Bioacoustics of Enoplognatha bryjai

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 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 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 ).