyère, 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 rRNA), respectively. The aligned sequences were concatenated 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 preferred 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: TVMe+G4 (18S rRNA) and TIM3e+G4 (28S rRNA). Maximum-likelihood (ML) topologies were constructed using IQ-TREE (Nguyen et al. 2015; Trifinopoulos et al. 2016). Strength of support for internal nodes of ML construction was measured using 1000 ultrafast bootstrap replicates (Hoang et al. 2018). Bootstrap (BS) support values ≥ 85% on the final tree were regarded as well supported and those > 70% as moderately supported. Bayesian inference (BI) marginal posterior probabilities were calculated 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 deviation 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 stationarity and to determine the correct ‘burn-in’ for the analysis 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 topologies and discarding the burn-in. In the BI consensus tree, clades recovered with posterior probability (PP) between 0.95 and 1 were considered well supported, those with PP between 0.90 and 0.94 were considered moderately supported and those with lower PP were considered unsupported. All final consensus trees were viewed and visualised 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 Register under www.tardigrada.net/register/0064.htm (B. australasiaticus sp. nov.), www.tardigrada.net/register/0065.htm (B. decoratus sp. nov.), www.tardigrada.net/register/0066.htm (B. nigripunctatus sp. nov.). Type DNA sequences are deposited in GenBank.

Results

Molecular phylogeny

Bayesian Inference and Maximum Likelihood trees shared identical topology (Fig. 1). Two lineages, each represented by four species, were recovered: the Oriental clade (B. arenosus Gąsiorek, 2018, B. australasiaticus sp. nov., B. decoratus sp. nov. and Bryodelphax sp. nov.) and the Western Palaearctic clade (B. instabilis Gąsiorek & Degma, 2018, B. maculatus Gąsiorek et al., 2017, B. nigripunctatus sp. nov. and B. parvulus Thulin, 1928).

Figure 1. 

BI and ML concatenated (18S rRNA + 28S rRNA) phylogenetic tree of Bryodelphax 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): 1 – ventral plates; 2 – granules on dorsal plates; 3 – males in population.

Descriptions of new species

Systematic account

Phylum: Tardigrada Doyère, 1840

Class: Heterotardigrada Marcus, 1927

Order: Echiniscoidea Richters, 1926

Family: Echiniscidae Thulin, 1928

Genus: Bryodelphax Thulin, 1928

Bryodelphax australasiaticus Gąsiorek, Vončina, Degma & Michalczyk, sp. nov.

http://zoobank.org/BE521B67-6769-4EF5-B3C9-56EB43BF2DFE
Figures 2, 3, 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 paratypes (2 females; slide MY.240.05) deposited in the Department 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, University 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.

Table 3.
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Measurements [in µm] 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 scapular plate expressed as a percentage).

Character N Range Mean SD Holotype
µm sp µm sp µm sp µm sp
Body length 16 91 – 119 584 – 737 108 667 7 41 111 666
Scapular plate length 16 15.2 – 17.9 – 16.2 – 0.8 – 16.6 –
Head appendages lengths
Cirrus internus 15 4.7 – 6.6 30.2 – 42.4 5.7 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 7.4 – 9.4 47.7 – 58.6 8.4 52.6 0.5 3.3 9.0 54.3
Clava 11 1.8 – 3.1 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 24.6 147.7
Cirrus A/Body length ratio 15 21% – 26% – 24% – 1% – 22% –
Claw heights
Claw I 13 4.5 – 5.7 26.2 – 33.9 5.1 31.2 0.4 2.2 5.1 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 – 5.5 26.5 – 34.2 4.8 29.6 0.3 2.1 5.0 30.2
Claw IV 13 4.9 – 6.2 30.7 – 38.4 5.4 33.3 0.4 2.7 5.4 32.2

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 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 barely-discernible intra-cuticular pillars (better visible under a 1000× 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, consequently, darker (Fig. 2A). Pores large and easily detectable (Figs 2A, 3A, B, 4A, B). Pores distributed unevenly, with the largest number present on the antero-central portion of the scapular plate (17–40 pores/100 μm2, x̅ = 29, N = 16, Fig. 4A) and the central portion of the caudal plate (7–43 pores/100 μm2, x̅ = 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 μm2, x̅ = 14, N = 16, Fig. 2A). Scapular plate gently faceted by transverse cuticular extensions (Figs 3A, 4A), with deep sutures separating lateral 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). Median 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 anterior pentagonal portion and poorly sclerotised posterior portion (Figs 2A, 3A). Median plate 3 with only the anterior portion fully developed, the posterior portion triangular 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 between the paired plates and a pair between the second pair of segmental plates and the caudal plate; Fig. 2A).

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 µm.

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 µm.

Venter with seven rows of faint, greyish plates (VII:4-4-2-4-2-2-1), of which two plates of the first, subcephalic row are located in a more ventrolateral position (Figs 2B, 3C, D, 10). Under SEM, only the central subcephalic 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 rectangular 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 IV toothless, but with a distinct dark margin (Fig. 2A). External claws spurless, but internal ones with minute spurs positioned close to the claw bases (Figs 2B [insert], 4C).

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 µm.

Juveniles

Body 73–101 μm long in the two found juveniles. Dorsal and ventral plates developed similarly to adults. Scapular plate 12–16 μm long. Claws 3.7–4.8 μm long.

Larvae

Body 80 μm long in a single found two-clawed specimen. Dorsal and ventral plates developed similarly to adults. Scapular plate 12.7 μm long. Claws 4.0–4.4 μm 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 weglarskae group, only B. decoratus sp. nov., B. sinensis and B. instabilis have seven plate rows, but B. olszanowskii Kaczmarek et al., 2018 exhibits peculiar ventrolateral plates in the subcephalic 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- 2-4-2-2-1 in B. decoratus sp. nov.) and by dorsal plate sculpturing (merged epicuticular ridges surrounding 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);
  • B. instabilis , currently considered endemic to the Tatras and northern Slovakia, by the ventral plate formula (VII:4-4-2-4-2-2-1 in the new species vs. VII/IX:(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 detectability of papilla IV 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. olszanowskii), the presence of dark epicuticular granules (ab sent in the new species vs. present and accumulated on ventral plates in B. olszanowskii) and the detectability of papilla IV 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 continents appeared identical in terms of morphology.

Bryodelphax decoratus Gąsiorek, Vončina, Degma & Michalczyk, sp. nov.

http://zoobank.org/B009F420-45BF-4B38-B752-E9CE578AB5A5
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 Department of Zoology, Comenius University in Bratislava; three paratypes (females; slide ID.548.11) deposited in the Natural History Museum of Denmark, University of Copenhagen; one paratype (female; slide ID.546.14) deposited in the Department of Animal Biology, University of Catania; one paratype (female; slide ID.546.16) deposited in the Raffles Museum of Biodiversity Research, National University of Singapore.

Table 4.
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Measurements [in µm] 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
µm sp µm sp µm sp µm sp
Body length 13 99 – 120 595 – 694 107 633 6 32 104 640
Scapular plate length 13 16.1 – 18.2 – 16.9 – 0.7 – 16.2 –
Head appendages lengths
Cirrus internus 13 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 1.7 3.3 20.5
Cirrus externus 11 8.1 – 12.2 50.2 – 70.6 9.6 57.2 1.3 7.3 9.0 55.4
Clava 9 2.2 – 3.0 13.1 – 18.4 2.6 15.3 0.2 1.8 ? ?
Cirrus A 12 24.1 – 27.7 137.6 – 169.5 25.9 153.4 1.2 9.5 27.4 169.3
Cirrus A/Body length ratio 12 22% – 28% – 24% – 2% – 26% –
Claw heights
Claw I 11 5.0 – 6.2 29.8 – 35.1 5.5 32.5 0.4 1.9 ? ?
Claw II 11 4.4 – 5.5 26.5 – 33.3 5.1 30.2 0.4 2.0 4.8 29.8
Claw III 13 4.6 – 5.7 28.1 – 34.1 5.2 30.8 0.4 1.7 4.9 30.1
Claw IV 13 5.2 – 6.4 32.2 – 39.2 5.8 34.6 0.4 2.4 5.2 32.2

Etymology

From Latin decoratus = beautified, embellished. 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 after 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, posterior 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 elements 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 μm2, x̅ = 32 and 23–47 pores/100 μm2, x̅ = 33, respectively; N = 12) and lower numbers on the central portions of the caudal plate and paired segmental plate II (1–38 pores/100 μm2, x̅ = 19 and 10–27 pores/100 μm2, x̅ = 17, respectively; N = 12). Scapular plate with lighter rectangles (pseudofacets) between three or four transverse rows of merged dark epicuticular granules (Figs 5C, 6). Paired plates divided into two equal anterior 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 anterior portion with dark epicuticular granules accumulated at the posterior edge and the posterior, unsculptured pentagonal portion with transverse suture (Fig. 5A, D). Median 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, triangular 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).

Figure 5. 

Habitus of adult females of Bryodelphax 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 µm.

Figure 6. 

Semi-schematic drawing of the arrangement of epicuticular granules on dorsal armour of Bryodelphax decoratus sp. nov.

Juveniles

Not found.

Larvae

Not found.

Eggs

Not found.

DNA sequences

Two 18S rRNA haplotypes (MT333469–70) and two 28S rRNA haplotypes (MT333462–3) and single ITS-1 haplotype (MT333478).

Phenotypic differential diagnosis

The new species belongs 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 μm in the new species vs. 76–95 μm in B. arenosus) and dorsal plate sculpturing (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 (VII: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 facets formed by the raised plate areas between two longitudinal 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)- 2/4-2-2/4-2-2-2-1 in B. instabilis), the absence of dentate collar IV (present in B. instabilis), the detectability of papilla IV 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 , by the ventral plate formula (VII:4-2-2-4-2-2-1 in the new species vs. VIII:4-1-1-2-2-2-2-2 in B. olszanowskii), the presence of dark epicuticular granules on the dorsum (absent in B. olszanowskii) and by the detectability of papilla IV 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, Gąsiorek, Vončina & Michalczyk, sp. nov.

http://zoobank.org/48DA4500-2806-491E-8AD8-D2C830AF39F4
Figures 7, 8, 9, 10, 11, 12, Tables 5, 6

Locus typicus and type material

39°57'00"N, 3°10'50"E, 160 m a.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) deposited in the Department of Zoology, Comenius University 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 Biomedical 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 Research, Jagiellonian University. Eight specimens used for DNA extraction. Bryodelphax nigripunctatus sp. nov. was not accompanied by other species in the sample.

Table 5.
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Measurements [in µm] 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 scapular plate expressed as a percentage).

Character N Range Mean SD Holotype
µm sp µm sp µm sp µm sp
Body length 15 114 – 144 562 – 689 126 645 9 38 131 657
Scapular plate length 15 18.0 – 21.7 – 19.6 – 1.1 – 19.9 –
Head appendages lengths
Cirrus internus 11 6.1 – 8.9 32.9 – 42.8 7.3 37.4 0.8 3.3 7.5 37.4
Cephalic papilla 14 2.6 – 3.7 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 – 3.1 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 7.2 – 12.5 1.8 9.5 0.3 2.0 1.5 7.6
Number of teeth on the collar 10 2 – 5 – 3.3 – 0.9 – 2 –
Claw heights
Claw I 12 7.0 – 8.3 35.1 – 44.4 7.8 39.8 0.5 3.1 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 3.1 7.6 38.1
Claw IV 9 7.4 – 8.9 38.1 – 48.0 8.4 43.0 0.5 3.5 ? ?
Table 6.
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Measurements [in µm] 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 scapular plate expressed as a percentage).

Character N Range Mean SD Allotype
µm sp µm sp µm sp µm sp
Body length 15 106 – 134 618 – 725 123 685 8 30 130 704
Scapular plate length 15 16.1 – 19.5 – 17.9 – 1.1 – 18.5 –
Head appendages lengths
Cirrus internus 13 6.3 – 9.9 35.8 – 51.7 7.7 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 3.9 5.2 28.1
Cirrus externus 13 12.3 – 15.9 70.6 – 85.8 14.2 78.6 1.0 4.3 13.9 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 29.3 163.8 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 1.7 – 2.4 9.7 – 12.8 2.0 11.4 0.3 1.0 2.0 10.9
Number of teeth on the collar 12 3 – 5 – 3.9 – 1.0 – 5 –
Claw heights
Claw I 11 7.3 – 8.7 37.6 – 50.3 8.1 45.1 0.5 3.4 8.3 44.8
Claw II 10 7.1 – 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 ? ?

Etymology

From Latin niger = black + punctum = dot, spot. The name underlines evident epicuticular granules appearing dark in PCM and contrasting with other elements of dorsal sculpture. Adjective in the nominative singular.

Adults

Body translucent without distinct colour and usually stout in females and more slender in males (Figs 7, 8), bs = 43.0–49.2% (x̅ = 45.6%, N = 8) in females and 37.5–44.7% (x̅ = 39.8%, N = 10) in males. Eyes absent or not visible after mounting in Hoyer’s medium. Cephalic papillae and clavae elliptical with rounded apex. Cephalic papilla is relatively broader in males than in females. Both cephalic papillae and primary clavae 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 (Tables 5, 6). Unappendaged. Cuticular sculpture consists of large epicuticular granules, true pores and intra-cuticular pillars (Fig. 10). In PCM, these structures appear, respectively, as conspicuous large dark spots, smaller bright spots and fine dark and dense punctuation (pseudogranulation). Epicuticular granules of irregular shape and size (up to ca. 1.6 μm) 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 posterior median plate 1 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. Granules 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 scapular 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 anterior m1–2 plates into left and right portions (Figs 7A, B, 8, 9B). Scattered granules also irregularly cover the surface of the cephalic, paired, caudal, anterior m1–2 and anterior parts of scapular plates (Figs 7A, B, 8, 9, 10A). Round, focusable pores (0.3–0.4 μm in diameter) are unequally distributed on dorsal plates in spaces between 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 μm2, x̅ = 29, 14–28 pores/100 μm2, x̅ = 22 and 18–31pores/100 μm2, x̅ = 26, respectively, N = 15 in females and 7–35 pores/100 μm2, x̅ = 29, 0–34 pores/100 μm2, x̅ = 25 and 11–33 pores/100 μm2, x̅ = 25, respectively; N = 15 in males) and lowest density on caudal plate (16–27 pores/100 μm2, x̅ = 22 in females and 7–28 pores/100 μm2, x̅ = 20 in males; N = 15). On the scapular plate (in the area delimited with lateral longitudinal 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 pillars (0.1–0.2 μm in diameter) reinforce the entire cuticle (also under the granules), but they are well-visible only in the cephalic, scapular, both paired segmental, caudal, anterior median 1 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.

Figure 7. 

Habitus of adults of Bryodelphax nigripunctatus sp. nov. (PCM): A – female (holotype, dorsolateral view); B – male (allotype, 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 II, arrowhead indicates spur on internal claw). Scale bars: 50 µm.

Figure 8. 

Habitus of adult male of Bryodelphax nigripunctatus sp. nov. (paratype, SEM): A – dorsal view; B – lateral view. Scale bars: 50 µm.

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 µm.

Figure 10. 

Details of cuticular sculpturing of Bryodelphax nigripunctatus sp. nov.: A – microscope focused on the epicuticular granules (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 µm.

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 narrower anterior (ca. 1/3–2/5 of the plate length) and the wider posterior portions, trapezoidal anterior median plate 1 is subdivided just behind its anterior margin, pentagonal anterior m2 (the largest amongst the median plates) is divided 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 rounded 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 caudal plate. Anterior platelets of each pair have very distinctly-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 III: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 distal 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 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 sculptured with distinct intra-cuticular pillars and scattered pores and distally hemmed with dentate collar. The collar with sharp teeth, always longer than the width of their bases and with the distance between teeth similar 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 slender, 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).

Figure 11. 

Variability of the dentate collar IV in Bryodelphax nigripunctatus sp. nov. (paratypes, PCM). Scale bars: 10 µm.

Juveniles

In appearance as adults, but smaller (111–112 μm) and with ventral plates just marked with rows of granules. Selected measurements of a shorter specimen: cephalic papilla 3.1 μm, scapular plate 14.9 μm long, claws I–III 5.1–5.6 and claws IV 6.5 μm long.

Larvae

83–85 μm long. Dorsal plates (especially median ones) mostly with poorly-delineated margins, supplementary 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 dentate collar IV well developed. Ventral plates not visible in laterally orientated larvae. Claws with spurs formed as in adults. Some measurements of shorter specimen: cephalic papilla 2.5 μm, claws II–III 4.6– 5.3 and claws IV 6.4 μm 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. amphoterus and B. maculatus have a reduced number of ventral plate rows to two or three, as in the new species. Consecutively, 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. parthenogenesis in B. amphoterus), the presence of lateral supplementary platelets (absent in B. amphoterus), the presence of epicuticular granules on dorsal and ventral plates (absent in B. amphoterus), a different number of ventral plate rows in females (ventral plate formula III: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 species 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 μm 2 in segmental II plate in the new species females vs. 48–61 pores per 100 μm 2 in the same plate in B. maculatus), a relatively longer internal peribuccal cirrus (sp is 33–43% in females of the new species vs. 21–30% in B. maculatus) and by relatively longer claws II–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, KY609137 and MT333471) 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).

Discussion

Phylogeny and evolution of traits in Bryodelphax

Inter-generic tardigrade relationships are constantly being unravelled (Bertolani et al. 2014; Fujimoto et al. 2016; Gąsiorek 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 (Gąsiorek et al. 2018a), but, importantly, the current phylogenetic evidence is 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 Echiniscini, i.e. the presence of peribuccal cirrophores, is biased and unreliable – Bryodelphax has weakly outlined cirrophores 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-standing problem of Bryochoerus (Kristensen 1987; Lisi et al. 2017; Gąsiorek 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 genus, based on the presence (weglarskae 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 intermingled. 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. amphoterus, formerly affiliated within the parvulus group, which corroborates the supposition by Gąsiorek et al. (2017a) that these structures have been previously overlooked. Peculiarly, the reduction of ventral armature to plesiomorphic subcephalic and genital plate rows is known in Bryodelphax only in three Mediterranean species (B. amphoterus, B. maculatus and B. nigripunctatus sp. nov.).

Secondly, since the description of B. maculatus, dark epicuticular granules have received attention of taxonomists (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. olszanowskii). Although this trait was not described in many previous descriptions, our analysis indicated no granules in ten spp. (B. amphoterus, B. arenosus, B. asiaticus, B. australasiaticus sp. nov., B. brevidentatus, B. mateusi (Fontoura, 1982), B. meronensis, B. parvulus, B. tatrensis (Węglarska, 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 reproduction are scattered on the tree. Two of the three known dioecious Bryodelphax spp., B. instabilis and B. nigripunctatus sp. nov., are not directly related (Fig. 1). This pattern, that is parthenogenetic and dioecious taxa mixed on the tree, is consistent with recent data for Paramacrobiotus 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 reproduction (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 Doyère, 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 restricted 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 considerably 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 relationship with other congeners). Deeper nodes in the Western Palaearctic clade may result from: (a) longer divergence 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 2000). If the last possibility is excluded, then the observed pattern may mean that speciation could be more rapid in tropical tardigrades, as postulated by the tropical 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 is required for Bryodelphax spp. in order to test this hypothesis.

Taxonomic key to the genus Bryodelphax

Since the last key by Fontoura et al. (2008), the number of described Bryodelphax species almost doubled (from 15 spp. considered valid in 2008 to 25 spp. gathered in the present contribution). Moreover, the 2008 key has several inadequacies: (I) three members of the weglarskae group (B. iohannis, B. sinensis and B. weglarskae) 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 delimited from B. parvulus by the absence of supplementary lateral platelets, but these structures are present in both species (Gąsiorek 2018). Our analysis of B. amphoterus 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 identifying its members. Importantly, B. lijiangensis Yang, 2002 is designated as nomen dubium due to the insufficient description and the general habitus not conforming to the characteristics of the genus (trunk cirri in all lateral positions suggest its affinity to Echiniscus) and hence it is omitted from our key.

Figure 12. 

Schematic arrangement of the ventral plates in all members of the weglarskae group (species are arranged in order of the increasing reduction of the ventral armature). Known cases of ontogenetic variability and sexual dimorphism are depicted. Following species taken from Kaczmarek et al. (2012): B. aaseae, B. iohannis, B. parvuspolaris, B. sinensis and B. weglarskae; Gąsiorek et al. (2017a): B. maculatus; Gąsiorek & Degma (2018): B. instabilis.

Key

1 Ventral plates present (the weglarskae group) 2
– Ventral plates absent (the parvulus group) 13
2(1) Two (subcephalic and genital) or three rows of ventral plates 3
– At least four rows of ventral plates 5
3(2) Ventral plate formula II:2-2, dark epicuticular granules absent, external claws with minute spurs B. amphoterus (Durante Pasa & Maucci, 1975)
– Ventral plate formula different, dark epicuticular granules present, external claws spurless 4
4(3) Ventral plate formula III:2-2-1, entire venter and ventral plates covered with stripes of dark epicuticular granules, typical, short and stout Bryodelphax claws B. maculatus Gąsiorek et al., 2017
– Ventral plate formula II/III:2-2-(1), only ventral plates covered with dark epicuticular granules, long and slender, Pseudechiniscus-like claws B. nigripunctatus sp. nov.
5(2) Ventral plate rows composed of 1–2 plates each 6
– Ventral plate rows composed of at least three plates each (excluding the subcephalic row) 7
6(5) Ventral plate formula VIII:1-1-2-2-2-2-2-1, dentate collar IV present B. parvuspolaris Kaczmarek et al., 2012
– Ventral plate formula VII:2-2-2-2-2-2-1, dentate collar IV absent B. sinensis (Pilato, 1974)
7(5) Four plates in the subcephalic row 8
– Two or no plates in the subcephalic row 10
8(7) Eight rows of ventral plates, plates typically developed and with dark epicuticular granules B. olszanowskii Kaczmarek et al., 20181
– Seven rows of ventral plates, all plates faint and devoid of dark epicuticular granules 9
9(8) Ventral plate formula VII:4-4-2-4-2-2-1, dorsal plate margins uniformly thick and dark in PCM B. australasiaticus sp. nov.
– Ventral plate formula VII:4-2-2-4-2-2-1, dorsal plate margins with separated epicuticular granules observable as dark points in PCM B. decoratus sp. nov.
10(7) Ventral plate formula IX:2-2-5-2-4-2-2-2-1, cephalic cirri bifurcated at their tips B. weglarskae (Pilato, 1972)
– Ventral plate formula different, cephalic cirri with a single tip 11
11(10) Ventral plate formula VII/IX:(2)-(1)-2/4-2-2/4-2-2-2-1, dioecious B. instabilis Gąsiorek & Degma, 2018
– Ventral plate formula different, parthenogenetic 12
12(11) Ventral rows immediately before legs II and III, composed of 2 plates each B. iohannis Bertolani et al., 1996
– Ventral rows immediately before legs II and III composed of 4 plates each 12
12(11) Ventral plate formula X:2-1-4-4-2-4-2-1-2-1, cirrus A/body length ratio at least 24% B. aaseae Kristensen et al., 2010
– Ventral plate formula IX/X:2-(1)-4-4-2-4-2-1-2-1, cirrus A/body length ratio below 24% B. kristenseni Lisi et al., 2017
13(1) Dentate collar IV present 14
– Dentate collar IV absent 19
14(13) Supplementary lateral plates absent B. brevidentatus Kaczmarek et al., 2005
– Supplementary lateral plates present 15
15(14) Six supplementary lateral plates, more than ten teeth in the dentate collar B. alzirae (du Bois-Reymond Marcus, 1944)
– Twelve supplementary lateral plates, fewer than ten teeth in the dentate collar 16
16(15) Papilla IV visible under LCM 17
– Papilla IV not visible under LCM 18
17(16) Endocuticular pillars in the scapular and the caudal (terminal) plate almost of the same size, pores evident and densely distributed on the anterior and lateral portions of the plates B. atlantis Fontoura et al., 2008
– Endocuticular pillars in the scapular plate clearly smaller than pillars in the caudal (terminal) plate, pores evident only in the central portion of the paired plates B. meronensis Pilato et al., 2010
18(16) Teeth of the dentate collar long and acute B. tatrensis (Węglarska, 1959)
– Teeth of the dentate collar short and blunt B. mateusi (Fontoura, 1982)
19(13) Pores/pseudopores absent, endocuticular pillars scarcely visible only in the caudal (terminal) plate, large depressions (fossae) in poorly defined rows B. dominicanus (Schuster & Toftner, 1982)
– Pores/pseudopores present, endocuticular pillars visible on all dorsal plates, depressions (fossae) absent 20
20(19) Lateral portions of dorsal plates ornamented either with dark epicuticular granules or elevations B. arenosus Gąsiorek, 2018
– Lateral portions of dorsal plates not ornamented 21
21(20) Supplementary lateral plates absent, internal claws spurless B. ortholineatus (Bartoš, 1963)
– Supplementary lateral plates present, internal claws with spurs 22
22(21) Papilla IV visible under LCM B. crossotus Grigarick et al., 1983
– Papilla IV not visible under LCM 23
23(22) The largest endocuticular pillars only in the central portion of the scapular plate B. parvulus Thulin, 1928
– The largest endocuticular pillars in the central portions of the scapular and the caudal (terminal) plate and posterior portions of paired segmental plates B. asiaticus Kaczmarek & Michalczyk, 2004

Acknowledgements

Reinhardt M. Kristensen (University of Copenhagen), Roberto Guidetti (University of Modena and Reggio Emilia), Roberta Salmaso (Museum of Natural History of Verona) and Giovanni Pilato (University of Catania) are acknowledged for the loan of type material of some species. Alicja Witwicka, Artur Oczkowski and Łukasz Krzywański provided the moss samples or assisted in their collection. We are grateful to Sandra Claxton who consulted the morphology of individuals of B. australasiaticus sp. nov. from Australia and provided valuable microphotographs. Two reviewers, Matteo Vecchi and Łukasz Kaczmarek, kindly commented on the manuscript. The study and sampling were supported by the grant from the European Commission’s (FP6) Integrated Infrastructure Initiative programme SYNTHESYS (grant no. DK-TAF-6332 to PG), National Science Centre (2019/33/N/NZ8/02777 to PG, supervised by ŁM), and by the Polish Ministry of Science and Higher Education via the Diamond Grant (DI2015 014945 to PG, supervised by ŁM). Some of the analyses were carried out with the equipment purchased from the Sonata Bis programme of the Polish National Science Centre (grant no. 2016/22/E/NZ8/00417 to ŁM). This work was also supported by the Slovak Research and Development Agency under the contract No. APVV-15-0147 (to PD). We owe our sincere thanks to the Museum für Naturkunde, Berlin, for covering the publication charge. The Authors declare no conflict of interest.

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1 Kaczmarek et al. (2018) defined the ventral plate formula as follows: VIII:2-1-1-2-2-2-2-2, i.e. not acknowledging another pair of ventrolateral plates as part of row I. These plates are added to the formula in the present contribution, resulting in the overall number of plates equal to four in the first ventral row, i.e. VIII:4-1-1-2-2-2-2-2.
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