Integrating museum collections and molecules reveals genus-level synonymy and new species in red devil spiders (Araneae, Dysderidae) from the Middle East and Central Asia

. This paper reviews little-known species of the dysderid spider genera Dysdera Latreille, 1804, and Dysderella Dunin, 1992 based on specimens collected in the Caucasus, Middle East, and Central Asia. After combining molecular phylogeny of five mitochondrial and three nuclear genes with morphological evidence, Dysderella is proposed as a junior synonym of Dysdera . In addition, three species are described as new to science: D . jaegeri Bellvert & Dimitrov sp. nov., D . naouelae Bellvert & Dimitrov sp. nov., and D . kourosh Bellvert, Zamani & Dimitrov sp. nov. Four combinations are proposed: Dysdera caspica Dunin, 1990 comb. rev., Dysdera transcaspica Dunin & Fet, 1985 comb. rev., Dysdera elburzica ( Zamani, Marusik & Szűts, 2023) comb. nov. and Dysdera sancticedri (Brignoli, 1978) comb. nov. (ex. Dasumia Thorell, 1875). Furthermore, we report a first record of D . festai Caporiacco, 1929 in Turkey and its male cheliceral polymorphism. Our results illustrate the deficiencies that undermine the current taxonomy of this genus. For example, many species are described based on only one or few specimens or limited locality data. The advancements in DNA sequencing technologies applied to museum specimens reduce the need for fieldwork collection and export of fresh specimens. This highlights the significance of museum collections for improving research in this field.


Introduction
Dysderidae C.L. Koch, 1837 is a large family of spiders, currently comprising 612 extant species in 25 genera, distributed in the West Palearctic (World Spider Catalog 2023).The taxonomy of Dysderidae remains problematic.Almost one-third of the genera are monotypic, many of which have not been even reported again since their original description, in some cases more than 100 years ago, and in at least one case the type specimen is immature (World Spider Catalog 2023).One-third of the species are known by only one sex.Some descriptions lack a proper diagnosis (see Dysderella Dunin, 1992 below) or are poorly illustrated -e.g., most of Eugène Simon's papers, who described over 100 dysderid species.The female vulva, which is the main source of specific diagnostic traits for females was not regularly documented until the 1960s by the pioneering works of Alicata (1964), over 150 years after the description of the first dysderid.With very few exceptions (e.g., Alicata 1964;Deeleman-Reinhold & Deeleman 1988), there has been no revisionary work conducted, and most studies describe either single or few species or are focused on restricted geographic areas (e.g., Řezáč et al. 2023).Similarly, molecular phylogenetic hypotheses are only available for some of the genera (e.g., Bidegaray-Batista & Arnedo 2011) and particular regions (e.g., Arnedo et al. 2001).
Most of these limitations are particularly acute for the species distributed in the easternmost regions of its range, such as Anatolia, the Levant, Caucasus, the Middle East and Central Asia.The genus Dysderella exemplifies this situation well.When describing the genus, Dunin (1992) did not provide any diagnosis.The original description only pointed out some similarities with Dysdera Latreille, 1804, but no specific differences were mentioned.Similarly, the two main eastern species groups of Dysdera -asiatica and aculeata -have never been properly diagnosed, as some authors already noted (Deeleman-Reinhold & Deeleman 1988;Dimitrov 2021).Many species have published non-valid diagnostic characters, poor illustrations, outdated descriptions, or their type specimens are lost.Accessing some type specimens kept in different collections in Russia and Turkey ranges from very difficult to impossible.Resolving taxonomic problems, due to unavailability of type material or missing information on one sex, requires the study of additional specimens.Similarly, expanding distribution ranges or gathering DNA sequence data for classification and testing evolutionary hypotheses relies on the availability of more specimens.Unfortunately, obtaining collection and exportation permits from certain countries can create obstacles for fieldwork.
Given the aforementioned limitations, museum collections remain the primary available source of material from these regions.It is essential to recognize that the use of museum specimens has certain weaknesses.The specimens currently held in museums were typically gathered several decades ago, rendering them unsuitable for sequencing using Sanger methodologies.However, advancements in sequencing technologies may greatly assist in resolving this issue (Raxworthy & Smith 2021).Moreover, they frequently lack important information about the specific locality, habitats, or even the collection date and collector.Despite these shortcomings, the study of museum collections can expose mistakes as well as inaccurate taxonomic (e.g., Scharff & Hormiga 2013;Holmgren et al. 2016;Ernst et al. 2022) and phylogenetic decisions (e.g., Twort et al. 2021), substantially enhancing our current knowledge of biodiversity.
In this study, we primarily examined museum specimens to revise several species of the dysderid subfamily Dysderinae from the Middle East and Central Asia.Based on morphological and molecular evidence, we synonymize a genus, describe three new species, propose four new combinations, and discover a new faunistic record (Fig. 1).

Material and methods
The studied material was primarily sourced from various museum collections and preserved in 70-75% ethanol.Specimens were examined using a LEICA MZ16A stereoscopic microscope equipped with a camera lucida.High-resolution digital images were captured using a LEICA DFC 450 digital camera and the Leica Application Suite ver.4.4 software program, and a FLIR digital camera equipped with a THORLABS C-mount CML15 lens and attached to a ZEISS Axio LAB.A1 microscope.Helicon Focus software program was used to stack the photographs.LAS ver.4.4 was used to take measurements from the scaled images, and both male palps and female vulvae were dissected to allow for further examination.The genitalia were enzymatically digested in a pancreatin and borax solution following the methodology of Alvarez-Padilla & Hormiga (2007) to remove membranous tissues and retain sclerotized parts.The description of the new species was recorded in DELTA format, as specified by Dallwitz (1980) and Dallwitz et al. (2020).The nomenclature of genitalic and copulatory structures follows Arnedo et al. (2000).Leg chaetotaxy was recorded according to the techniques utilized in previous studies that focused on the revision and description of species of Dysdera from the Canary Islands (Arnedo & Ribera 1996, 1997, 1999;Arnedo et al. 2007;Macías-Hernández et al. 2010).All measurements are in mm and coordinates in decimal format.
The sequences were edited, and the matrices were manipulated using Geneious Prime ver.2022.2.2 (www.geneious.com).Ribosomal genes were aligned using the automatic alignment algorithm G-INS-i as implemented in the online version of MAFFT ver.7 (Katoh & Standley 2013).COI, NAD1, and H3 were aligned using the translation align option with the cost matrix BLOSUM62.The single gene matrices were then concatenated into a single supermatrix using the program SequenceMatrix ver.1.7.8 (Vaidya et al. 2011).The supermatrix was analyzed under maximum likelihood, Bayesian inference, and parsimony.Maximum likelihood tree was inferred using the program IQTREE ver.2.1.2(Minh et al. 2020), which selected the best partition scheme and evolutionary models with MODELFINDER (Kalyaanamoorthy et al. 2017) and assessed nodal support with 1000 iterations of ultrafast bootstrap (Hoang et al. 2018).Bayesian inference was conducted with MrBayes ver.3.2.6 (Ronquist et al. 2012), and the best partition scheme and evolutionary models were estimated using the program Partition Finder ver.2.1.1 (Lanfear et al. 2017).The analysis was run for 10 million generations, sampling every 1000, with eight simultaneous Markov Chain Monte Carlo (MCMC) chains, a heating temperature of 0.15, and a relative initial burn-in of 25%.Support values were calculated as posterior probabilities.
Convergence of the chains, correct mixing, and the number of burn-in generations were monitored using Tracer ver.1.7 (Rambaut et al. 2018).Parsimony analysis of the concatenated matrix was conducted with the program TNT ver.1.5 (Goloboff & Catalano 2016).For parsimony analyses, gaps were recoded as absence / presence characters using the simple coding method proposed by Simmons & Ocheterena (2000) implemented in the computer program SeqState (Müller 2005).Following Soto et al. (2017), we use a combination of the 'New Technologies' search strategies in TNT, namely sectorial searches, tree fusing, drift and ratchet.Tree searches were driven to hit independently 10 times the optimal scoring, followed by tree bisection and reconnection (TBR) branch swapping.Support values were estimated by jack-knifing frequencies derived from 1000 resampled matrices using 15 random addition sequences, retaining 20 trees per replication, followed by TBR, and TBR collapsing to calculate the consensus.All trees were rooted assuming Caponiidae as the sister group to the remaining families (Michalik et al. 2019).The phylogenetic trees were edited using FIGTREE (http://tree.bio.ed.ac.uk/software/figtree/).

Molecular phylogeny
One specimen each of Dysderella caspica (Dunin, 1990) and Dysdera kourosh sp.nov.yielded DNA extractions of enough quality for subsequent target amplification.In addition, a specimen of Dysdera longirostris Doblika, 1853 was also sequenced for the target gene fragments to test the morphological affinities of D. caspica with the longirostris species group.A list of sequences generated and retrieved from GenBank along with accession numbers for the newly generated sequences are available in Supp.file 1.The final supermatrix included 166 taxa and 5711 characters.Results of the phylogenetic analyses under alternative inference methods are summarized in Fig. 2. The best trees with supports for maximum likelihood, Bayesian inference and parsimony are available in Supp.file 2. Preferred partition schemes and corresponding evolutionary models are available in Supp.file 3.All analyses agreed on including D. caspica within the genus Dysdera, as the sister of D. longirostris and related to other representatives of the longirostris species group.Surprisingly, D. kourosh was not recovered as closely related to other eastern species.Instead, all analyses agreed, with low support in parsimony, to place it within a clade exclusively including representatives from the westernmost part of the genus distribution range.One possible explanation could be that no other representative of the aculeata group was available for comparison.This illustrates well the importance of performing an integrative study of the genus east of the Balkans.

Taxonomy
Class Arachnida Cuvier, 1812 Order Araneae Clerck, 1757 Family Dysderidae C.L. Koch, 1837 Genus Dysdera Latreille, 1804Dysderella Dunin, 1992: 67 (type species: Dysdera transcaspica Dunin & Fet, 1985), syn.nov.Dunin (1992) described the genus Dysderella and transferred the species Dysdera caspica and Dysdera transcaspica Dunin & Fet, 1985 to it.He designated D. transcaspica as the type species of this genus.However, he did not explicitly transfer it as a new combination, nor did he provide diagnostic differences to distinguish Dysderella from Dysdera.Zamani et al. (2023a) diagnosed and redescribed the genus Dysderella and described one more species, namely D. elburzica.The diagnosis of the genus they proposed, in our opinion, is not correct.According to the authors, it differs from Dysdera by the smaller size (i.e., carapace < 2.1 mm vs > 4 mm) and the spineless legs I and II.We think, the size is not a reliable diagnostic character for Dysderidae.There are some small-sized species of Dysdera, like D. zonsteini Dimitrov, 2021 with size of the carapace 1.65 mm.The spineless legs I and II are typical for the Dysdera longirostris species group (Deeleman-Reinhold & Deeleman 1988) and this character does not separate the two genera either.The molecular analysis shows that D. caspica belongs to the longirostris group of Dysdera (sensu Deeleman-Reinhold & Deeleman 1988).We could not sequence D. transcaspica and D. elburzica, but due to their high morphological resemblance to D. caspica, they should also fall in this group.Additionally, the three species share morphological features with Dysdera, such as scopulae on the posterior metatarsi, claw tufts on all tarsi, and a notched anterior edge of the labium.They also share the spineless legs I and II with D. longirostris species group which further supports the results from the molecular analyses.Thus, we propose to transfer the three of them back to Dysdera and consider Dysderella as a junior synonym of Dysdera.Dunin, 1990 comb. rev. Figs 3-5 Dysdera caspica Dunin, 1990: 143, fig. 4.1-4.

Description
Male (Figs 3-5) Prosoma.1.88 long; maximum width 1.47; minimum width 0.95.Uniformly red; slightly wrinkled, anterior region smooth.Frontal border roughly round, ca ½ of carapace length; anterior lateral borders convergent; rounded at maximum dorsal width, posterior lateral borders rounded; posterior margin narrow, rounded.Eye diameters: AME 0.12; PLE 0.11; PME 0.10; AMEs on edge of frontal border, separated from one another by ca 1 diameter, touching PLEs; PMEs ca one-quarter of diameter apart, less than ¼ of PME diameter from PLEs.Labium trapezoid-shaped, its base wider than its distal part, borders slightly curved; longer than wide at base; semi-circular notch at tip.Sternum orange, frontally darker, becoming lighter posteriorly; smooth; uniformly covered with slender black setae.
Chelicerae. 0.84 long, ca ½ of carapace length in dorsal view; fang 0.80 long; paturon proximal dorsal and ventral side scantly covered with piligerous granulations.Cheliceral inner groove short, ca ⅓ of cheliceral length; armed with three teeth and lamina at base; B = M > D; D triangular, located roughly at centre of groove; B close to basal lamina; M close to B.
For diagnosis and description see Zamani et al. (2023a).

Etymology
This species is named after our colleague and reputed German arachnologist Peter Jäger.

Type material
Holotype SYRIA • 1 ♂; Homs Province, west of Homs, ruins and abandoned cemetery NW of the castle; 18 Mar.1973; R. Kinzelbach leg.; SMF.6-8) Prosoma.3.13 long; maximum width 2.32; minimum width 1.61.Reddish, uniformly; with small dark grains mainly in frontal part; frontally covered with small white setae.Frontal border roughly round, ca ½ of carapace length; anterior lateral borders parallel; rounded at maximum dorsal width, back lateral borders rounded; back margin narrow, bilobulated.Eye diameters: AME 0.17; PLE 0.15; PME 0.14; AMEs on edge of frontal border, separated from one another by ca ½ diameter, close to PLEs; PMEs very close to one another, ca ⅓ of PME diameter from PLEs.Labium trapezoid-shaped, base wider than distal part; as long as wide at base; semicircular groove at tip.Sternum orange-red, frontally darker, becoming lighter posteriorly; smooth; uniformly covered with slender black setae.

Description Male (Figs
Chelicerae. 1.13 long, ca ⅓ of carapace length in dorsal view; fang medium-sized, 0.88 long; paturon dorsal and ventral side completely covered with piligerous granulations.Cheliceral inner groove short, ca ⅓ of cheliceral length; armed with three teeth and lamina at base; B > M > D; D triangular, located roughly at centre of groove; B close to basal lamina; M close to B.

Female
Unknown.

Distribution
Known only from the type locality in Homs Province, central Syria.

Remarks
Dysdera jaegeri sp.nov.along with D. argaeica Nosek, 1905, D. sultani, D. galinae Dimitrov, 2018and D. yozgat Deeleman-Reinhold, 1988 form a complex of species within the diverse asiatica group that can be characterized by the long cylindrical T, entirely fused IS and ES, and the sclerotized processus-like lateral sheet.This complex is distributed in Turkey and Syria.Unfortunately, we were unable to sequence any of these species and their position in the Dysderinae tree remains unclear.

Etymology
The new species is named after Naouel El Jaarani Oualkadi, partner of the first author, for all her patience and support.
Vulva .DA wider than long, fused to VA (Fig. 13); DF wide in dorsal view.MF margins fused, sheet-like, well developed, and completely sclerotized.VA rectangular, transparent (Fig. 14); frontal region completely sclerotized; posterior region sclerotized except for most internal area; AVD absent.S attachment projected under VA; arms as long as DA (Fig. 12), distinctly curved; tips not projected; neck as wide as arms.Laterals of TB directed forward.

Etymology
The specific epithet is a noun in apposition after Cyrus the Great -Kourosh in Persian, which translates as Lord of the Sun -the founder of the first Persian empire.

Description
Male  Prosoma.2.77 long; maximum width 2.11; minimum width 1.37.Red, frontally darker, becoming lighter towards the back; smooth with some small black grains mainly at the front.Anterior border roughly round, about ½ of carapace length; anterior lateral borders convergent; rounded at maximum dorsal width, posterior lateral borders rounded; posterior margin narrow, straight.Eye diameters: AME 0.13; PLE 0.10; PME 0.10; AMEs on edge of the anterior border, separated from one another by about 1 diameter, close to PLEs; PMEs very close to one another, less than ¼ of PME diameter from PLEs.Labium trapezoidshaped, its base wider than its distal part, borders slightly curved; longer than wide at base; semicircular groove at the tip.Sternum orange, darkened on borders; very slightly wrinkled, mainly between legs and anterior border; uniformly covered in slender black setae.
Palp .T slightly shorter than ED; external distal border straight; internal sloped backward.ED the same T axis in lateral view, internal distal border not expanded.IS wider than ES, continuous to tip (Fig. 19).ED tip sloped towards back in lateral view.C present, long, slightly concave at the middle, seen from lateral view (Figs 18,20); distal border round, the posterior border with a small sclerotized apophysis, markedly expanded, perpendicular to ED. AC absent.LF absent.L well developed; external border sclerotized, not folded, with processus-like basal lateral apophysis.LA absent.F absent.AL present, very poorly developed; proximal border in posterior view fused with DH.P not fused to T; perpendicular to T in lateral view; lateral length from ⅖ to ½ of T width; ridge present, parallel to T; not expanded, upper margin smooth, distally ridge-like expanded, back margin slightly folded towards the internal side.

Distribution
Known only from the type locality in Fars Province, southern Iran.
Palp .T short and rounded, slightly longer than ED, bearing small knob on the anterior side (arrowed in Fig. 25); external distal border sloped forwards; internal sloped backward.ED not bent, same T axis in lateral view, internal distal border not expanded.ES wide markedly sclerotized, IS reduced to whip-like sclerotization (Fig. 24).ED tip reduced, only the sclerotized structures are visible.C absent.AC absent.L lateral margin distally projected, split into two spine-like apophyses (Fig. 24).L is a single well-developed apophysis, curved mesally.LA absent.F absent.AL absent.P absent (Figs 23,25); DH sloped, forming an angle of ca 135° to T in lateral view; lateral length ca ¼ of T width; ridge ca 45° to T in lateral view, not sclerotized; not expanded, upper margin smooth; not distally projected; back margin not folded.

Distribution
Known only from the type locality in the North Governorate, northern Lebanon.

Remarks
The complete absence of a posterior apophysis is a unique feature of this species.Because of the overall resemblance of the male palp to that of D. festai, it most likely belongs to the festai group.However, it differs from the other two species in this group by the elongated chelicerae.To determine its correct position in the phylogeny of Dysdera, it is necessary to collect fresh material and describe the female too.Caporiacco, 1929 Figs

Distribution
Known from Greece (Rhodes), and the new record in Turkey reported herein.

Remarks
The chelicerae of the male collected from Turkey are unmodified (Fig. 26) while all the male specimens from the type locality bear a distinct projection at the middle part of the prolateral margin of the paturon (Fig. 27).There are two possible explanations for this phenomenon: either it is due to trait polymorphism, or the population from mainland Anatolia belongs to a different species altogether.The presence of a tooth-like prolateral projection on the male chelicera has been reported in other, unrelated species of Dysdera such as Dysdera dentichelis Simon, 1882 from Lebanon or Dysdera mucronata Simon, 1911 from Morocco, and in those species it is considered a diagnostic trait.However, because of the identical male palps (Figs 28-30 vs 31-33) and the small sampling size (single male), here we tentatively assign the Turkish specimen to D. festai, pending more thorough analyses.

Discussion
The red devil spiders are mostly restricted to the Western Palearctic, a region with a long taxonomic tradition and arguably with the best-catalogued biodiversity (Coddington & Levi 1991).In fact, the first description of a dysderid, Harpactea hombergi (Scopoli, 1763), was contemporary to Linnaeus.Additionally, red devil spiders are conspicuous, easy to distinguish from other spiders, relatively abundant, and easy to collect.Despite this, the taxonomy of Dysderidae remains contentious and descriptions usually lack an assessment of the intraspecific variability of diagnostic features, usually because they are either based on single specimens or single localities.
The combination of morphological evidence with a quantitative analysis of the phylogenetic relationships based on molecular data, provides unequivocal evidence that the genus Dysderella nests within Dysdera, and hence should be considered as its junior synonym.Additionally, the former species of Dysderella are shown to be closely related to species within the longirostris group, as already suggested by Deeleman-Reinhold & Deeleman? (1988).Interestingly, species of the longirostris group are mostly restricted to the Eastern Mediterranean and the Caucasus, with the single exception of Dysdera ferghanica Dunin, 1985 from Kyrgyzstan.The species formerly included in Dysderella expand the range of the bulk of the group towards the east, effectively bridging the biogeographic gap within the group.Surprisingly, the phylogenetic analyses supported the placement of D. kourosh sp.nov.as a sister to species inhabiting the westernmost part of the distribution range of Dysdera.Judging from the morphology (e.g., distal segment of the bulb clearly longer than the proximal one, the well-developed crest, and the leg spination formula), the new species belongs to the aculeata group (sensu Deeleman-Reinhold & Deeleman 1988) which is mostly circumscribed to Central Asia.Unfortunately, the sparse species sampling precludes a quantitative biogeographic analysis of the genus.A more thorough sampling will be necessary to tackle this interesting biogeographic and evolutionary conundrum.
In conclusion, although undoubtedly increasing collecting efforts in poorly known and sparsely sampled regions will help to complete our understanding of the diversity of the red devil spiders, natural history collections across Europe hold large amounts of unsorted material from the western Palearctic.Brignoli's collection, now in the Museo Civico di Storia Naturale, Verona, keeps types of rare species from the Balkan Peninsula, Anatolia and Levantine region, many of which known only from the holotype (e.g., D. sancticedri comb.nov.).The collection of the Naturhistorisches Museum, Vienna, contains a lot of material from Turkey (incl.the Pontic Mountains), collected last century and still unidentified.In the present work, we have been able to prove that the species previously included in the genus Dysderella are nested inside Dysdera, the former being a junior synonymy, and describe three new species.Our study provides further evidence that the required information to tackle specific constraints in the family's classification might already exist within museum collections.

Fig. 1 .
Fig. 1.Map of the geographical distribution of the studied specimens.Blurred area represents the species with unknown exact locations.

Fig. 2 .
Fig. 2. Preferred maximum likelihood tree of Dysderoidea C.L. Koch, 1837.Species and clades are boxed according to taxonomic groupings or geographic regions.Node support results from analyses under alternative inference methods are summarized using pie charts on branches.Upperleft pie = Bayesian inference (BI; MrBayes), Upperright pie = Maximum Likelihood (ML; IQTree2), Lower pie = Parsimony (MP; TNT).Support levels are color coded as follows: black = node recovered with high support (PP > 0.95, ML ultrafast bootstrap support > 95, Parsimony jackknife > 80); gray = node recovered but support values below thresholds indicated before; white = node not recovered.GenBank accession numbers in Supp.file 1.
Palp (Figs 3-5).T markedly shorter than ED; external distal border straight; internal sloped backward.ED slightly bent in lateral view; internal distal border not expanded.IS and ES equally developed, fused basally; IS and ES continuous to tip, bent ventrally.ED tip straight in lateral view.C present, long; distal border rounded, smooth, slightly expanded, projected over ED external part.AC absent.LF absent.L poorly developed; external border sclerotized, not folded.LA absent.F present, distally curved.AL present, very poorly developed; proximal border in posterior view fused with DH.Base of P fused to T; slightly sloped upwards; lateral length from ⅓ to ⅖ of T width; ridge present, perpendicular to T; not expanded, upper margin smooth; not distally projected; posterior margin not folded.FemaleNot examined.SeeDunin, 1990.

Chelicerae. 1 .
36 long, about ⅖ of carapace length in dorsal view; fang medium-sized, 0.99 long; paturon dorsal and ventral side completely covered with piligerous granulations.Cheliceral inner groove short, about ⅓ of cheliceral length; armed with three teeth and lamina at base; B > D = M; D triangular, located roughly at the centre of the groove; B close to basal lamina; M close to B.
, and (4) the long chelicerae, about ⅗ of carapace length in dorsal view in D. sancticedri, vs significantly smaller in D. festai.AMEs on edge of the anterior border, separated from one another by about ¾ diameter, touching PLEs; PMEs very close to one another, about ⅓ of PME diameter from PLEs.Labium trapezoid-shaped, base wider than the distal part; as long as wide at base; triangular groove at the tip.Sternum dark orange, darkened on borders; very slightly wrinkled.Chelicerae.1.62long,about ⅗ of carapace length in dorsal view; fang 1.865 long; paturon smooth, with no granulations.Cheliceral inner groove not visible; armed with slender long setae at the base.