Redescription of type species of the genus Cytaea Keyserling, 1882 (Araneae: Salticidae) – an integrative approach

Abstract The jumping spider genus Cytaea is an iconic member of the Australo-Pacific region. The genus as recognized today is reportedly not monophyletic, most of the forty-two species described here in the 19th century lacking modern diagnoses and adequate illustrations. The genus clearly needs a throughout revision. To avoid future synonyms, species redescriptions based on integrative taxonomy are necessary. Several species of the genus Cytaea are studied here in terms of species taxonomy. Cytaea alburna Keyserling 1882, the type species, is re-described. We propose Cytaea severa (Thorell, 1881) and Cytaea barbatissima (Keyserling, 1881) are as junior synonyms of the new combination for Ascyltus asper (Karsch, 1878), Cytaea asper (Karsch, 1878) comb nov. as we find to belong to the genus as well. The integrative approach was used based on diagnostic morphological characters (presented as images and drawings), DNA barcodes and a barcode gap analysis which tested the species distinctness. Genitalic structures are redefined and discussed in terms of function. The conclusion for the 4 species were based on 22 specimens, 14 DNA barcodes, publication supplemented with 46 digital micrographs and 7 drawings.http://LSIDurn:lsid:zoobank.org:pub:001FB3A0-074E-4290-B2D8-AD246D629E93


Introduction
Spiders are one of the most successful groups of arthropods and an interesting model in biological studies (Mammola et al. 2017). So far, nearly 50 thousand species in 120 families have been described (WSC 2021), but recent estimates range from 76 to 170 thousand species (Agnarsson et al. 2013;Platnick & Raven 2013). A large part of species described in XIXth and early XXth centuries lack proper diagnoses, documentation, were placed in wrong genera, need verification and revision (Paquin & Dupérré 2009;Dimitrov & Hormiga 2010;Sudhin et al. 2016). Although studies of the type specimens are pivotal in revisional works, several hindering circumstances like the poor condition of the specimens, unknown depository, being destroyed or lost, could be managed (Arnedo 2003;Nentwig et al. 2020). Molecular markers also can help support species delimitation (Hebert et al. 2003). The DNA barcoding has variety of applications in spider research, while is apply for estimate country scale species diversity (Astrin et al. 2016;Coddington et al. 2016), identification species in tropical regions (Gaikwad et al. 2017;Tahir et al. 2019;Tyagi et al. 2019) in few cases problems arose (Ortiz et al. 2020). With over 6200 species and 646 genera (WSC 2021), salticids are the most diverse family of spiders. Still however, the actual numbers may be 2-3 times higher, especially in tropical regions (Żabka 1991). Almost half of the species have been described from single sex and 367 species still lack illustrations (Metzner 2021). The genus Cytaea Keyserling (1882) well illustrates these issues. It includes 42 nominal species (WSC 2021) from the Australian and Oriental Regions (Żabka 1991;Patoleta & Trębicki 2015). Many species described over a century ago lack clear diagnoses and documentation (Trębicki et al. 2016). The phylogenetic studies on Salticidae family indicate that genus in its present composition is not monophyletic and has to be revised (Zhang & Maddison 2013, 2015Maddison 2015). Some Cytaea species have already been studied by Prószyński (1976Prószyński ( , 1984, Davies and Żabka (1989), Żabka (1991), Berry et al. (1998), Patoleta and Gardzińska (2010), Deeleman-Reinhold (2010, 2013), Cao et al. (2016), Wang and Li (2020); however, the status of type species, of the genus and its closest relatives remains unclear. Cytaea alburna Keyserling 1882 is the generic type. In 1991 Żabka synonymized it with C. severa (Thorell 1881), while Prószyński (2017) reinstated the species. Here we revisit the problem based on type and new material for four nominal species: Cytaea alburna Keyserling 1882, Cytaea severa (Thorell 1881), Cytaea barbatissima (Keyserling 1881) and Ascyltus asper (Karsch 1878) -all being previously involved in the problem. The taxonomical decisions were supported by DNA barcoding analysis.
The goals of this study are as follows: (I) to redescribe the type species of the genus Cytaea and its relatives, using integrative methods, (II) to provide a new reliable documentation enabling species identification, (III) to propose new characters for the genus.

Morphological methods
The study was based on type and new specimens; the latter being collected during various biodiversity surveys in Australia and preserved in 75% or in 96% ethanol. The specimens used in taxonomic and phylogenetic studies belong to the following institutions: Zoologisches Institut und Zoologisches Museum, Universität Hamburg (ZMH), Museo Civico di Storia Naturale Giacomo Doria, Genoa (MCSN), Zoologisches Museum der Humboldt-Universität, Berlin (ZMB), Muséum National d'Histoire Naturelle, Paris (MNHN), Queensland Museum, Brisbane (QMB). Specimens were examined with Nikon SMZ800, SMZ1000 and Ci microscopes and photographed with Nikon D5100 digital camera. Pictures were digitally processed with HeliconFocus and Adobe Photoshop software. The drawings were made from digital photographs. Epigynes were cleared in 10% potassium hydroxide (KOH) and in methyl salicylate (C 8 H 8 O 3 ). All measurements are given in millimetres. Photographs of live spiders and fresh specimens were provided by Robert Whyte, through Queensland Museum Brisbane, Australia. Terminology follows Zhang and Maddison (2015). For further explanations see Figures 3-8. Abbreviation used: CL: cephalothorax length, CW: cephalothorax width, CH: cephalothorax height, AL: abdomen length, AW: abdomen width, AH: abdomen height, EFL: eye field length, AEW: anterior eye row width, PEW: posterior eye row width, DAM: diameter of anterior median eye. The embolic base and course have been described using clock position.

Molecular methods
Specimens used in phylogenetic analysis are listed in Table I. New sequence data were generated from 14 Cytaea specimens. Total genomic DNA was extracted from single leg of each specimen, using the DNeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) according to manufacturer's instructions. The COI gene fragment was amplified with bcdF01 and bcdR04 primers (Dabert et al. 2010). PCRs were conducted in 10 μL reaction volumes containing 5 μL of the Type-it Microsatellite PCR Kit (Qiagen), 0.5 μM of each primer and 4 μL of DNA template. Thermocycling profile was as follows: one cycle of 5 min at 95°C followed by 35 steps of 30 s each at 95°C, 60 s at 50°C, 1 min at 72°C, and with a final step of 5 min at 72°C.

TL124
Bellthorpe, QLD, Australia M --I). In addition, comparative pictures of specimens preserved in ethanol are attached to sequences deposited in BOLD.

Results
The final alignment for species delimitation comprised 644 nucleotide positions (nps) for COI gene sequences representing 14 specimens (Table I). The nucleotide sequences could be translated into amino acid sequences without any stop codons. In the dataset, 77 nps out of 644 were variable, and transition-totransversion ratio (R) amounted to 1.47. Neighbour-Joining (NJ) analysis clustered Cytaea COI sequences into two maximally supported clades ( Figure 1) grouping members of C. alburna and C. asper.
Genetic distance between C. alburna and C. asper COI sequences amounted to 12.13%. Mean intraspecific distances were 0.91% (SD = 0.27%) and 0.84% (SD = 0.33) for C. alburna and C. asper, respectively. Using ABGD method, we found a 3-10% barcoding gap between the species of C. alburna and C. asper ( Figure 2), supporting species distinctiveness. Closer examination of the type specimens of C. severa, C. barbatissima and Ascyltus asper has revealed their copulatory organs do not differ to put them into separate species; all are here transferred to Cytaea as Cytaea asper comb nov. The analysis (Figures 1, 2)

Female S103096
Cephalothorax dark orange, eye surroundings black. Whole surface covered with brown numerous setae and white scales, the latter form a belt along the lower margin (Figures 3(a-c), 4(a-f)). Eye field wider than long, its length 54% of CL. PME halfway between PLE and ALE. Fovea located between PLE (Figure 4(a,b)). Abdomen elongate, whitish, covered with sparse brown hairs and orange scales, the latter numerous on sides (Figure 4(a-d)). Spinnerets orange, not distinctive. Clypeus narrower (33%) than AME diameter, pale orange, covered with long, white scales (Figure 4(e)). Chelicerae orange, massive, elongate, inclined downwards, promargin with four teeth, retromargin with a single bicuspidate tooth (Figure 4(e,f)). Endites and labium orange, with pale chewing margins. Sternum yellowish (Figure 4(c)). Legs I orange, others lighter, all legs covered with numerous spines and setae (Figure 4(a-d)). Leg formula: I-IV-III-II. Epigyne with two circular windows separated by narrow median septum (Figure 5(a-c)) and with sclerotized lateral atrial margins. Copulatory openings located posteriorly and oriented laterally (Figure 5(a-c)). Copulatory ducts short. Spermathecae channel-like, long and twisted, with 2 accessory glands: one located near the copulatory duct entry and another close to the fertilization duct ( Figure 5(a-c)).

Male S103079
Cephalothorax orange, eye surroundings dark. Whole surface covered with numerous setae and black scales, the latter form two longitudinal thoracic stripes (Figures 3(d-i), 4(g,i)). White scales present on eye field and on sides (Figures 3(d-i), 4(g,i)). Eye field wider than long, narrowing posteriorly (Figure 4(g)), its length 54% of CL. Fovea short, located between PLE. Abdomen elongate, pale, with dark orange and brown scales on sides, covered with dark brown setae (Figures 3(d-i), 4(g,i)). Spinnerets orange. Clypeus light orange, narrower (44%) than AME diameter (Figures 3(d,g), 4(j-k)), covered with long whitish scales. Chelicerae vertical, elongate, orange with black spots and transverse stripes of white scales at the base (Figure 4(k)). Promargin with three teeth, retromargin with a single bicuspidate tooth. Endites and labium slender, not distinctive, orange, with lighter chewing margins. Sternum longer than wide, pale (Figure 4 (h)). Venter dark brown, pale on sides. All legs covered by numerous spines and setae (Figure 4(g-j)). Proximal parts of tibiae pale, the rest of legs pale covered with red shimmering scales. Leg formula: I-III-II-IV.

Diagnosis
C. asper is grater in size than C. alburna, covered with metallic shimmering scales and is dull in colour (Figures 6-7) vs. red, in alburna (Figures 3-4). Also, the first pair of legs longer in C. severa. Palpal tibia twice longer than in C. alburna, embolus shorter by 0.5 loop length, spermatecae differ by course of ducts (Figure 8(a-g)).

Female S103029
Cephalothorax dark brown, eye surroundings black. Whole surface covered with numerous setae and white scales, the later more numerous along the central part and on sides (Figures 6(a), 7(a-d)). Eye field wider than long, its length 54% of CL. PME halfway between PLE and ALE. Fovea located between PLE (Figure 7b). Abdomen elongate, dark brown, covered with brown setae and white scales (Figure 7a-b. Spinnerets brown. Clypeus brown, covered with long, white scales, narrower (36%) than AME diameter. Chelicerae brown, elongate, inclined downward (Figure 6a, 7e). Promargin with three teeth, retromargin with bicuspidate tooth. Endites and labium dark brown, with pale chewing margins, sternum brown (Figure 7c). Ventral abdomen dark brown. Legs brown, covered with numerous spines and setae (Figure 7a-d). Leg formula: I-III-II-IV.
Epigyne with two circular windows separated by narrow median septum (Figure 8(a-c)) and sclerotized lateral atrial margins. Copulatory openings located posteriorly and oriented laterally (Figure 8  (a-c)). Copulatory ducts short. Spermathecae long and twisted, channel-like, with two accessory glands; one located near the copulatory duct, second close to fertilization duct (Figure 8(a-c)).

Discussion
The nominal type species of the genus Cytaea has been redescribed and 8 COI sequences have been obtained for molecular characterization of the species. Integrated morphological and molecular data allowed us to distinguish several species of the genus Cytaea and to propose 3 synonyms. Cytaea alburna,

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Ł. Trębicki et al. type species of the genus is well separated by morphology and DNA barcodes. Ascyltus asper, Cytaea severa and C. barbatissima were transferred and synonymized as Cytaea asper comb nov. Diagnostic illustration and DNA barcoding data proved species distinctiveness and clarified that Cytaea alburna is not the junior synonym of Cytaea severa (Żabka 1991;Prószyński 2017) -here proposed as Cytaea asper comb nov. Additionally, genitalic structures of studied species were redefined. To delimitate the boundaries between animal species, Hebert et al. (2003) proposed a cytochrome c oxidase subunit 1 (COI) segment of approximately 650 nucleotides and the COI gene fragment has proven to be a good and most widely used marker for animal species delimitation (Hebert et al. 2003;Ratnasingham and Hebert 2007; DeSalle and Goldstein 2019), including spiders (Barrett & Hebert 2005). To establish the boundaries between species, for most taxa, the genetic discrepancy of 3% proved sufficient (Sbordoni 2010). However, due to different rates of evolution, the value may differ between groups of animals and even closely related species. To support species redescription in Salticidae, DNA barcoding method was used by Yamasaki et al. (2018). The 28S gene was additionally sequenced to support the COI results (Vink et al. 2011;Macías-Hernández et al. 2020). The gene 16S was used to designate the species (Rezác et al. 2008;Marusik et al. 2018), while COI was also used in generic revisions (Richardson & Gunter 2012;Pekar et al. 2017). In our study, we applied DNA barcoding for studied species delimitation.
Result of Neighbor-joining and ABGD analysis support distinctiveness of C. alburna and C. asper. COI DNA barcoding revealed that the mean nearestneighbour distance based on species was 10 times higher than the mean intraspecific divergence in Canadian spiders (7.85% vs. 0.78%), and 7 times higher in Canadian Salticid spiders (7.57% vs. 1.18%) (Blagoev et al. 2016). Other studies on various arthropod taxa including spiders (Hebert et al. 2003;Barrett & Hebert 2005;Robinson et al. 2009) suggested that the interspecific divergence values of COI are usually greater than 2-3%, or the intraspecific divergence values of COI are usually less than 2-3%. Applying these thresholds showed that the distinctiveness of Cytaea alburna and C. asper comb nov. is strongly supported, it has to remembered though that some authors considered the DNA barcoding gap analyze as not conclusive (Puillandre et al. 2012) and suggest using morphological, distributional, environmental, microhabitat and behavioral data. In this research we analyze all available data, including DNA and morphology.
We redefined morphology of genitalic structures in studied species. Long channel-like spermathecae in Cytaea have been so far considered copulatory ducts (Zhang & Maddison 2015;Trębicki et al. 2016). This may be important defining the genus as a monophyletic group. Both Cytaea alburna and C. asper comb nov. have two spermathecal accessory glands, previously only the one nearby copulatory opening was known in those species. We discovered second accessory gland nearby fertilization duct. Accessory glands located in spider epigyne play a nutritive and protective function (desiccation and pathogens) for sperm cells stored in (Michalik & Uhl 2005). Female secretory products also have been suggested to control sperm activation. The accessory glands in Cytaea located nearby fertilization ducts may play similar role. Indentation at the embolus base correspond with anterior lateral atrium in epigyne window are probably part of lock and key mechanismwell known in many arthropods and plays a role of effective reproductive barrier (Masly 2012). All those aspects suggest need for future study on biology of C. alburna, C. asper comb nov. as well as on other species in the genus.