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Corresponding author: Ivan L. F. Magalhaes ( ivanlfmagalhaes@yahoo.com.br ) Academic editor: Lorenzo Prendini
© 2022 Ivan L. F. Magalhaes, Abel Pérez-González, Facundo M. Labarque, Martín Carboni, Jörg U. Hammel, Robin Kunz, Martín J. Ramírez, Mónica M. Solórzano-Kraemer.
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Recluse or violin spiders in the genus Loxosceles (Scytodoidea: Sicariidae) are a diverse group (~140 extant species) including medically important species and distributed mainly in the Americas, Africa, and the Mediterranean region. In addition, this genus includes three fossil species from Miocene Dominican amber. Here we revise the taxonomy of these fossil species by examining, imaging and re-describing their type specimens. We find that L. defecta Wunderlich, 1988 and L. deformis Wunderlich, 1988 are bona fide members of the genus and report additional characters overlooked in their original descriptions. We further study the holotype of L. aculicaput Wunderlich, 2004 using synchrotron radiation micro-computed tomography to reveal previously unknown morphological details hidden by fissures in the amber. We found several characters inconsistent with Loxosceles but consistent with Drymusa (false violin spiders; Scytodoidea: Drymusidae), such as three claws, well-developed podotarsite, and a broad colulus. This suggests the species is misplaced in Loxosceles. To test this hypothesis, we estimated a total-evidence phylogeny of the superfamily Scytodoidea including extant and fossil taxa, morphological data, traditional molecular markers, and sequences of ultra-conserved elements. The results show unambiguously that L. aculicaput belongs to Drymusa and is a close relative of extant species of the genus inhabiting the Greater Antilles. Therefore, we here transfer this species to Drymusa, establishing a new combination and new family assignment. Drymusa aculicaput comb. nov. represents the first known fossil Drymusidae and shows that crown members of this genus already existed in the Miocene.
Drymusa, micro-CT, UCE, Miocene
Spiders are a speciose clade of predators containing ~50000 species (
Miocene amber from the Dominican Republic is the major source of spider fossils in the Neotropical region. Around 170 currently valid species have been named from this deposit (
Dominican amber yields the only fossils known for a few spider families, such as Sicariidae. Sicariids include six-eyed sand spiders (Sicarius Walckenaer, 1847 and Hexophthalma Karsch, 1879) and recluse or violin spiders (Loxosceles Heineken et Lowe, 1832). The family belongs to Synspermiata, a major spider clade that is well-represented in Cretaceous amber (
During a preliminary examination of the holotype of L. aculicaput, we found that some of its characters are inconsistent with a placement in Loxosceles, such as the presence of three tarsal claws and a well-delimited podotarsite (the genus has only two tarsal claws and a poorly delimited podotarsite; see
Recently, systematic advances in spiders have relied heavily upon genomic-scale molecular data (e.g.,
The aims of this contribution are: (1) to revise the taxonomy of Dominican amber Loxosceles, re-illustrate the type specimens and re-assess their morphology; and (2) to test the phylogenetic placement of the fossils using a dataset containing morphological and sequence data for representatives of all extant Scytodoidea families.
Examined specimens, fossil or extant, are housed in the following collections (curators in parentheses):
IBSP – Instituto Butantan, São Paulo, Brazil (A. D. Brescovit);
Amber pieces were re-polished at the SMF using a Phonix Beta polishing machine with grinding paper for metallography, wet and dry: Grip 2500 and 4000. After polishing, pieces were embedded in Araldite 2020® Epoxy resin following
The following specimens have been examined for the new scorings of the morphological matrix, or to discuss the morphology of scytodoids. We scored Ochyrocera diablo Pérez-González, Rubio et Ramírez, 2016 using the data from the original description (
Althepus maechamensis Li et Li, 2018 (Psilodercidae): THAILAND • 1 ♂ 1 ♀; Chiang Mai, Doi Inthanon National Park; 18.53°N 98.5025°E; 6 Oct 2003; ATOL Expedition 2003 leg.;
Drymusa spectata Alayón, 1981 (Drymusidae): CUBA • 1 ♂ 1 immature; Cienfuegos; Comanaqua (Cumanayagua); cueva del Canto; 750 m a.s.l.; 21.8930°N 80.1486°W; 20 August 2002; J. M. Ramos leg.; MNHNCu.
Drymusa armasi Alayón, 1981 (Drymusidae): CUBA • 1 ♂ 1 ♀; Santiago de Cuba, Gran Piedra; under rocks; 1100 m a.s.l.; 20.083333°N 75.623611°W; 21 June 1982; L. F. Armas leg.; MNHNCu.
Drymusa simoni Bryant, 1948 (Drymusidae): HAITI • 1 ♂ (holotype) 1 ♀; Nord-Ouest, LaHotte; 16–17 Oct 1934; P. J. Darlington leg.;
Loxosceles caribbaea Gertsch, 1958 (Sicariidae): CUBA • 2 ♂ 4 ♀; Santiago de Cuba, Reserva Ecológica Siboney-Jutici, Cueva La Virgen; 19.96083°N 75.7144°W; 04 May 2010; A. Pérez González leg.;
Loxosceles cubana Gertsch, 1958 (Sicariidae): CUBA • 2 ♂ 2 ♀; Pinar del Río; Viñales; Cueva del Cable; 22.6677°N 83.7094°W; A. Sánchez leg.; 20 April 2012; IBSP 164702.
Loxosceles deserta Gertsch, 1973 (Sicariidae): USA • 1 ♂ 1 ♀; Arizona, Tucson; F. E. Russell leg.;
Loxosceles hirsuta Mello-Leitão, 1931 (Sicariidae): ARGENTINA • 1 ♂; San Luis, Junín, Establecimiento La Clotilde; 32.19955°S 65.59549°W; 10 January 2017; M. Ramírez et J. Faivovich leg.;
Loxosceles laeta (Nicolet, 1849) (Sicariidae): BRAZIL• 3 ♂ 3 ♀; São Paulo, Osasco; 23.5325°S 46.7916°W; 26 Jul. 1982; J. R. Bettinazzi leg.; IBSP 34948.
Loxosceles rufescens (Dufour, 1820) (Sicariidae): ISRAEL • 1 ♂ 1 ♀; HaZafon, near Elon, Nahal Bezet Nature Reserve; 33.0740°N 35.2379°E; 17 February 2020; I. L. F. Magalhaes, E. Gavish-Regev, Z. Ganem, S. Aharon, N. Givon et M. Arnedo leg.;
Loxosceles simillima Lawrence, 1927 (Sicariidae): NAMIBIA • 2 ♂ 2 ♀; Waterberg; 20.352701°S 17.337579°E; M. Stockmann leg.;
Loxosceles taino Gertsch et Ennik, 1983 (Sicariidae): DOMINICAN REPUBLIC • 1 ♂ 2 ♀; Pedernales; no collecting date; A. Sánchez leg.; IBSP 164710.
The photographs and Z-stacks images of fossil species were taken under a Nikon SMZ25 microscope, using Nikon SHR Plan Apo 0.5× and SHR Plan Apo 2× objectives with a microscope camera Nikon DS-Ri2 and the NIS-Element software (version 4.51.00; www.microscope.healthcare.nikon.com). In some cases, to prevent diffraction caused by irregular surfaces of the amber piece, we placed a coverslip and a drop of water with sugar on top of the piece to flatten the surface to be photographed.
Morphological observations on extant specimens were made using Leica M165 C and Leica M125 stereomicroscopes. Pictures were taken with Nikon DXM1200 digital camera mounted on a stereoscopic microscope Nikon SMZ1500 and on a microscope Leica DM4000 M, and with a Leica DFC 500 digital camera mounted on a stereoscopic microscope Leica M165 C or Leica M216. Extended focal range images were composed with the Leica Application Suite version 3.6.0. or Helicon Focus 3.10.3–4.62 (https://www.heliconsoft.com, Ukraine). Preparations were carefully cleaned using fine brushes and a thin jet of alcohol from a thinned pipette; some setae were removed to expose structures, especially those on legs, palps, spinnerets, and chelicerae.
For scanning electron microscope (SEM), all preparations were dehydrated in a series of increasing concentrations of ethanol (80%, 90%, 95%, 100%), and critical-point dried. After drying and brushing, they were mounted on adhesive copper tape (Electron Microscopy Sciences, EMS 77802) affixed to a stub and secured with a conductive paint of colloidal graphite on isopropyl alcohol base (EMS 12660). Prior to SEM examination under a high vacuum with a FEI XL30 TMP or a LEO 1450VP, the structures were sputter-coated with Au-Pd.
The imaging of the holotype of L. aculicaput was performed at the Imaging Beamline – IBL P05 - PETRA III at Deutsches Elektronen Synchrotron (DESY) in Hamburg, operated by the Helmholtz-Zentrum Hereon (
We carried out the segmentation and volume rendering of the pedipalps in AMIRA5.4.5 (FEI Visualization Science Group, Burlington, MA, USA). All images were post-processed to adjust contrast and sharpness using Photoshop CS6 (Adobe Inc., San Jose, CA, USA).
We augmented the morphological matrix of Scytodoidea families (
We added the following characters to accommodate the morphological diversity brought by newly incorporated taxa:
(102) Chelicerae promarginal lobe, shape: (0) small and rounded, (1) large and detached, (2) with a distal prolongation (Fig.
(103) Chelicerae medial lamina, apex shape: (0) simple, (1) bifid (Fig.
Loxosceles defecta Wunderlich, 1988, holotype (
(104) Colulus, shape: (0) lobe-like, (1) broad, plate-like (Fig.
(105) Male, leg I, femur, prolateral macrosetae: (0) absent, (1) present (Fig.
(106) Labium, apex, shape: (0) straight, (1) pointed, (2) notched (Fig.
(107) Podotarsite, divisions: (0) single unit, (1) subdivided by additional articulation (see
(108) Podotarsite, cuticle on dorsal side: (0) membranous, open podotarsite; (1) sclerotized, closed podotarsite.
(109) Podotarsite, distal dorsal hood: (0) absent, (1) present.
(110) Chelicerae, promarginal lobe, distal prolongation, shape: (0) curved, rounded; (1) straight, pointed (Fig.
We added legacy genetic data from six target-gene markers from the mitochondrial (12S rRNA, 16S rRNA, COI) and nuclear (histone H3, 18S rRNA, 28S rRNA) genomes, and genomic ultra-conserved elements (Table 1). To maximize morphological and genetic data overlap, we merged data from closely related species into a single terminal in the outgroup genera Stedocys, Ariadna, Althepus, and Ochyrocera (Table 1). The alignment of target-gene markers was made with the online version of MAFFT (Katoh et al. 2019) with the L-INS-I option, which resulted in a total length of 6458 sites (12S 333 sites; 16S 469; 18S 1746; 28S 2348; COI 1235; H3 327).
Published UCE and transcriptome sequences were downloaded from the Sequence Read Archives using fastq-dump v. 2.11 (SRA Toolkit Development Team: https://hpc.nih.gov/apps/sratoolkit.html) with the flags –gzip –clip –split-files –qual-filter to remove adapters and trim low-quality base calls. Sequence reads were trimmed using TRIMMOMATIC v. 0.39 (
The phylogenetic analysis under maximum likelihood was done with IQ-TREE v. 2.1.3 (
ALS = anterior lateral spinneret, Bu = bulb, Ch = chelicera, Co = colulus, Cy = cymbium, CF = cheliceral fang, e = embolus, Fe = femur, Pa = patella, PLS = posterior lateral spinneret, PMS = posterior median spinneret, SF = stridulatory file, Ti = tibia.
The supplementary figures and data underpinning the analyses reported in this paper are deposited in the Zenodo repository at https://zenodo.org/record/6954956 under www.doi.org/10.5281/zenodo.6954956.
We present new photographs under light microscopy of the holotypes of Loxosceles defecta (Fig.
Loxosceles deformis Wunderlich, 1988, holotype (
Drymusa aculicaput (Wunderlich, 2004) comb. nov., holotype (
After scanning the amber piece containing the holotype of L. aculicaput, we rendered a 3D reconstruction of the specimen (Figs
Drymusa aculicaput (Wunderlich, 2004) comb. nov., holotype (
Drymusa aculicaput (Wunderlich, 2004) comb. nov., holotype (
The target-gene and UCE datasets produced similar trees (Figs S1, S2), with higher support in the last case, as expected. The combination of all sequence data (Fig. S3) produced a tree with all the groups in agreement with the UCE analysis. The morphological dataset produced trees with the same resolution for Sicariidae, Scytodidae, Periegopidae, and Drymusidae, but discordant with the molecular data in the more basal splits, also differing between maximum likelihood (ML) and maximum parsimony (MP) analyses (Fig. S4). The ML and MP analyses of the complete dataset produced very similar results, only differing in three polytomies of the MP tree, that were resolved in the ML tree (Fig.
Relationships among Scytodoidea inferred under maximum likelihood using a combined dataset (morphology + target genes + ultraconserved elements). Taxa with “ch” in their names are chimeras formed by combining morphological data of a species with sequence data of a congener. Bootstrap percentages are above branches, apomorphies below branches (state 0 in regular font, state 1 in bold font, states 2–3 indicated below character number).
The total evidence analysis (Fig.
Sicariidae is supported by five unambiguous morphological apomorphies, only one of which is observable in the fossils (third tarsal claw absent; Fig.
Extant drymusids from the Greater Antilles: Drymusa spectata Alayón (A, D) Drymusa armasi Alayón (B, C, F), and Drymusa simoni Bryant (E, G–I). A: male habitus, ventral (MNHNCu 51). Arrow to femoral macrosetae. B: female, mouthparts, ventral (MNHNCu 28). Arrow to notch in labium. C: female (MNHNCu 28), cheliceral lobe, arrow to distal prolongation. D: left palp, prolateral (MNHNCu 51). E: holotype male, left palp, prolateral (
All three species are redescribed and re-diagnosed below. Based on the phylogenetic results, we propose the transfer of Loxosceles aculicaput from Sicariidae to Drymusa in Drymusidae, resulting in Drymusa aculicaput (Wunderlich, 2004) comb. nov. and new family assignment.
The phylogenetic results we obtained are consistent with those of the most recent phylogeny with a broad sampling of Synspermiata families (
We tested the position of Loxosceles defecta in the phylogeny of Scytodoidea and found it to be a true member of Loxosceles, supported in our analysis by the bifid cheliceral lamina (Fig.
We could not include Loxosceles deformis in the phylogeny, since the only known individual is poorly preserved and details of the legs, chelicerae, and spinnerets cannot be observed. However, the genitalia is similar to that of L. defecta, and it has the flattened, ribbon-shaped embolus that is common in the Loxosceles reclusa species group (see
We here transfer Loxosceles aculicaput to Drymusa. This species presents three tarsal claws (Fig.
Our phylogenetic and taxonomic revision of spider taxa preserved in Dominican amber and previously assigned to Loxosceles allows us to conclude:
(1) Loxosceles defecta and L. deformis are bona fide members of this genus, and L. defecta can be further placed in the L. reclusa species group based on the prolaterally expanded palpal cymbium; this species also presents an incrassate first tibia bearing macrosetae. Tibial macrosetae are also present in the extant species Loxosceles cubana, suggesting that L. defecta may be closely related to extant taxa from the Antilles.
(2) Loxosceles aculicaput presents three claws and shares a broad colulus, femoral macrosetae, and a notched labium with extant members of Drymusa inhabiting the Antilles, and thus was misplaced in Loxosceles; we here propose the new combination Drymusa aculicaput, representing the first known fossil of Drymusidae. This extends the stratigraphic range of crown Drymusa to the Miocene and indicates these spiders had already reached the islands by then.
[Family Sicariidae Keyserling, 1880]
Loxosceles citigrada Heineken et Lowe [junior synonym of Loxosceles rufescens (Dufour)] (extant, Mediterranean region to Middle East).
Loxosceles defecta Wunderlich, 1988: 69, figs 92–94.
Adult male holotype and adult male paratype in the same Dominican amber piece, holotype SMF Be 970a, paratype SMF Be 970b, deposited in SMF, examined. No other specimens are known.
Both spiders are preserved in the same reddish yellow amber piece. Both individuals are partly incomplete and preserved in moderate condition. In both spiders the abdomen is severely shrunk. The holotype has both palps (right palp slightly deformed, with a bubble trapped in the tibia), while the paratype has none. The holotype is missing right leg I, right leg IV and parts of right legs II and III; left leg I is detached from the body and preserved to the left of the animal. The paratype has all the legs on the right side but they are distinctly deformed and covered by emulsion; only part of femur IV remains in the left side. Syninclusions: a large leg of an entelegyne spider, a leaf, and a Collembola.
Loxosceles defecta can be diagnosed from the other Loxosceles in Dominican amber (L. deformis), as well as from extant Antillean species, by the sinuous, distally incrassate first tibia bearing macrosetae (Fig.
Male holotype (
Loxosceles deformis Wunderlich, 1988: 68, fig. 90.
Holotype adult male in Dominican amber, SMF Be 968a, deposited in SMF, examined. No other specimens are known.
The spider is incompletely preserved in a reddish yellow piece of amber. The abdomen is severely shriveled. The anterior portion of the prosoma is covered by emulsion. Only left legs I and III and right femur II are preserved. The left palp, as well as the right palpal tibia, are strongly deformed. Syninclusions: two small Lepidoptera, a nematoceran Diptera, an Acari, a Hymenoptera: Formicidae, another unidentified spider with fungi, and a Bryophyta.
Loxosceles deformis can be diagnosed from the Loxosceles in Dominican amber (L. defecta) by the unmodified first tibia (Fig.
Male holotype (
[Family Drymusidae Simon, 1893]
Drymusa nubila Simon (extant, Saint Vicent).
Loxosceles aculicaput Wunderlich, 2004: 703, fig. 10b–d, photo 36.
Holotype adult male in Dominican amber, deposited in
The spider is completely and well-preserved in an orange piece of amber. The abdomen is slightly shrunken and shriveled but no other structures are obviously deformed. The amber piece has large fissures in the portion ventral to the spider. The piece has been embedded in artificial resin. There are no syninclusions other than some pieces of unidentifiable plants and detritus.
Drymusa aculicaput comb. nov. can be distinguished from other scytodoids preserved in Dominican amber, as well as from extant congeners, by the palp with short, incrassate tibia and by the bulb with a thin, straight, needle-like embolus (Fig.
Male holotype (
The authors have declared that no competing interests exist.
We are indebted to curators and curatorial staff from the collections holding the material studied in this revision. We thank Alejandro Valdez Mondragón for sharing information on the morphology of members of the Loxosceles reclusa species group, Solanlly Carrero for sharing information on extant Drymusa from the Dominican Republic, and the late Norman Platnick for organizing a collecting expedion with APG in Cuba. We thank Guilherme Azevedo and Rodrigo Monjaraz-Ruedas, from San Diego State University, for help with processing genomic data. Earlier versions of the text benefited from comments by Michael Rix, Charles Griswold and the editors, Lorenzo Prendini and Klaus Klass. This research received support from the SYNTHESYS+ Project www.synthesys.info, which is financed by the European Commission via the H2020 Research Infrastructure programme (grant DE-TAF-1356 to ILFM). ILFM was supported by a CONICET postdoctoral fellowship. This study was financially supported by Fondo para la Investigación Científica y Tecnológica (PICT 2015-0283, PICT 2017-2689, PICT-2019-2745), Consejo Nacional de Investigaciones Científicas y Técnicas (PUE 098), and the German VolkswagenStiftung [Project N. 90946]. We acknowledge DESY (Hamburg, Germany), a member of the Helmholtz Association HGF, for the provision of experimental facilities. Parts of this research were carried out at PETRA III [BAG-20190010].
Figures S1–S5
Data type: .pdf
Explanation note: Fig. S1. Maximum likelihood tree estimated from target-gene molecular markers. — Fig. S2. Maximum likelihood tree estimated from ultraconserved elements. — Fig. S3. Maximum likelihood and maximum parsimony trees estimated from sequence data (target genes + ultraconserved elements). — Fig. S4. Maximum likelihood and maximum parsimony trees estimated from morphology. — Fig. S5. Maximum likelihood and maximum parsimony trees estimated from the total evidence dataset.
Morphological matrix
Data type: .tnt
Explanation note: Morphological matrix in TNT format (http://www.lillo.org.ar/phylogeny/tnt).
DNA alignments and partition file
Data type: .zip
Explanation note: DNA alignments and partition file for the maximum likelihood analysis.
Tree from the total evidence analysis
Data type: .nwk
Explanation note: Tree from the total evidence, maximum likelihood analysis in Newick format.