Enoplognatha bryjai Řezáč, 2016

Řezáč, Milan, Řezáčová, Veronika & Heneberg, Petr, 2016, Enoplognatha bryjai new species, a bizzare cobweb spider of the Pannonian swamps (Araneae, Theridiidae), Zootaxa 4147 (1), pp. 92-96 : 92-96

Enoplognatha bryjai Řezáč new species

( Figure 1 , Table 1 )

Enoplognatha sp. Bryja et al. 2005: 32.

Enoplognatha caricis Bryja et al. 2005: 32 . Misidentification.

Types. Czechia: Holotype: Tvrdonice, nature reserve Stibůrkovská jezera, on Typha sp. growth in littoral zone, 48°44'60"N, 17°0'10"E, 29 May 2002, 1♂, leg. Vítězslav Bryja, coll. Národní muzeum Praha (specimen NMP P6A- 5949) . 4♂ and 15♀. Paratypes: Lednice-Nejdek, 48°49'18.5"N, 16°47'13.6"E, littoral zone, 17 July 1996, 2♀, leg. & coll. Vlastimil Růžička; Hlohovec, national nature reserve Lednické rybníky, Hlohovecký pond, 48°46'53"N, 16°45'39"E, water-fringing reed beds in littoral zone, 3 June 2003, 1♂ 1♀, leg. Josef Chytil, coll. Robert Bosmans; Hlohovec, Výtopa pond, 48°45'59"N, 16°44'19"E, water-fringing reed beds in littoral zone, 9 July 2003, 1♀, leg. Josef Chytil, coll. Národní muzeum Praha ; Lednice, national nature reserve Lednické rybníky, Prostřední rybník pond, 48°46'60"N, 16°47'25"E, on flooded Carex sp. growth in littoral zone, 3 August 2003, 2♀, leg. Vítězslav Bryja, coll. Národní muzeum Praha, 3 August 2003, 3♀, leg. & coll. Pavel Kasal, 8 August 2013, 3♀, leg. Tomáš Krejčí, coll. Milan Řezáč, 28 May 2014, 2♂ 2♀, leg. Milan Řezáč, Jan Dolanský & Vítězslav Bryja, coll. Národní muzeum Praha, 1♂ 1♀, leg. Jan Dolanský, coll. Východočeské muzeum Pardubice .

Etymology. Named after our friend Vítězslav Bryja, a Czech molecular biologist and enthusiastic arachnologist, who discovered this species.

Diagnosis. Enoplognatha bryjai n. sp. differs from all other European species by very prominent outgrowths on the basal cheliceral segments in males. It shares this character with E. monstrabilis from the Russian region of Lake Baikal ( Marusik & Logunov 2001). Enoplognatha bryjai n. sp. is distinguished from E. monstrabilis by the details in the morphology of its genitalia. In particular in Enoplognatha bryjai n. sp. the cymbium is more distally pointed, the median apophysis is thinner, with parallel margins, and the prolateral apical edge of the accessory apophysis is rounded. The distal edge of the epigynum is rounded, the copulatory ducts first slightly converge before they bend in arc to the sides towards the spermathecae, the spermathecae are closer to together. Among the European Enoplognatha spp., E. bryjai n. sp. is most similar to E. mordax , but differs, besides its heavily armored male chelicerae, also by its more pale colouration, relatively shorter embolus and median apophysis. In addition, the median apophysis anteriorly carries only one tooth, and the accessory apophysis is wider than in E. mordax . Concerning the epigynum, the copulatory ducts first run anteriad parallel to one another and then they bend in arc laterally towards the spermathecae. The lateral parts of the copulatory ducts do not exceed the extent of the lateral edges of the spermathecae. The spermathecae of E. bryjai n. sp. are higher than those of E. mordax .

Description. Male (holotype) ( Fig. 1 A). Total length 5.7. Carapace 2.4 long, 1.7 wide, light brown, with an indistinct dark longitudinal band, especially around the fovea. Located on the posterior edge of the carapace there are two areas with ca. 10 transversal stridulatory furrows with a deep petiolar groove between them. Sternum and labium light brown, darker than carapace. Chelicerae ( Fig. 1 C) light brown, with five large tooth-like outgrowths, two being anterior, two posterior (proximal one blunt and hirsute), fifth tooth close to cheliceral groove and largest, bearing one (right side) or two additional teeth. Cheliceral fang equipped with a one tooth on the basal half of its ventral side. Pedipalpal femur 1.1 long. Tibia and cymbium 0.5 and 0.6 long, respectively. Theridiid tegular apophysis ( Figs 1 D, 1E and 1H) thin, with parallel margins, rounded at its base, and anteriorly carries one tooth. Median apophysis terminally rounded, the conductor has parallel margins that are terminally pointed; embolus relatively long. Legs light brown. Femur I 2.9 long (the ratio femur I/carapace length is 1.3), and slightly swollen compared to the other femora. Tibia I 3.1 long (ratio tibia I/carapace length 1.3). Metatarsi I and II slightly curved, their ventral sides bearing a row of low sharp spines. Abdomen grey, with white spots. Lateral and posterior parts of the abdomen decorated with a brown, lobated folium, whose center gradually fading, the outer side has a white margin. Cardiac mark darker than adjacent regions, with a white margin; ventral side with a wide dark longitudinal stripe and a dark ring around the spinnerets. Above the petiolus there is a semicircular sclerotised rim with two stridulatory teeth.

Female paratype (from Lednice, national nature reserve Lednické rybníky, Prostřední rybník pond, 48°46'60"N, 16°47'25"E,) ( Fig. 1 B). Colour pattern resembling that of male, but darker. Total length 5.5 (5.5–6.3). Carapace 1.8 (1.8– 2.1) long, 1.5 (1.5–1.7) wide. Chelicerae unmodified, with two anterior teeth (the proximal one larger) and one posterior. Length ratios tibia I: carapace, and femur I: carapace are both 1.1. The epigynum ( Fig 1 F) with a small transverse oval pit, 0.06 wide. Lateral loops of the copulatory ducts visible through the integument as a pair of black spots. Epigynum as in Figs 1 F, 1G and 1I. Copulatory ducts relatively long, first curving outwards, then returning in a sharp angle to the median pit.

Analysis of DNA variability. Specimens used for the DNA isolation: E. bryjai n. sp.: Czechia, Lednice , Prostřední rybník pond, reed bed, 48°46'60"N, 16°47'25"E, 8 August 2013, 4♀, leg. Tomáš Krejčí, coll. Milan Řezáč; E. mordax : Czechia, Sedlec, national nature reserve Slanisko u Nesytu , salt marsh, 48°46'33"N, 16°41'56"E, 28 May 2014, 1♂ 1♀, leg. et coll. Milan Řezáč; E. latimana : Czechia, Koněprusy , 49°55'12"N, 14°03'36"E, 29 July 2011, 1♀, leg. Petr Dolejš, coll. Národní muzeum Praha ; Poland, województwo Mazowieckie, banks of the river Bug , 52°36'0"N, 21°42'0"E, 14 July 2010, 1♀, leg. Petr Dolejš, coll. Národní muzeum Praha .

The spiders stored in 96% ethanol were washed twice for 15 min using 1 ml of 10 mM Tris-HCl (pH 7.5) and 5 mM EDTA buffer. Following the wash, the DNA was extracted with the NucleoSpin Tissue XS kit ( Macherey Nagel , Düren , Germany) according to the manufacturer’s instructions. The extracted DNA was amplified using the primers targeting the following loci: 1) CO1: Fw ( LCO 1490 _t1 (Folmer tailed)): TGT AAA ACG ACG GCC AGT GGT CAA CAA ATC ATA AAG ATA TTG G; Rv (HCO2198_t1 (Folmer tailed)): CAG GAA ACA GCT ATG ACT AAA CTT CAG GGT GAC CAA AAA ATC A; 2) ITS2 and the flanking 5.8S rDNA and 28S rDNA: Fw (ITS5.8): GGG ACG ATG AAG AAC GCA GC; Rv (ITS4): TCC TCC GCT TAT TGA TAT GC; 3) 28S rDNA: Fw (D3B): TCG GAA GGA ACC AGC TAC TA; Rv (28Sc): GAA ACT GCT CAA AGG TAA ACG G; 4) 18S rDNA: Fw (18Sa): ATT AAA GTT GTT GCG GTT A; Rv (18Sb): GAG TCT CGT TCG TTA TCG GA. The obtained DNA fragments were purified using USB Exo-SAP-IT (Affymetrix, Santa Clara, CA), were subjected to bidirectional Sanger sequencing using ABI 3130 DNA Analyzer (Applied Biosystems, Foster City, CA) and edited in CLC Main Workbench 6. The resulting consensus DNA sequences were submitted to the GenBank database under the accession numbers KJ643251 - KJ643261 and KP223721 - KP223726 .

The obtained sequences of mitochondrial and nuclear DNA, and all the relevant publicly available sequences of Enoplognatha spp. revealed by NCBI Blast were analysed as described in Řezáč et al. (2014). As outgroups, were utilized the corresponding sequences of the other Theridiidae genera ( Latrodectus geometricus and Anelosimus studiosus ). For CO1, 384 positions were analysed, corresponding to the specimen GenBank ID GU682764.1, position 236–619. For the

ITS2 locus, 298 positions were analysed, corresponding to the specimen GenBank ID AF084942.1, position 583–867. For 28S rDNA, 645 positions were analysed, corresponding to the specimen GenBank ID GU338590.1, position 125– 762. For 18S rDNA, 194 positions were analysed, corresponding to the specimen GenBank ID GU338525.1, position 876–1069. Sequence alignment and phylogenetic analyses were performed in MEGA5. Maximum likelihood fits of 24 alternative nucleotide substitution models were performed. For each analysis the model with the lowest Bayesian information criterion score was chosen, and further used to construct a tree and calculate divergence rates. For the CO1 data, we used the Tamura-Nei model corrected for the non-uniformity of evolutionary rates among sites by using a discrete Gamma distribution. For the ITS2 and 28S rDNA loci, we used the Tamura 3-parameter model. For the 18S rDNA locus, we used the Jukes-Cantor model. Bootstrap procedure (1,000 replicates) was employed. Gene trees were inferred using the maximum likelihood method. Heuristic searches used a neighbor joining starting tree subsequently improved via nearest-neighbor-interchange. Estimates of inter- and intraspecific evolutionary divergence in Enoplognatha spp. were inferred using the maximum likelihood method. The number of base differences per site was by averaging over all sequence pairs between groups.

Sanger sequencing of mitochondrial (CO1) and nuclear DNA (ITS2, 5.8S, 18S, 28S rDNA) revealed that E. bryjai n. sp. can be easily distinguished from any other Enoplognatha species with sequences publicly available in GenBank or newly sequenced ( E. caricis , E. mordax , E. margarita , E. latimana , E. ovata and E. intrepida ). Whereas the sequencing data clearly dstinguishes E. bryjai n. sp. from other sampled species ( Tab. 1 ), the precise taxonomic position of E. bryjai n. sp. as well as of the other closely related species, such as E. mordax , requires further research because the data obtained by the analysis of the CO1 and 18S rDNA loci contradict each other ( Tab. 1 ). It is worth mentioning that E. latimana and E. ovata showed 100% sequence similarity in both the CO1 and the ITS2 ( Figs 1 K and 1L, Tab. 1 ) despite that the two species are clearly distinguished morphologically1.

Ecology. Spiders were collected from Phragmites australis , Carex sp. and Typha sp. growing in water in the littoral zone of lakes in the lowlands ( Fig. 1 J). The spiders mature in late May, males can be found till June, females up to August. Enoplognatha bryjai n. sp. was found in an area that had been known for the presence of naturally occurring brackish lakes and salt marshes until the middle of the 19th century (see, Břízová 2009). Therefore, E. bryjai n. sp. could represent a relic of these habitats, which are endangered across the whole Pannonian region (the species is considered to be critically endangered in Czechia - Řezáč et al. 2015). Notably, the two closest relatives, E. mordax and E. monstrabilis , occur in salt marshes ( E. monstrabilis in salt marshes with Acanthemum splendens grass stands, Marusik & Logunov 2001; E. mordax is found in salt marshes and on sandy coasts, Bosmans & Van Keer 1999).

Distribution. Several lakes (Lednické fishpond system, Stibůrkovská jezera oxbow lake) in southern Moravia (southeastern part of Czechia). This area lies within the northwestern part of the Pannonian region. The presence of this species is thus expected in adjacent Pannonian parts of Austria and perhaps also Slovakia and Hungary.

Comments. The material mentioned by Miller & Obrtel (1975: 274–275) under the name E. maritima (junior synonym of E. mordax ) (Sedlec, national nature reserve Slanisko u Nesytu, 48°46'35"N, 16°42'9"E, reed swamp, 1 specimen, 17 April–18 November 1969) might refer to E. bryjai n. sp. It was collected in the characteristic habitat of E. bryjai n. sp. Moreover, the site is only three kilometers from Výtopa pond, where the new species occurs. However, E. mordax occurs on a salt marsh (48°46'33N, 16°41'51"E) only 400 meters far from the site where Miller & Obrtel (1975) collected. This record was later reinterpreted as E. cf. caricis ( Buchar & Růžička 2002: 34) , probably erroneously considering the habitat. The material is not available in Miller's collection ( Buchar & Růžička 2002).

Figures

FIGURE 1. Enoplognatha bryjai new species. A, male. B, Female. C, Male chelicera, lateral view. D – E, Left palp. D, Ventral. E, Retrolateral. F – G, Vulva. F, Ventral. G, Dorsal. H, Left palp, ventral. I, Vulva, ventral view. J, Prostřední rybník fishpond, a paratype locality. K – M, Maximum likelihood phylogenetic trees of Enoplognatha spp. inferred from the sequences of CO 1 (K), ITS 2 (L), 28 S rDNA (M) loci. The evolutionary models were selected according to the results of maximum likelihood fits of 24 nucleotide substitution models. Abbreviations: MA, median apophysis; C, conductor; CD, copulatory duct; CO, copulatory opening; E, embolus; FD, fertilisation duct; TTA, theridiid tegular apophysis; S, spermatheca. Photos by Jan Dolanský (A), Tomáš Krejčí (J) and Milan Řezáč (B – G).

Tables

TABLE 1. Estimates of the pairwise evolutionary divergence in hitherto sequenced Enoplognatha spp. based on sequences of the CO 1, 18 S rDNA, 28 S rDNA and ITS 2 loci. Codon positions included were 1 st + 2 nd + 3 rd + Noncoding. All positions containing gaps and missing data were eliminated. The number of base substitutions per site from between sequences are shown.

CO1 locus:
Enoplognatha bryjai n. sp. KJ643255
Enoplognatha bryjai n. sp. KJ643256	0.003
Enoplognatha margarita GU338699	0.070	0.073
Enoplognatha mordax KP223723	0.077	0.082	0.090
Enoplognatha caricis AY231040	0.098	0.102	0.128	0.148
Enoplognatha latimana KP223722	0.119	0.124	0.079	0.125	0.159
Enoplognatha ovata GU682764	0.119	0.124	0.079	0.125	0.159	0.000
Enoplognatha intrepida GU684411	0.146	0.151	0.125	0.147	0.154	0.104	0.104
Latrodectus geometricus FJ607567	0.167	0.172	0.157	0.186	0.207	0.200	0.200	0.205
18S rDNA locus:
Enoplognatha bryjai n. sp. KP223724
Enoplognatha bryjai n. sp. KP223725	0.000
Enoplognatha caricis AY230909	0.010	0.010
Enoplognatha latimana KJ643260	0.016	0.016	0.005
Enoplognatha latimana KJ643261	0.016	0.016	0.005	0.000
Enoplognatha margarita GU338525	0.021	0.021	0.010	0.005	0.005
Enoplognatha mordax KP223726	0.021	0.021	0.010	0.016	0.016	0.021
Anelosimus studiosus EF050200	0.021	0.021	0.010	0.016	0.016	0.021	0.021
28S rDNA locus:
Enoplognatha bryjai n. sp. KJ643251
Enoplognatha bryjai n. sp. KJ643252	0.000
Enoplognatha bryjai n. sp. KJ643253	0.000	0.000
Enoplognatha bryjai n. sp. KJ643254	0.000	0.000	0.000
Enoplognatha margarita GU338590	0.061	0.061	0.061	0.061
Enoplognatha mordax KP223721	0.036	0.036	0.036	0.036	0.041
Latrodectus geometricus FJ607532	0.162	0.162	0.162	0.162	0.186	0.162
ITS2 locus:
Enoplognatha bryjai n. sp. KJ643257
Enoplognatha bryjai n. sp. KJ643258	0.0000
Enoplognatha latimana KJ643259	0.0563	0.0563
Enoplognatha ovata AF084942	0.0563	0.0563	0.0000
Anelosimus studiosus HM584879	0.2231	0.2231	0.2279	0.2279