# Using morphological and molecular tools for the description of a new generalist-nematophagous species of Macrocheles (Acari: Mesostigmata: Macrochelidae)

Diana Rueda-Ramírez, Gerald W Krantz, Eric Palevsky, Liliane Ruess

► To cite this version:

Diana Rueda-Ramírez, Gerald W Krantz, Eric Palevsky, Liliane Ruess. Using morphological and molecular tools for the description of a new generalist-nematophagous species of Macrocheles (Acari: Mesostigmata: Macrochelidae). Acarologia, 2025, 65 (2), pp.417-447. 10.24349/wc1d-p9la. hal-05059215

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Acarologia

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# Using morphological and molecular tools for the description of a new generalist-nematophagous species of *Macrocheles* (Acari: Mesostigmata: Macrochelidae)

Diana Rueda-Ramírez IDª, Gerald W. Krantz IDᵇ, Eric Palevsky IDᶜ, Liliane Ruess IDª

## Original research

### ABSTRACT

As part of a project aimed at harnessing soil food webs for the biological control of root-knot nematodes, specimens of a new species of the gamasine mite genus *Macrocheles* were found in vegetable fields in Israel. Specimens of this new species were used to initiate laboratory colonies using several species of free-living nematodes and a root-knot nematode as food sources. Females, males, deutonymphs and protonymphs were taken from the colony and morphologically analysed after mounting on slides. Some females and males were also subjected to scanning electron microscopy. Furthermore, 18 female specimens underwent DNA extraction for genetic analysis. The newly discovered species is described and illustrated based on its morphological characteristics and on molecular data obtained from the Barcode of Life Database (BOLD). Finally, an update to the key to distinguish species within the *scutatus* complex is included.

**Keywords** glaber-group; scutatus-subgroup; scutatus morphology; DNA barcodes; edaphic mites

**Zoobank** http://zoobank.org/C8348491-7B0B-405E-A1F4-7358ED45CE75

### Introduction

*Macrocheles* Latreille, 1829 is the largest and most diverse genus of the gamasine mite family Macrochelidae, comprising more than 300 described species of the approximately 500 species presently recognized in the family (Azevedo *et al.* 2015; Beaulieu *et al.* 2011; Emberson 2010). The great preponderance of known *Macrocheles* species exploit temporary, often insular organic substrates such as animal or plant dung or the nests of small mammals, birds, or social insects, feeding largely on nematodes, enthytraeid worms and microarthropods that they encounter there. When their ephemeral habitat deteriorates or desiccates, most of these species utilize phoretic attachment of adult females to co-occurring flying insects to carry them to fresh substrates (Krantz 1998; Krantz 2009; Lindquist, Krantz and Walter 2009).

The great diversity displayed by *Macrocheles* has led to the recognition of several species groups in the genus, based largely on shared morphological traits. However, the classification of these groups remains poorly defined and incomplete. Among the first of these to be established was the *glaber* group, proposed by Filipponi and Pegazzano (1962) and including *M. glaber* (Mueller), *M. perglaber* Filipponi and Pegazzano, and *M. scutatus* (Berlese). Walter and Krantz

Received 12 November 2024

Accepted 24 April 2025

Published 05 May 2025

Corresponding author

Diana Rueda-Ramírez ID: ruedadia@hu-berlin.de

Academic editor

Faraji, Farid

https://doi.org/10.24349/wc1d-p9la

ISSN 0044-586X (print)

ISSN 2107-7207 (electronic)

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Rueda-Ramírez D. *et al.*

Licensed under
Creative Commons CC-BY 4.0

OPEN ACCESS

<footnote>ªInstitute of Biology, Ecology Group, Humboldt-Universität zu Berlin, Philippstraße 13, 10115 Berlin, Germany.</footnote>

<footnote>ᵇIntegrative Biology, Oregon State University, Corvallis, OR 97331, USA.</footnote>

<footnote>ᶜNewe Ya'ar Research Center, Agricultural Research Organization (ARO), Israel.</footnote>
(1986) further divided an expanded *glaber* group into subgroups, with the *scutatus* subgroup being distinguished by a reduction in overall size and a distinctive decrease in sclerotisation of the ventral shields. Later, Walter and Krantz (1992) recognized four species complexes in the *scutatus* subgroup, differentiating them on morphological and distributional grounds. The *scutatus* complex, with seven described species, is the largest of these assemblages.

Species of the *glaber* group, and particularly the *scutatus* subgroup, are known for their close association with scarabaeoid beetles and the dung they visit (Krantz 1983; Walter 1985; Walter and Krantz 1986). Members of the subgroup have received special attention as potential biological control agents against flies breeding in these habitats (Krantz 1983; Azevedo *et al.* 2015). Predation by *glaber*-group mites on free-living nematodes (FLN) is believed to promote mite fecundity in such environments (Rodriguez, Wade and Wells 1962; Filipponi and Dojmi di Delupis 1963; Ito 1973; Azevedo *et al.* 2017; Azevedo *et al.* 2018). Fertility enhancement of mites by nematodes being a part of their diet has been thought to promote more efficient levels of biological control (Rueda-Ramírez *et al.* 2023).

During the course of a German Research Foundation project (DFG Grant RU 780/20-1) aimed at promoting the indigenous soil food web (microorganisms, FLN, and predatory mites) for the biological control of root-knot nematodes (*Meloidogyne* spp.), specimens of a *scutatus* subgroup species of *Macrocheles* were frequently collected from nematode-infested vegetable field soil in Israel. Mite specimens from different collection sites were used to initiate laboratory colonies that were fed primarily on FLN species. The mites also were found to feed readily on *Meloidogyne incognita* (Kofoid & White, 1919) Chitwood, 1949 (Rueda-Ramírez *et al.* 2023), a serious nematode pest of diverse cultivated crops worldwide (Moens, Perry and Starr 2009).

Morphological examination of the mites from Israel revealed a strong resemblance to *M. scutatus* (Berlese 1904), but with discrete differences in female sternal shield ornamentation and in male ventral shield shape, leg armature, and cribral development. Herein, we propose and describe a new *Macrocheles scutatus*-subgroup species, *Macrocheles karkomensis* n. sp. Rueda-Ramírez & Krantz. The main objectives of this paper are to describe the new species, update the existing dichotomous key to the *scutatus* complex (Walter and Krantz 1992), and provide information on DNA barcoding of related taxa based on COI data.

## Material and methods

### Mite collection

Specimens used in this study were obtained from laboratory colonies maintained by the Ecology Group, Institute of Biology, Humboldt-Universität zu Berlin (Germany), and from the Department of Entomology, Newe-Ya'ar Research Center, Agricultural Research Organization, Raman Yishay (Israel). The colonies were initiated with females extracted from soil samples collected by Eric Palevsky from two greenhouses cultivating organic cucumber (*Cucumis sativus* L.), and three open-field plots of organic lettuce (*Lactuca sativa* L.) (Table 1).

Samples were collected with a core sampler (5 cm diameter and 5 cm depth). Soil was removed from the cores, gently mixed and then poured onto a sieve (6 cm high and 10 cm

<table><caption>**Table 1** Characterization of organic crops from which specimens of *Macrocheles karkomensis* n. sp. were collected for the establishment of lab colonies. ? = unknown information.</caption><thead><tr><th>Region</th><th>Soil type</th><th>Farm</th><th>Coordinates (DMS)</th><th>Crop</th><th>Date of collection (month-year)</th></tr></thead><tbody><tr><td rowspan="2">Coastal plain</td><td rowspan="2">Typic Chromoxeret</td><td rowspan="2">Hodaya</td><td rowspan="2">31.67896 N, 34.64847 E</td><td>Cucumber greenhouse</td><td>03-2021</td></tr><tr><td>Lettuce open-field plot</td><td>03-2021</td></tr><tr><td>Jezreel Valley</td><td>Typic Chromoxeret</td><td>Kfar Yehoshua</td><td>32.67353 N, 35.15206 E</td><td>Lettuce open-fields plot</td><td>04-2021</td></tr><tr><td>Upper Galilee - Korazim</td><td>Typic Chromoxeret</td><td>Karkom</td><td>32.91319 N, 35.58449 E</td><td>Cucumber greenhouse</td><td>11-2022</td></tr><tr><td>Beit Shean Valley</td><td>?</td><td>Mehola</td><td>32.36478 N, 35.51763 E</td><td>Lettuce open-fields plot</td><td>03-2021</td></tr></tbody></table>
in diameter) to extract the mites using a Galen-Berlese funnel (Rueda-Ramírez et al. 2022). Extraction was carried out for ~ 8 days, starting immediately after sample collection. Mites were collected in 7 cm plastic cups (microcosm) whose floors were covered with a mixture of nine parts gypsum and one part activated charcoal. Colonies were initiated after separating and confirming that all of the specimens represented the new species. Some colonies were kept separate based on the localities in which mites were collected, while other colonies consisted of mixed populations. Each colony was maintained in plastic units as described above. Each microcosm was filled to half capacity with vermiculite, which was kept moist by daily addition of sterile water. The mites were fed with a mixture of different free-living nematode species and, on some occasions, with a mixture of all developmental stages of the oribatid mite, *Carpoglyphus lactis* (Linnaeus, 1758) (Astigmatina: Carpoglyphidae).

## SEM examination

Mites taken directly from colonies were fixed and maintained for at least 24 hours in 70% ethanol. Subsequently, specimens were dehydrated in ethanol solutions of increasing concentration (80%, 90%, 95%, 100%), changing the concentration at least every 15 minutes. The samples were then critical point dried in a Balzers Union CPD 030, coated with gold on a Balzers Union SCD 040 sputter coater, and visualized with a LEO 1430 scanning electron microscope (Carl Zeiss AG, Jena, Germany).

## DNA barcoding

Eighteen mite specimens were sent for molecular analysis to the Canadian Centre for DNA Barcoding (CCDB). The barcode region of cytochrome c oxidase I (COI) was sequenced with a cocktail (1:1 ratio) of LepF1/LepR1 (Hebert et al. 2003) and LCO1490/HCO2198 (Folmer et al. 1994) primers using standard protocols modified to allow voucher recovery following DNA extraction (Ivanova, de Waard and Hebert 2007; Ivanova and Grainger 2007a; Ivanova and Grainger 2007b; Porco, Bedos and Deharveng 2010). DNA extracts were archived in -80 °C freezers at the Centre for Biodiversity Genomics (CBG; biodiversitygenomics.net), and the vouchered specimens were retained in 96-well microplates with 95% ethanol for subsequent morphological preparations.

COI sequences were assembled from forward and reverse chromatograms using CodonCode Aligner v. 4.2.7 and uploaded to BOLD. They were inspected for potential contamination or misidentification by examining their placement in a Neighbor-Joining tree and by querying each record against BOLD's complete reference library using the BOLD Identification Engine. To assess the COI sequences similarity between the species described here and other *Macrocheles* species, we followed the procedure and short phylogeny published by Knee (2017). We used the same COI sequences as Knee (2017), except that instead of seven sequences from *Macrocheles willowae* Knee, 2017, we used only three. Similarly, two species of *Ornithonyssus* (Macronyssidae), *Ornithonyssus bacoti* (Hirst, 1913) and *O. sylviarum* (Canestrini & Fanzago, 1877), were used as outgroup. In addition, we included two unidentified *Macrocheles* species that were recognised as closely related to the new species described here based on comparisons with the BOLD and GenBank databases (Table 2). Only sequences with verifiable morphological identity were used.

Sequence alignment, pairwise distance calculation, determination of the best-fit model of molecular evolution and phylogenetic reconstruction followed the procedures described by Knee (2017), with modifications only to adjust the latest versions of the software. Briefly, sequence alignments were performed based on their translation into proteins in Mesquite version 3.81 (Maddison and Maddison 2023). Pairwise distances (p-distance) were calculated using neighbour-joining (NJ) analysis with the Kimura 2-parameter (K2P) model in PAUP* v4.0a (Swofford 2003). The best-fit model of molecular evolution was determined to be GTR+I+G using MrModeltest v2.4 (Nylander 2004). Phylogenetic reconstructions of the COI sequences were performed using Bayesian inference (BI) in MrBayes v3.2.7a (Huelsenbeck
and Ronquist 2001). In our case, the set of COI sequences was run for 10 million generations in two runs, producing 20002, of which 19502 were sampled for the consensus tree. The consensus tree was constructed in MrBayes and visualised with FigTree v 1.4.4 (Rambaut 2018), with the posterior probability at each node of the tree.

## Mite identification and description

Morphological identification was based on examination of 30 mites, including the 18 specimens from which DNA had earlier been extracted. All of the specimens were mounted on microscope slides using Hoyer's mounting medium. Measurements are based on six females (two of which underwent DNA extraction), six males, three deutonymphs, and three protonymphs. Mites were imaged with a digital AxioCam MRc5 connected to an Axiophot Zeiss microscope. Photos and accompanying illustrations were then processed with a digital tablet using the Adobe Illustrator® program (version 21.0.0, 2017).

Measurements were taken with ZEN 2012 software (version 1.1.1.0). Measurements of each structure are given in micrometres (µm), with the average measurement for the specimens examined followed (in parentheses) by respective size ranges for variable structures.

Shield length is the maximum distance between anterior and posterior margins, whereas shield width generally refers to the maximum width. Sternal shield width is the width at the median level of coxeae II. Leg lengths are based on distances between coxal bases and tarsal termini. Idiosomatic setal nomenclature is based on Lindquist and Evans (1965), leg chaetotaxy on Evans (1963; 1969), and poroidotaxy on Athias-Henriot (1975), revised and complemented by Moraza (2025). In this work, we designate two previously unnamed lyrifissures with the abbreviation 'c' which comes from the Latin 'canalis' meaning 'groove' and refers to the location of the lyrifissures within the groove of the peritreme. In leg ornamentation, the term "tubercle" refers to a protuberance on the cuticle which bears a seta, and the term "spur" to a protuberance without a seta. The number of teeth indicated for cheliceral digits does not include the terminal hook.

Abbreviations of sternal shield lines are as follows:

<table><caption>**Table 2** Specimens of *Macrocheles* species and Macronyssidae *O. bacoti* and *O. sylviarum* (outgroup) included in the Bayesian analysis with their corresponding voucher collection, museum ID and GenBank accession numbers. NA = information not available.</caption><thead><tr><th>Species</th><th>BOLD Sequence ID</th><th>Museum ID</th><th>GenBank Accession number</th></tr></thead><tbody><tr><td>Macrocheles karkomensis n. sp.</td><td>PASMB578-22</td><td>HUJINVACA546</td><td>PP835193</td></tr><tr><td>Macrocheles karkomensis n. sp.</td><td>PASMB632-22</td><td>HUJINVACA600</td><td>PP835195</td></tr><tr><td>Macrocheles karkomensis n. sp.</td><td>PASMB633-22</td><td>HUJINVACA601</td><td>PP835198</td></tr><tr><td>Macrocheles karkomensis n. sp.</td><td>PASMB635-22</td><td>ZMB-Arach 55438</td><td>PP835205</td></tr><tr><td>Macrocheles karkomensis n. sp.</td><td>PASMB636-22</td><td>ZMB-Arach 55439</td><td>PP835199</td></tr><tr><td>Macrocheles kaiju Knee, 2017</td><td>GACAC1728-18</td><td>CNC829481</td><td>MF192750.1</td></tr><tr><td>Macrocheles nataliae Bregetova & Koroleva, 1960</td><td>GACAC1730-18</td><td>NA</td><td>MF192752.1</td></tr><tr><td>Macrocheles nataliae</td><td>GACAC1731-18</td><td>NA</td><td>MF192753.1</td></tr><tr><td>Macrocheles praedafimetorum Richards & Richards, 2012</td><td>GACAC1732-18</td><td>NA</td><td>MF192754.1</td></tr><tr><td>Macrocheles pratum Knee, 2017</td><td>GACAC1729-18</td><td>CNC829450</td><td>MF192751.1</td></tr><tr><td>Macrocheles subbadius (Berlese, 1904)</td><td>MBIOE1677-13</td><td>BIOUG08802-F02</td><td>MG414975</td></tr><tr><td>Macrocheles subbadius</td><td>MBIOE1699-13</td><td>BIOUG08802-G12</td><td>MG415493</td></tr><tr><td>Macrocheles willowae Knee, 2017</td><td>GACAC1721-18</td><td>NA</td><td>MF192743.1</td></tr><tr><td>Macrocheles willowae</td><td>GACAC1722-18</td><td>CNC829440</td><td>MF192744.1</td></tr><tr><td>Macrocheles willowae</td><td>GACAC1726-18</td><td>NA</td><td>MF192748.1</td></tr><tr><td>Macrocheles aff. scutatus (Berlese, 1904)</td><td>NA</td><td>NA</td><td>PQ095586.1</td></tr><tr><td>Macrocheles sp. South Africa</td><td>KMPDJ3158-19</td><td>BIOUG48715-B09</td><td>NA</td></tr><tr><td>Ornithonyssus bacoti (Hirst, 1913)</td><td>GBCH4049-09</td><td>NA</td><td>FM179677.2</td></tr><tr><td>Ornithonyssus sylviarum (Canestrini & Fanzago, 1877)</td><td>CNPEG510-14</td><td>BIOUG10499-A07</td><td>KR103486.1</td></tr></tbody></table>
*l ang.*: linea angulata, an anterior transverse line, deeply concave medially, terminating anterolaterally at *lyrifissures iv1*.

*l arc.*: linea arcuata, a deeply concave line whose anterior extremities approach or touch the lineae oblique anteriores (see below).

*l.m.t.*: linea media transversa, a median line terminating at or near the insertions of setae *st2*.

*l.o.a.*: lineae oblique anteriores, paired lines extending posterolaterally from the *l.ang.* through the insertions of *st2*.

*l.o.p.*: lineae oblique posteriores, paired lines extending posterolaterally from *l.m.t.* through *lyrifissures iv2*.

*a.p.*: areae punctatae, punctate areas along posterior margin of shield.

Voucher specimens of *M. karkomensis* are deposited in the Museum für Naturkunde (MfN), Berlin, Germany, the Hebrew University of Jerusalem, National Collections of Natural History (HUJ), Jerusalem, Israel and Oregon State Arthropod Collection (OSAC), Corvallis, Oregon, USA. DNA vouchers are deposited in the Centre for Biodiversity Genomics (CBG), Guelph, Ontario, Canada. Type depositories of the new species are indicated in the "Material examined" section.

## Results

**Macrocheles karkomensis Rueda-Ramírez & Krantz n. sp.**

Zoobank: A7FE0431-8346-491E-9D3A-59651C492D41

### Diagnosis

Female dorsal shield with a weak procurved line posterior to *z6* and *s5*, absent in some specimens; bearing 28 pairs of setae, all aciculate simple, except *j1*, which are appressed and distally pilose, *j4* and *z4*, distally pilose and *J5*, weakly serrate; sternal shield longer than wide, with ill-defined faint punctate lines: *l.m.t.* undulate, *l.arc.* strongly procurved and almost touching *l.o.a.*, *l.o.p.* barely visible, without ramus and free from *l.m.t.*, *l.ang.* procurved, and *a.p.* reduced to a few small scattered punctae; epigynal shield bearing a pair of accessory sclerites and *st5*; opisthogaster with six pairs of setiform setae (*Jv4* – 5, *Zv1*, *Zv2*, *Zv4* and *Zv5*) on unsclerotised cuticle; with a pair of elongated metapodal plates; ventrianal shield longer than wide, punctate-reticulate. Male with cheliceral spermatodactyl curved downward, extending beyond the base of the dorsal seta and progressively tapering in diameter to a blunt tip; dorsal shield bearing 31 pairs of setae (the same as those present in the female, plus *r5*, *R1* and *R2*), all aciculate simple, except *j1*, *j4* and *z4* which are distally pilose, *J5* weakly serrate; sternogenital shield separate from ventrianal shield, which is peltate, slightly broader than long, trochanter IV without spurs; femur IV with three tubercles, the most proximal of which with a terminal small spiniform seta (*v1*); genu IV with *av1* on a barely visible tubercle.

### Adult female

(Figures 1 – 5, six specimens measured).

**Gnathosoma** — Anteromedian region of epistome (Figure 1a) with a median with a median, distally bifurcate element flanked by pair of shorter, broader flag-like elements, whose bases are joined to weakly serrate epistomatic margins. Deutosternal groove medial, longitudinal (Figure 1b), located partially in the anterior hypostome (distal to insertions of hypostomatic setae *h2*–3 and adjacent palptrochanteral junctions) and partially in the more posterior basis gnathosomatica (Evans & Till 1979, Bowman 2023); with six transverse rows of denticles delimited laterally by subparallel grooved borders of deutosternal field, row 1 located anteriorly on the hypostome and weakly ornamented, rows 2–6 on the basis gnathosomatica, each with 10–15 distinct denticles. Internal malae (Figure 1b, *im*) narrow, paired, extending anteriorly
<figure><img src="image_3.png" /><figcaption>**Figure 1** *Macrocheles karkomensis* n. sp. adult female. Gnathosoma. **a** – Epistome; **b** – Ventral view of gnathosoma; **c** – SEM image of chelicera; **d** – Antiaxial view of chelicera. AL = antiaxial lyrifissure, ap = apotele, co = corniculum, DL = dorsal lyrifissure, DS = dorsal setae, *h1* – 3 = hypostomatic setae 1–3, *im* = internal malae, IAB = long external arthrodial brush, *pc* = palpcoxal seta, *pl* = *pilus dentilus*, sAB = short external arthrodial brush, *sst* = salivary stylus.</figcaption></figure>

almost to tips of adjacent corniculi external margins lightly fimbriate. Corniculi (Figure 1b, *co*)
elongate, horn-shaped, over twice as long as their basal width, each with a closely associated
flanking salivary stylus (*sst*) originating posterior to insertion level of hypostomatic setae
*h1* (Figure 1b). Hypostomatic and capitular setae (*h1*–3, *pc*) smooth, aciculate, insertions of
*h3* mesad and adjacent to those of *h2*, capitular setae shorter than hypostomatics, flanking
denticular row 6. Subcapitular setal measurements: *h1* 47 (46–48), *h2* 26 (23–31), *h3* 67
(61–73), *pc* 21 (19–22). Chelicerae with a long internal arthrodial brush reaching the mid-level
of movable digit and a short external arthrodial brush, both arising ventrally at the articulation
of the movable digit. Fixed cheliceral digit 69 (66–71) long, with an apical hook, a subterminal
recess fitting the tip of the movable digit when closed, and where two tiny teeth are also
found, with a more proximal spine-shaped *pilus dentilis* and a large tooth; dorsal cheliceral
seta thick, dorsal lyrifissure longitudinal, internal and anterior to the dorsal seta, antiaxial
lyrifissure external at the level of the arthrodial brushes; movable digit 60 (58–63) long, with an
<figure><img src="image_4.png" /><figcaption>**Figure 2** *Macrocheles karkomensis* n. sp. adult female. **a** – Dorsal idiosoma of adult female; **b** – LTSEM image of dorsal view of adult female.</figcaption></figure>

apical hook, a large, bidentate tooth flanked distally by two small proximal teeth (Figure 1c,d). Number of setae on palp trochanter-tarsus: 2-5-6-14-15, aciculate and smooth, except al of femur and *al1* and *al2* of palpgenu, which are stout and spine-like (Figure 1b); palptrochanter with outer seta (*pv*) shorter than inner seta (*av*); tarsal apotele 3-tined (Figure 1c).

**Idiosoma** — Length 654 (600–707), width 455 (363–528).

**Dorsal idiosoma** — (Figure 2, 3). Holodorsal shield, 590 (571–623) long and 357 (335–376) wide at level of dorsal setae *r3*, ovate-reticulate, reticulation less evident in sigillate region of setae *z5–s6*, usually with a weak procurved line posterad *z6-s5*, line occasionally absent. Holodorsal shield (Figure 2) bearing 28 pairs of setae, 16 pairs of lyrifissures and seven pairs of pores; shield setae smooth, aciculate except *j1*, which are appressed, distally flattened and pilose (Figure 3a), and *j4* and *z4*, which are distally pilose, *J5* lightly pectinate; distances *j2–j3* 49 (42–52), *j4–j4* 102 (99–107), *j5–j5* 54 (52–56), *J5–J5* 27 (26–27). Unsclerotised dorsal cuticle lateral and posterior to shield with 13 pairs of setae (*r5*, *R1–6*, *UR* 1–6) and lyrifissure *idRp*. Setal measurements are shown in Table 3.

**Ventral idiosoma** — (Figure 4). Tritosternum with columnar base, 33 (31–35) long and 17 (15–18) wide, with long pilose laciniae 96 (87–103), separated for ~92% total length. Sternal shield 150 (144–154) long and 121 (118–128) wide at level of coxae II, with faint punctate ornamentation (Figure 4, 15a): *l.m.t.* undulate, *l.arc.* strongly procurved and almost
<figure><img src="image_5.png" /><figcaption>**Figure 3** SEM image of *Macrocheles karkomensis* n. sp. adult female, with details of: **a** – setae *j1*, **b** – setae *j4* and *z4*, and **c** – dorsal setation of femur IV. *pm* = peritreme.</figcaption></figure>

touching *l.o.a.*, *l.o.p.* barely visible with weak ramus or without ramus and free from *l.m.t.*, *l.lang.* procurved, and *a.p.* reduced to a few small scattered punctae; with three pairs of setae (*st1–st3*) and two pairs of *lyrifissures* (*iv1*, slightly posteriad and laterad *st1*, and *iv2*, posteriad and laterad *st2*); distances *st1–st3* 143 (138–151), *st2–st2* 104 (101–112), *st3–st3* 108 (103–117); shield with broad anterolateral corners extending between coxae I and II, distally abutting anterior exopodal. Setae *st4* and *lyrifissure* *iv3* on metasternal platelets. Epigynal shield bearing *st5*, abutting a pair of accessory sclerites, ornamented with four weak, granulate recurved lines; shield 133 (122–144) long, 131 (117–152) wide at widest level, truncate posteriorly; distance *st5–st5* 115 (105–124), posteriorly truncate; *iv5* on unsclerotised cuticle, posterolaterad *st5*. Metapodal platelets elongate, somewhat irregular. Ventrianal shield subcordate, reticulate; shield 203 (189–232) long and 185 (171–197) wide at widest level; with three pairs of preanal setae (*Jv1*, *Jv2* and *Jv3*), a pair of *lyrifissures* posteromesad *Jv2*, cribral glands *gv3* on posterolateral shield margins and posterior to paranal setae; anal opening 26 (24–29) long and 20 (18–21) wide; cribrum narrow, confined to posterior margin of the shield. Opistogastric unsclerotised cuticle with six pairs of setae (*Jv4*, *Jv5*, *Zv1*, *Zv2*, *Zv4* and *Zv5*), three pairs of *lyrifissures*, *gv2* pore posterolaterad coxa IV. Endopodal and exopodal elements as illustrated in Figure 4.

Anterior section of endopodal shield contiguous with sternal shield, with section between
coxea III and IV boomerang-shaped. Exopodal shield free, fragmented into two pieces, the anterior surrounding acetabula II–III and the posterior (parapodal) an arch surrounding acetabulum IV. All ventral setae setiform. Setal measurements shown in Table 4.

**Peritreme and peritrematic plate** — (Figures 2a, 3 and 4). Peritreme extending anteriorly to level of dorsal lyrifissure *id1*, with two pairs of lyrifissures (*ipc1*, *ipc2*) on or within peritrematic groove, with *ipc2* ventral at level of coxea III (Figure 4) and *ipc1* anterodorsal at level of setae *z2* (Figure 2a); peritrematic shield fused with dorsal shield at level of *r2–r3*, free and recurved posteriorly, extending no further than anterior margin of coxea III, bearing a pore

<table><caption>**Table 3** Lengths of dorsal setae of *Macrocheles karkomensis* n. sp.; mean (minimum–maximum). - = seta absent.</caption><thead><tr><th></th><th>Female<br>(N = 6)</th><th>Male<br>(N = 5)</th><th>Deutonymph<br>(N = 3)</th><th>Protonymph<br>(N = 3)</th></tr></thead><tbody><tr><td>j1</td><td>21 (19–22)</td><td>22 (20–24)</td><td>21 (20–21)</td><td>17 (16–18)</td></tr><tr><td>j2</td><td>30 (26–36)</td><td>24 (21–25)</td><td>20 (18–23)</td><td>13 (12–14)</td></tr><tr><td>j3</td><td>35 (31–37)</td><td>25 (23–27)</td><td>22 (22–23)</td><td>17 (16–18)</td></tr><tr><td>j4</td><td>33 (31–35)</td><td>31 (28–34)</td><td>30 (30–31)</td><td>24 (23–25)</td></tr><tr><td>j5</td><td>31 (29–35)</td><td>23 (20–25)</td><td>20 (19–21)</td><td>16 (16–17)</td></tr><tr><td>j6</td><td>32 (30–35)</td><td>24 (23–24)</td><td>20 (19–20)</td><td>18 (17–18)</td></tr><tr><td>J2</td><td>39 (36–43)</td><td>27 (26–28)</td><td>20 (19–20)</td><td>12 (11–12)</td></tr><tr><td>J5</td><td>22 (21–25)</td><td>16 (14–18)</td><td>13 (11–14)</td><td>8 (7–8)</td></tr><tr><td>z1</td><td>11 (10–13)</td><td>8 (7–8)</td><td>10 (9–11)</td><td>-</td></tr><tr><td>z2</td><td>35 (33–37)</td><td>24 (23–26)</td><td>22 (22–22)</td><td>16 (16–17)</td></tr><tr><td>z4</td><td>41 (40–44)</td><td>30 (25–38)</td><td>34 (30–39)</td><td>31 (29–33)</td></tr><tr><td>z5</td><td>31 (26–35)</td><td>23 (22–24)</td><td>21 (20–21)</td><td>16 (16–17)</td></tr><tr><td>z6</td><td>35 (29–39)</td><td>25 (23–27)</td><td>18 (18–18)</td><td>-</td></tr><tr><td>Z1</td><td>40 (39–41)</td><td>28 (26–30)</td><td>24 (22–26)</td><td>15 (15–16)</td></tr><tr><td>Z2</td><td>44 (42–47)</td><td>29 (28–30)</td><td>22 (21–22)</td><td>14 (13–14)</td></tr><tr><td>Z4</td><td>42 (39–45)</td><td>31 (28–34)</td><td>22 (21–23)</td><td>11 (11–12)</td></tr><tr><td>Z5</td><td>26 (25–28)</td><td>22 (21–22)</td><td>15 (13–18)</td><td>13 (13–14)</td></tr><tr><td>s2</td><td>34 (32–36)</td><td>24 (22–28)</td><td>17 (16–18)</td><td>-</td></tr><tr><td>s4</td><td>38 (35–41)</td><td>28 (24–30)</td><td>26 (25–27)</td><td>18 (17–18)</td></tr><tr><td>s5</td><td>39 (38–41)</td><td>30 (28–31)</td><td>23 (23–23)</td><td>16 (15–16)</td></tr><tr><td>s6</td><td>38 (34–41)</td><td>28 (27–31)</td><td>21 (20–22)</td><td>15 (15–16)</td></tr><tr><td>S1</td><td>39 (34–43)</td><td>26 (25–29)</td><td>17 (16–19)</td><td>-</td></tr><tr><td>S2</td><td>36 (31–43)</td><td>29 (27–31)</td><td>19 (16–21)</td><td>13 (12–13)</td></tr><tr><td>S4</td><td>37 (34–40)</td><td>28 (27–29)</td><td>20 (19–21)</td><td>16 (15–16)</td></tr><tr><td>S5</td><td>29 (28–29)</td><td>21 (19–24)</td><td>16 (15–17)</td><td>12 (12–13)</td></tr><tr><td>r2</td><td>33 (29–36)</td><td>30 (27–32)</td><td>24 (24–24)</td><td>15 (14–16)</td></tr><tr><td>r3</td><td>34 (29–37)</td><td>28 (27–30)</td><td>23 (23–23)</td><td>16 (15–17)</td></tr><tr><td>r4</td><td>34 (31–37)</td><td>29 (26–31)</td><td>20 (17–23)</td><td>-</td></tr><tr><td>r5</td><td>15 (14–16)</td><td>15 (14–15)</td><td>17 (16–18)</td><td>14 (12–16)</td></tr><tr><td>R1</td><td>20 (17–24)</td><td>17 (16–19)</td><td>14 (12–16)</td><td>14 (13–14)</td></tr><tr><td>R2</td><td>22 (20–23)</td><td>16 (15–17)</td><td>12 (11–14)</td><td>-</td></tr><tr><td>R3</td><td>22 (20–24)</td><td>15 (14–17)</td><td>13 (12–14)</td><td>-</td></tr><tr><td>R4</td><td>22 (20–24)</td><td>16 (14–17)</td><td>11 (11–11)</td><td>-</td></tr><tr><td>R5</td><td>22 (18–23)</td><td>16 (14–17)</td><td>13 (12–14)</td><td>-</td></tr><tr><td>R6</td><td>23 (18–25)</td><td>17 (16–18)</td><td>12 (12–12)</td><td>-</td></tr><tr><td>UR1</td><td>17 (14–21)</td><td>-</td><td>12 (12–13)</td><td>-</td></tr><tr><td>UR2</td><td>18 (17–21)</td><td>-</td><td>12 (12–12)</td><td>-</td></tr><tr><td>UR3</td><td>20 (19–21)</td><td>-</td><td>11 (11–11)</td><td>-</td></tr><tr><td>UR4</td><td>20 (18–23)</td><td>-</td><td>12 (10–14)</td><td>-</td></tr><tr><td>UR5</td><td>21 (20–23)</td><td>-</td><td>13 (13–14)</td><td>-</td></tr><tr><td>UR6</td><td>21 (20–22)</td><td>-</td><td>14 (13–15)</td><td>-</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Figure 4** *Macrocheles karkomensis* n. sp. adult female. Ventral Idiosoma. *l.lang.*: linea angulata; *l.arc.*: linea arcuata; *l.m.t.*: linea media transversa; *l.o.a.*: linea obliqua anterior; *l.o.p.*: linea obliqua posterior; *w.rm*: weak ramus of *l.o.p.*</figcaption></figure>

(gvi) and two lyrifissures (ips and ivi) at the level of each stigma, with pore gd3 and lyrifissure ip dorsad of the peritreme and between coxae II–III.

**Spermathecal apparatus** — Not clearly seen in available specimens.

**Legs** — (Figure 5). Tarsi II–IV with claws and ambulacra; tarsus I without pretarsus, claw and ambulacrum. Leg lengths: I: 459 (427–486.2); II: 405 (390–425); III: 348 (335–365); IV: 525 (506–544). Pretarsus lengths: II: 32 (30–36); III: 31 (28–36); IV: 32 (32–33). Setation (legs I–IV): coxae: 0-0/1 0/1-0, 0-0/1 0/1-0, 0-0/1 0/1-0, 0-0/1 0/0-0; trochanters: 1-0/1 0/2-1, 1-0/1 0/2-1, 1-0/2 0/1-1, 1-1/2 0/1-0; femora: 2-3/1 2/3-2 (*ad2* and *ad3* thickened and distally pilose), 2-3/1 2/2-1 (*ad1* thickened and distally pilose), 1-2/1 1/0-1, 1-2/1 1/0-1 (*ad2* and *pd1* thickened and distally pilose); genua: 2-3/1 2/1-2, 2-3/1 2/1-2, 1-2/1 2/0-1 (*pd2* small spine-like), 1-2/1 2/0-0; tibiae: 2-3/2 2/1-2 (*pd2* thickened and distally pilose), 2-2/1 2/1-2, 1-1/1 2/1-1, 1-1/1 2/1-1; tarsi: not counted, 18 (distal setae thickened), 18 (ventral setae thickened), 18 (ventral setae thickened).

**Adult male**

(Figures 6–9, five specimens measured)

**Gnathosoma** — Epistome, deutosternal groove, position and shape of hypostomatic setae, internal malae, and corniculi as in adult female. Subcapitular setal measurements: *h1* 45 (39–
49), *h2* 16 (14–18), *h3* 61 (55–64), *pc* 15 (13–17). Fixed cheliceral digit 49 (45–54) long, with blunt apical hook, followed proximally by three small teeth and a large, weakly bidentate tooth; spine-shaped *pilus dentilis* inserted slightly anteriad the large tooth; movable digit 42 (36–47) long, with a large tooth behind terminal hook; spermatodactyl 49 (45–54) long, curved upward and posteriorly, extending beyond insertion of dorsal seta, tapering progressively distally to a blunt tip; dorsal seta thick; dorsal and antiaxial lyrifissures distinct, the former with longer axis longitudinal, on internal face of digit (Figure 6). Arthrodial process of chelicera, palp chaetotaxy, and apotele as in adult female. Palpal setae aciculate and smooth, except for stout and spinose *al* setae on the femur and *al1* and *al2* on the genu.

**Idiosoma** — Length 501 (458–534), width 319 (281–358).

**Dorsal idiosoma** — (Figure 7). Holodorsal shield, 493 (450–523) long and 311 (278–340) wide, virtually covering dorsal surface of idiosoma, narrowing slightly posteriad *r5*, with ornamentation similar to adult female, less evident, between *j3-j4-z2* and *j5-j6-z5*; shield with 31 pairs of setae (the same as those of female, plus *r5*, *R1* and *R2*); distances *j2-j3* 41 (37–43), *j4-j4* 87 (83–90), *j5-j5* 44 (41–46), *J5-J5* 22 (20–23). Poroidotaxy, adenotaxy and shape of setae as in adult female. Setal measurements shown in Table 3.

**Ventral idiosoma** — (Figure 8). Tritosternum similar to that of adult female; base 21 (18–23) long and 14 (13–14) wide, and long pilose laciniae 84 (80–89), separated for about 94% of their total length. Sternogenital shield with anterior half reticulate, with punctate-reticulate ornamentation; posterior half with granulate ornamentation and scant wavy lateral lines oriented more or less longitudinally; 217 (199–227) long and 92 (85–98) wide at the level of coxeae II; with five pairs of setae (*st1*–3, 5; *st4* absent on at least one side in most observed specimens, sometimes completely absent; figure 8b,c), with three pairs of lyrifissures (*iv1*–3); distances *st1-st3* 132 (126–135), *st2-st2* 80 (78–82), *st3-st3* 81 (75–85). Endopodal shield fused with sternogenital shield. Ventrianal shield peltate, without anterolateral excavations around coxeae IV, ornamentation similar to that of anterior half of sternal shield, reticulation absent anteriad anal opening; slightly broader than longer; 171 (153–181) long and 181 (165–190) wide at widest level; with six pairs of setae (*Jv1*–4, *Zv1* and *Zv2*) in addition to circumanal setae, *iv5* lyrifissures at anterior corners of shield, with additional pair lyrifissures posterolaterad to *Zv1* and cribral glands *gv3* at shield margin, slightly anteriad level of postanal seta; anal opening 20 (18–22) long and 19 (18–19) wide. Cribrum wider than in female and with a pair of strap-like paranal extensions on either side of postanal seta. Metapodal plates elongate, irregular. Opistogastric unsclerotised cuticle with a pair of setae (*Jv5*), three pairs of lyrifissures, and

<table><caption>**Table 4** Lengths of hypostomatic and ventral setae of *Macrocheles karkomensis* n. sp.; mean (minimum–maximum). - = seta absent.</caption><thead><tr><th></th><th>Female<br>(N = 6)</th><th>Male<br>(N = 5)</th><th>Deutonymph<br>(N = 3)</th><th>Protonymph<br>(N = 3)</th></tr></thead><tbody><tr><td><i>st 1</i></td><td>36 (33–39)</td><td>25 (25–26)</td><td>26 (25–27)</td><td>21 (21–22)</td></tr><tr><td><i>st 2</i></td><td>36 (34–37)</td><td>31 (29–33)</td><td>25 (23–27)</td><td>19 (19–20)</td></tr><tr><td><i>st 3</i></td><td>33 (30–34)</td><td>30 (29–32)</td><td>23 (23–23)</td><td>19 (18–19)</td></tr><tr><td><i>st 4</i></td><td>30 (27–32)</td><td>28 (27–28)</td><td>17 (16–17)</td><td>-</td></tr><tr><td><i>st 5</i></td><td>31 (29–33)</td><td>23 (22–25)</td><td>21 (21–21)</td><td>8 (8–9)</td></tr><tr><td><i>JV 1</i></td><td>30 (28–34)</td><td>25 (24–26)</td><td>25 (24–25)</td><td>19 (18–19)</td></tr><tr><td><i>JV 2</i></td><td>29 (27–30)</td><td>24 (23–25)</td><td>18 (17–18)</td><td>13 (13–14)</td></tr><tr><td><i>JV 3</i></td><td>28 (25–32)</td><td>23 (21–25)</td><td>15 (15–15)</td><td>-</td></tr><tr><td><i>JV 4</i></td><td>21 (19–25)</td><td>13 (12–13)</td><td>-</td><td>-</td></tr><tr><td><i>JV 5</i></td><td>22 (19–23)</td><td>15 (14–15)</td><td>12 (11–14)</td><td>10 (9–10)</td></tr><tr><td><i>ZV 1</i></td><td>13 (11–14)</td><td>-</td><td>-</td><td>-</td></tr><tr><td><i>ZV 2</i></td><td>21 (18–23)</td><td>-</td><td>15 (14–17)</td><td>12 (12–13)</td></tr><tr><td><i>ZV 4</i></td><td>20 (18–21)</td><td>-</td><td>10 (10–11)</td><td>-</td></tr><tr><td><i>ZV 5</i></td><td>23 (20–26)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>paranal</td><td>28 (26–30)</td><td>20 (18–22)</td><td>19 (18–21)</td><td>19 (17–20)</td></tr><tr><td>postanal</td><td>20 (17–23)</td><td>12 (9–14)</td><td>13 (13–14)</td><td>11 (10–11)</td></tr></tbody></table>
<figure><img src="image_7.png" /><figcaption>**Figure 5** *Macrocheles karkomensis* n. sp. adult female. Legs. a – Leg I; b – Leg II; c – Leg III; d – Leg IV.</figcaption></figure>

gv2 pore posterolaterad coxae IV. Exopodal shield surrounding acetabula II–IV. Idiosomatic setae similar to those of adult female. Setal measurements shown in Table 4.

**Peritreme and peritrematic plate** — (Figure 7, 8). Peritreme similar to that of female lysrifissures anterodorsally expressed *ipc1* (Figure 7), and ventral *ipc2* (Figure 8). Peritrematic plate fused with dorsal shield for its entire length; bearing pore (*gvi*) and two lysrifissures (*ips* and *ivi*) in close association with stigmata, with pore *gd3* and a lysrifissure *ip* dorsad of peritreme and between coxae II-III.

**Legs** — (Figure 9a–c). Leg lengths: I: 416 (390–439); II: 378 (350–399); III: 321 (302–338); IV: 491 (447–528). Pretarsus lengths: II: 29 (27–32); III: 25 (21–26); IV: 29 (26–31). Setae aciculate; femur II bears a thick spur ventrally between *pv1* and *pv2*, and with setae *ad1* and *pd1–2* somewhat thickened and distally pilose; *ad3* absent. genu and tibia II with *av1* represented by small triangular spur (Figure 9a); genu II with setae *ad2*, *ad3*, *pd1*, and *pd2* distally pilose. Podomeric fissure between basi- and telofemur of leg III (Figure 9b) with series of small, rounded ventral excrescences; *ad1* of femur III, *ad1*, *ad2*, *pd1* and *al1* of genu III, *ad1*, *pd1*, *pd2*, *al1* and *pl1* of tibia III, and *ad4* and *pd4* of genu III distally pilose; *v1* of femur III thickened, distally pilose (Figure 9b). Femur and telotarsus of legs IV (Figure 9c) strongly ornamented with spurs and tubercles: femur IV with large, ventral curval setiferous tubercle carrying a small spiniform setae (*v1*) and two smaller adjacent spurs as illustrated, *ad1* and
<figure><img src="image_8.png" /><figcaption>**Figure 6** *Macrocheles karkomensis* n. sp. adult male. Chelicera. AL= antiaxial lyrifissure, DL = dorsal lyrifissure, DS = dorsal setae, IAB = long external arthrodial brush, pc = palpcoxal seta, pl = *pilus dentilus*, sAB = short external arthrodial brush, spd = spermatodactyl.</figcaption></figure>

*al1* slightly thickened, distally pilose, *ad2* and *pd1* strongly thickened, distally pilose; genu IV with *av1* on barely visible tubercle and with *ad1*, *ad2*, *pd1*, *pd2* and *al1* distally pilose; tibia IV with *ad1*, *pd1*, *pd2*, *al1* and *pl1* distally pilose; tarsus IV with a rounded dorsal spur distally, *ad4*, *pd3-4* distally pilose (Figure 9c).

**Deutonymph**

(Figure 10 – 11, three specimens measured).

**Gnathosoma** — Anteromedian region of epistome, deutosternal groove, position and shape of hypostomatic setae, internal malae, and corniculi as in adult female. Subcapitular setal measurements: *h1* 37 (36–38), *h2* 13 (11–15), *h3* 61 (60–63), *pc* 18 (18–19). Fixed cheliceral digit 45 (44–46) long, with subapical tooth, a spine-like *pilus dentilis*, and two adjacent teeth; movable digit 41 (40–43) long, with two large teeth medially; dorsal seta thick, dorsal and antiaxial lyrifissures distinct. Arthrodial process of chelicera, corniculus, palp chaetotaxy, and apotele similar to those of adult female. Palpal setae aciculate and smooth, except for thickened *al* on femur and *al1* and *al2* on genu.

**Idiosoma** — Length 639 (614–664), width 421 (418–423).

**Dorsal idiosoma** — (Figure 10). Dorsal shield with deep, curved lateral incisions behind insertions of *s6* and approaching those of *z6*; shield 457 (454–460) long and 263 (262–265) wide, covering only central portion of idionotum, tapering behind *S2* and with weak punctate ornamentation posteriorly. Shield setae, lyrifissures and pores as in adult female, but *lyrifissure idl4* not distinguishable; distances *j2–j3* 35 (33–37), *j4–j4* 74 (71–76), *j5–j5* 44 (43–44), *J5–J5* 21. Idiosomatic setae similar to those of female. Setal measurements shown in Table 3.

**Ventral idiosoma** — (Figure 11). Tritosternum similar to that of adult female; base 29 (28–30) long and 15 (14–15) wide, long pilose laciniae 78 (77–78), separated for about 92% of their total length. Sternal shield lightly sclerotised, without ornamentation, tapering posteriad *st3*; 206 (198–214) long and 77 (73–81) wide, with four pairs of aciculate setae (*st1–st4*) and
<figure><img src="image_9.png" /><figcaption>**Figure 7** *Macrocheles karkomensis* n. sp. adult male. a – Dorsal idiosoma of adult male; b – LTSEM image of dorsal view of adult male.</figcaption></figure>

three pairs of lyrifissures (*iv1–iv3*); distances *st1–3* 134 (133–135), *st2–st2* 87 (86–87), *st3–st3* 85. Seta *st5* and lyrifissure *iv5* on unsclerotised cuticle, the latter posterolaterad *st5*; distance *st5–st5* 92 (89–95). Opisthogastric unsclerotised cuticle with eight pairs of setae (*Jv1–Jv5*, *Zv2*, *Zv4* and *Zv5*), three pairs of lyrifissures, *gv2* pore posterolaterad coxa IV and *gv3* near anal shield margin, lateral and slightly posterior to postanal seta. Exopodal shield reduced to a narrow arched strip surrounding acetabulum IV (parapodal plate). Anal shield small, semicircular, lightly sclerotised and lightly reticulate, 77 (75–79) long and 73 (71–75) wide; anal opening 23 (23–24) long and 15 (14–15) wide. Cribrum wider than in female with a pair of strap-like paranal extensions on either side of postanal seta. Idiogastric setae as in adult female. Setal measurements shown in Table 4.

**Peritreme and peritrematic plate** — (Figure 10, 11). Peritremes extending anteriorly to level of dorsal setae *z1*, recurving posteriorly at level of anterior margin of coxae II; peritrematic plate lightly sclerotised, reduced to narrow strip bordering peritreme, bearing pores *gp* and *gd3* at levels of coxae II-III respectively, and lyrifissures *ipc2* at level between coxae III-IV; lyrifissure *ips* on peritrematic plate terminus adjacent and mesad to stigmata, *gvi* and *ivi* on unsclerotised cuticle behind stigmata.

**Legs** — Leg lengths: I: 379 (379–380); II: 328 (324–331); III: 288 (287–289); IV: 435 (434–436). Pretarsus lengths: II: 22 (21–22); III: 21 (21–22); IV: 22 (20–23). Setation as in
<figure><img src="image_10.png" /><figcaption>**Figure 8** *Macrocheles karkomensis* n. sp. adult male. Ventral Idiosoma. a. Ventral idiosoma of adult male; b. and c. Phase-contrast images of mid-ventral view of two adult males.</figcaption></figure>

adult female. Setae aciculate and smooth, except *ad1*, *ad2* and *pd1* of femora IV, *ad1* and *pd1* of genua IV, and *al1* and *pl1* of tibiae IV distally spatulate, ventral setae of tarsus II–IV thickened.

### Protonymph

(Figure 12 – 13, three specimens measured).

**Gnathosoma** — Epistomatic margin lightly denticulate, bifurcate anteromedian extension divided only distally. Deutosternal groove, position and shape of hypostomatic setae, internal malae, and corniculi as in adult female. Setal measurements: *h1* 32 (31–34), *h2* 14 (13–15), *h3* 50 (49–51), *pc* 17 (16–17). Fixed cheliceral digit 36 (35–36) long, with two subapical teeth, at the same level of the spine-shaped *pilus dentilis*; movable digit 29 (28–29) long, with two teeth. Numbers of setae on palp trochanter–tarsus: 1, 4, 5, 12, 15. Arthrodial process of chelicera and apotele as in adult female. Palpal setae aciculate and smooth, except for thickened *al* on the femur and genu.
<figure><img src="image_11.png" /><figcaption>**Figure 9** *Macrocheles karkomensis* n. sp. adult male. Legs. a – Leg II; b – Leg III; c – Leg IV.</figcaption></figure>

**Idiosoma** — Length 433 (428–440), width 278 (261–294).

**Dorsal idiosoma** — (Figure 12). With separate podonotal and opisthonotal shields. Podonotal shield 194 (191–197) long and 173 (164–182) wide; with setation similar to that of deutonymphs and adults, but without *z6*, *s2*, *s6* and *r2–4*; with four pairs of lyrifissures (*id1*, *id4–6*), and one pair of pores (*gd2*); all setae aciculate simple, except *j1*, *j4* and *z4*, which are distally pilose; unsclerotised cuticule laterad podonotal shield with four pairs of setae (*s6*, *r2*, *r3* and *r5*) and a pair of lyrifissures (*id2*); distances *j2–j3* 13 (13–14), *j4–j4* 57 (56–59), *j5–j5* 42 (39–45). Opisthonotal shield 120 (114–126) long and 92 (89–94) wide, with an inconspicuous curved punctate line between setae *Z4*; with setation similar to that of deutonymphs and adults, but without *S1* and with *S2* on unclerotized cuticle, ten pairs of distinguishable lyrifissures (*idm1–5*, *idl1* and *idl3–5*), and a pair of pores (*gd9*); all setae aciculate simple except for serrate *J5* and distally pilose *Z4*, *Z5*, *S4* and *S5*; unsclerotised cuticle laterad opisthonotal shield carries setae *R1* and *S2*, and *lyrifissures* *idl2* and *idRp*; distance *J5–J5* 15 (14–15). Setal measurements shown in Table 3.

**Ventral idiosoma** — (Figure 13). Tritosternum similar to that of adult female, base 22 (21–22) long and 12 (11–13) wide, and a long pilose laciniae 60 (57–63), separated for about 87% of their total length. Sternal shield lightly sclerotised, smooth, with the widest region at the level of *iv2*, tapering posteriad *st3*; 138 (134–142) long and 51 (47–55) wide, with three
<figure><img src="image_12.png" /><figcaption>**Figure 10** *Macrocheles karkomensis* n. sp. deutonymph. Dorsal Idiosoma.</figcaption></figure>

pairs of setae (st1–st3) and two pairs of lyrifissures (iv1–iv2); distances st1–3 112 (110–113), st2–st2 68 (66–71), st3–st3 60 (58–62). Seta st5 on unsclerotised cuticle; distance st5–st5 62 (61–63). Unsclerotised opistogastric cuticle with four pairs of setae (Jv1, Jv2, Jv5, Zv2), two pairs of lyrifissures, and two pairs of pores (gv3 adjacent to posterior margin of anal shield, gv2 posteriad coxa IV). Without endopodal or exopodal sclerites. Anal shield small, semi-ovoid, 50 (49–52) long and 42 (38–45) wide; anal opening 16 (15–16) long and 11 (10–12) wide. Cribrum wider than in female, with a pair of strap-like paranal extensions on either side of postanal seta. Shape of ventral idiosomatic setae as in adult female. Setal measurements shown in Table 4.

**Peritreme and peritrematic plate** — (Figure 12). Peritreme anlage short, reaching mid-level of coxa III. Peritrematic shield represented by a weakly sclerotised fragment at terminus of peritreme; lyrifissure *id3* in the exterior margin of anterior plate; lyrifissure *id7* in exterior margin of posterior portion of peritreme; pores *gp*, *gd3* and *gdp* on unsclerotised cuticle.

**Legs** — Leg lengths: I: 286 (279–293); II: 242 (235–248); III: 209 (201–218); IV: 303 (297–308). Pretarsus lengths: II: 18 (17–19); III: 17 (17–18); IV: 21 (21–22). Setation (legs I–IV): coxae: 0-0/1 0/1-0, 0-0/1 0/1-0, 0-0/1 0/1-0, 0-0/1 0/0-0; trochanters: 1-0/1 0/1-1, 1-0/1 0/1-1, 1-0/1 0/1-1, 1-1/1 0/1-0; femora: 2-2/1 2/1-2, 1-2/1 2/1-1, 1-2/1 1/0-0, 1-2/0 1/0-0; genua: 1-2/0 2/1-1, 1-2/0 2/0-1, 1-2/0 2/0-1, 1-2/0 2/0-0; tibiae: 1-2/1 2/1-1, 1-1/1 2/1-1, 1-1/1 2/1-1, 1-1/1 2/1-1; tarsi: not counted, 17 (distal setae thickened), 17 (ventral setae thickened), 17 (ventral setae thickened).
<figure><img src="image_13.png" /><figcaption>**Figure 11** *Macrocheles karkomensis* n. sp. deutonymph. Ventral Idiosoma.</figcaption></figure>

**Material examined**

Holotype: female (ZMB-Arach 55437) from a laboratory colony deposited in MfN. Paratypes: two females (ZMB-Arach 55438 – 55439) collected in Karkom (Upper Galilee – Korazim, Israel), three females (ZMB-Arach 55440 – 55442) from a laboratory colony; four males (ZMB-Arach 55443 – 55446) from a laboratory colony, three deutonymphs (ZMB-Arach 55447 – 55449) from a laboratory colony, and three protonymphs (ZMB-Arach 55450 – 55452) from a laboratory colony deposited in MfN; a female (HUJINVACA540) from Hodaya (Coastal plain, Israel) deposited in HUJ; a female (OSAC collection number 0001418396) collected in Kfar Yehoshua (Jezreel Valley, Israel) and a male (OSAC collection number 0001418397) from a laboratory colony deposited in OSAC (Type deposition detailed in table 5).

Other material: two females (HUJINVACA541 – HUJINVACA542) collected in Hodaya (Coastal plain, Israel), two females (HUJINVACA548 – HUJINVACA549) collected in Kfar Yehoshua (Jezreel Valley, Israel), four females (HUJINVACA554 – HUJINVACA557) collected in Mehola (Beit Shean Valley, Israel), and five females (HUJINVACA600 – HUJINVACA602, HUJINVACA605 and HUJINVACA606) collected in Karkom (Upper Galilee – Korazim, Israel) deposited in HUJ. A female (ZMB-Arach 55453) collected in Kfar Yehoshua (Jezreel Valley, Israel) deposited in MfN. A female (OSAC collection number 0001418398) collected
<figure><img src="image_14.png" /><figcaption>**Figure 12** *Macrocheles karkomensis* n. sp. protonymph. Dorsal Idiosoma.</figcaption></figure>

in Kfar Yehoshua (Jezreel Valley, Israel) deposited in OSAC.

Specimens with genetic information and sequence data are on BOLD in the dataset DS-MACROSPN (doi: 10.5883/DS-MACROSPN) and in GenBank (Table 5).

## DNA barcodes

COI DNA barcodes were recovered from 15 specimens, including three female paratypes (BOLD:AEV3149, dx.doi.org/10.5883/DS-MACROSPN). It was not possible to recover genetic material from two of the specimens, and one specimen showed signs of contamination during processing. However, the three paratype females were confirmed as belonging to the new species based on morphological characteristics.

COI Sequences of *M. karkomensis* n. sp. ranged in length from 332 to 658 bp and displayed an average intraspecific *p*-distance of 0.21% (range: 0–1.61%), probably due to the homogeneity of the specimens obtained from a laboratory colony. Comparison of these sequences to the BOLD reference library and GenBank indicated that these are the first public sequence records for *M. karkomensis* n. sp. The closest available match is a species from South Africa tentatively identified only as *Macrochelidae*, which we verified as belonging to *Macrocheles*, with 11.4% divergence (*p*-distance).

The minimal intraspecific variation observed in *M. karkomensis* n. sp., compared to
<figure><img src="image_15.png" /><figcaption>**Figure 13** *Macrocheles karkomensis* n. sp. protonymph. Ventral Idiosoma.</figcaption></figure>

the sizable variations among other *Macrocheles* species, suggests that COI-DNA barcodes
effectively distinguish between *Macrocheles* species. The highest intraspecific distance was
observed in *M. willowae* (max 5%), but it remained lower than the interspecific divergence
among *Macrocheles* species (13–30%). All COI sequences of *Macrocheles* species analysed
showed high divergence from outgroup species (22–35%).

The consensus tree derived from the BI analysis of COI sequences was relatively well-
supported, with most nodes having high posterior probabilities (11 nodes with supports greater
than 85% and nine nodes with 100% support). However, two nodes had lower posterior
probabilities of around 50% or less (Figure 14). Expanding sampling efforts with accurate
morphological identification and more biomarkers will refine our understanding of differences
between species and enhance the accuracy of future phylogenetic studies.

Remarks

*Macrocheles karkomensis* n. sp. is similar to *Macrocheles scutatus* (Berlese, 1904) described from specimens collected in Europe. *M. scutatus* is broadly distributed in the Old World (Berlese 1904; Filipponi and Pegazzano 1962; Walter and Krantz 1992).

A comparison between the new species herein described and *M. scutatus* was greatly
facilitated by a loan of *M. scutatus* specimens from Dr. Bruno Cicolani (Department of
Environmental Sciences, University of L'Aquila, Italy) (Figure 15), and by reference to published descriptions (Filipponi and Pegazzano 1962; Özbek, Bal and Doğan 2015; Walter and Krantz 1992). Adult female *M. scutatus* differs from *M. karkomensis n. sp.* in having a distinct procurved line on the dorsal shield and more pronounced ornamentation on the sternal shield. Filipponi and Pegazzano (1962) stated that the procurved line is only evident medially in some specimens of *M. scutatus*, but it appears to be present and complete in all of the specimens that we have examined. Most of the specimens in our collections of the new species have a weak procurved line, which in some of the type series is totally absent. In *M. scutatus*, sternal shield sutures *l.o.p.* are clearly united with *l.m.t.* anteriorly and joined to well-developed posterolateral rami medially, whereas in *M. karkomensis n. sp.* the sternal sutures are mostly represented by intermittent punctae (Figures 4 and 15a) that show neither clear *l.o.p/l.m.t.* convergence nor the presence of evident rami.

Differences between *M. karkomensis n. sp.* and *M. scutatus* tend to be clearer in males than in females. For example, the anterolateral margins of the ventrianel shield of *M. scutatus* males is concave adjacent to the insertions of coxae IV (Filipponi and Pegazzano 1962), while in *M. karkomensis n. sp.* males, anterolateral margins of the shield are slightly convex (Figure 8). Posterior dorsal setae Z4, Z5, S4 and S5 of *M. scutatus* males are elongate and distally pilose (Filipponi and Pegazzano 1962, Fig. VIII, 1), while in *M. karkomensis n. sp.* they are slightly longer than the small, smooth marginal *R* setae (Figure 8). Although not illustrated in the original description of *M. scutatus*, the posterior margin of the male dorsal shield of *M. scutatus* is crenulate, but is smooth in males of *M. karkomensis n. sp.* Males also differ in the pattern of ventral spurs on the legs, especially on legs IV (see Figures 16c,d and Table 6). Trochanter IV of male *M. scutatus* has a large rounded ventral spur (Fig. 16d), while femur IV has a complex of a large curved tubercle carrying terminal seta *v1* and two adjacent, underlying spurs; genu IV has a single small tubercle that carries seta *av1*. Trochanter IV of male *M. karkomensis n. sp.* (Fig. 16c) lacks a ventral spur, and seta *av1* is terminal on a barely perceptible tubercle.

<table><caption>**Table 5** Summary of the specimens, and COI barcode data for the specimens of *Macrocheles karkomensis n. sp.* collected from different locations in Israel and from laboratory colonies. Dashes indicate absence of genetic information.</caption><thead><tr><th>Museum ID</th><th>BOLD Sequence ID</th><th>Length</th><th>Identification method</th><th>Origin</th><th>Voucher Status</th><th>Stage</th><th>GenBank Accession Number</th></tr></thead><tbody><tr><td>ZMB-Arach 55437 **</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>♀</td><td>-</td></tr><tr><td>ZMB-Arach 55438 (before HUJINVACA603) *</td><td>PASMB635-22</td><td>658</td><td>BIN Match and morphology</td><td>Karkom</td><td>Vouched at MfN; DNA Vouched at CBG</td><td>♀</td><td>PP835205</td></tr><tr><td>ZMB-Arach 55439 (before HUJINVACA604)*</td><td>PASMB636-22</td><td>658</td><td>BIN Match and morphology</td><td>Karkom</td><td>Vouched at MfN; DNA Vouched at CBG</td><td>♀</td><td>PP835199</td></tr><tr><td>ZMB-Arach 55440 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>♀</td><td>-</td></tr><tr><td>ZMB-Arach 55441 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>♀</td><td>-</td></tr><tr><td>ZMB-Arach 55442 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>♀</td><td>-</td></tr><tr><td>ZMB-Arach 55443 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>♂</td><td>-</td></tr><tr><td>ZMB-Arach 55444 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>♂</td><td>-</td></tr><tr><td>ZMB-Arach 55445 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>♂</td><td>-</td></tr><tr><td>ZMB-Arach 55446 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>♂</td><td>-</td></tr><tr><td>OSAC 0001418397 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at OSAC</td><td>♂</td><td>-</td></tr><tr><td>ZMB-Arach 55447 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>DN</td><td>-</td></tr><tr><td>ZMB-Arach 55448 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>DN</td><td>-</td></tr><tr><td>ZMB-Arach 55449 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>DN</td><td>-</td></tr><tr><td>ZMB-Arach 55450 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>PN</td><td>-</td></tr><tr><td>ZMB-Arach 55451 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>PN</td><td>-</td></tr><tr><td>ZMB-Arach 55452 *</td><td>-</td><td>-</td><td>Morphology</td><td>Lab colony</td><td>Vouched at MfN</td><td>PN</td><td>-</td></tr><tr><td>HUJINVACA540*</td><td>PASMB572-22</td><td>-</td><td>Morphology</td><td>Hodaya</td><td>Vouched at HUJ</td><td>♀</td><td>-</td></tr><tr><td>HUJINVACA541</td><td>PASMB573-22</td><td>-</td><td>Morphology</td><td>Hodaya</td><td>Vouched at HUJ</td><td>♀</td><td>-</td></tr><tr><td>HUJINVACA542</td><td>PASMB574-23</td><td>-</td><td>Morphology</td><td>Hodaya</td><td>Vouched at HUJ</td><td>♀</td><td>-</td></tr><tr><td>ZMB-Arach 55453 (before HUJINVACA546)</td><td>PASMB578-22</td><td>658</td><td>BIN Match</td><td>Kfar Yehoshua</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835193</td></tr><tr><td>OSAC 0001418396 (before HUJINVACA547)*</td><td>PASMB579-22</td><td>658</td><td>BIN Match</td><td>Kfar Yehoshua</td><td>Vouched at OSAC; DNA Vouched at CBG</td><td>♀</td><td>PP835206</td></tr><tr><td>HUJINVACA548</td><td>PASMB580-22</td><td>658</td><td>BIN Match</td><td>Kfar Yehoshua</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835196</td></tr><tr><td>HUJINVACA549</td><td>PASMB581-22</td><td>332</td><td>Morphology</td><td>Kfar Yehoshua</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835191</td></tr><tr><td>HUJINVACA554</td><td>PASMB586-22</td><td>658</td><td>BIN Match</td><td>Mehola</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835202</td></tr><tr><td>HUJINVACA555</td><td>PASMB587-22</td><td>658</td><td>BIN Match</td><td>Mehola</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835192</td></tr><tr><td>HUJINVACA556</td><td>PASMB588-22</td><td>658</td><td>BIN Match</td><td>Mehola</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835207</td></tr><tr><td>HUJINVACA557</td><td>PASMB589-22</td><td>658</td><td>BIN Match</td><td>Mehola</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835201</td></tr><tr><td>HUJINVACA600</td><td>PASMB632-22</td><td>658</td><td>BIN Match</td><td>Karkom</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835195</td></tr><tr><td>HUJINVACA601</td><td>PASMB633-22</td><td>658</td><td>BIN Match</td><td>Karkom</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835198</td></tr><tr><td>HUJINVACA602</td><td>PASMB634-22</td><td>658</td><td>BIN Match</td><td>Karkom</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835190</td></tr><tr><td>HUJINVACA605</td><td>PASMB637-22</td><td>658</td><td>BIN Match</td><td>Karkom</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835197</td></tr><tr><td>HUJINVACA606</td><td>PASMB638-22</td><td>658</td><td>BIN Match</td><td>Karkom</td><td>Vouched at HUJ; DNA Vouched at CBG</td><td>♀</td><td>PP835200</td></tr></tbody></table>

<footnote>**Holotype, *Paratype, ♀: female, ♂: male, DN: deutonymph, PN: protonymph, MfN: Museum für Naturkunde, HUJ: Hebrew University of Jerusalem - National Collections of Natural History, OSAC: Oregon State Arthropod Collection, CBG: Centre for Biodiversity Genomics.</footnote>
<figure><img src="image_16.png" /><figcaption>**Figure 14** Consensus tree generated by Bayesian MCMC analysis (10 million generations) of COI fragment from available and confirmed *Macrocheles* including *Macrocheles karkomensis* n. sp. (indicated by labels colorized in blue and an asterisk) and two species of *Ornithonyssus*, *Macronyssidae* (*O. bacoti* and *O. sylviarum*) as an outgroup. Branch labels include the GenBank accession number followed by the specimen's Process ID (BOLD) of each specimen in parenthesis. Phylogeny constructed based on and supplemented from that published by Knee (2017). NA = information not available.</figcaption></figure>

The femur IV spur complex is similar to that of *M. scutatus*, but the curved tubercle carries a much smaller spiniform terminal seta than that present in *M. scutatus*. Finally, the cribra of *M. scutatus* and *M. karkomensis* n. sp. males are primarily marginal, but attached rows of paranal spicules may be seen in some *M. scutatus* specimens and in all of the male *M. karkomensis* n. sp. in the material at hand.

## Etymology

The specific name "karkomensis" to "Karkom", one of the type localities of the new species, and "ensis" denoting origin. Karkom is also the Hebrew name for the flowers of *Crocus*, also known as saffron.
<table><caption>Table 6 Morphological comparison between species of the *scutatus* complex, *scutatus* subgroup. - = unknown, WI = without information.</caption><thead><tr><th>Characteristic</th><th>Macrocheles agnosticus Walter & Krantz, 1992</th><th>Macrocheles dispar (Berlese, 1910)</th><th>Macrocheles medialis Berlese, 1918</th><th>Macrocheles neoscutatus Krantz, 1972</th><th>Macrocheles neovernalis Ryke & Meyer, 1958</th><th>Macrocheles subscutatus Walter & Krantz, 1992</th><th>Macrocheles scutatus (Berlese, 1904)</th><th>Macrocheles karkomensis n. sp.</th></tr></thead><tbody><tr><td>Female dorsal shield setae ornamentation</td><td>Thickened and distally pilose-pectinate (z1, z5 and j6 smooth in some specimens; J5 serrate)</td><td>Most smooth (j4, z4, r2, r3 usually, and S5 and Z5 always distally pectinate/pilose)</td><td>Most smooth (j1 long, slender, and distally pilose, j4 and z4 distally pectinate, J5 acicular and serrate)</td><td>Most smooth (j1, r3 <named as r5 in original description>, z4 <named as z1> distally plumose/pectinate; J5 weekly pectinate; j4 lost <named as i3>)</td><td>Most smooth (j1 distally pectinate, j4 sparsely pilose, J5 serrate)</td><td>Most smooth (j1, j3-4, z4, s2, r2-3 distally pectinate)</td><td>Most smooth (j1, j4 and z4 distally pilose; J5 finely pilose). According to Özbek et al. (2015), S5 and Z5 also distally pilose*</td><td>Most smooth (j1, j4 and z4 distally pilose; J5 slightly serrate)</td></tr><tr><td>Length of female dorsal shield (µm)</td><td>667-759</td><td>644-886</td><td>650</td><td>780</td><td>621-719</td><td>699-828</td><td>463-707</td><td>571-623</td></tr><tr><td>Female procurved line</td><td>Present</td><td>Present</td><td>Present, strongly produced</td><td>Poorly defined behind insertion of z6 (named as z3)</td><td>Not shown in figure (fig. 13) of original description, but present in all the species of the glaber species group according to Walter (1985)</td><td>Present</td><td>Present, weak in some specimens. Evident in the middle portion according to Filipponi and Pegazzano (1962).</td><td>Weak or lost in some specimens. When present, as a faint line and only in the middle portion (posterior to z6 and s5).</td></tr><tr><td>Female postanal and opisthogastric setae in un-sclerotised</td><td>distally pilose-pectinate</td><td>smooth?</td><td>smooth</td><td>smooth</td><td>smooth</td><td>smooth</td><td>smooth</td><td>smooth</td></tr><tr><td>Ornamentation of female sternal shield</td><td>l.o.p. disjunct from l.m.t. and without ramus; a.p. lost; l.arc. faint</td><td>l.o.p. disjunct from l.m.t., and without ramus; a.p. reduced to a few punctae (or lost), l.arc. weak</td><td>l.o.p. united to l.m.t and with ramus; a.p. distinct, coarsely punctate; l.arc. as two parallel lines, weakly procurved medially</td><td>l.o.p. united to l.m.t and with ramus; a.p. distinct; distinctly reticulate anterior to l.m.t.; a.p. finely punctate; l.arc. indistinct</td><td>l.o.p. disjunct from l.m.t. and without ramus; a.p. reduced to a few punctae (or lost), l.arc. lost; smooth posterior to l.o.p.</td><td>l.o.p. united to l.m.t and without ramus; a.p. reduced to a few small scattered punctae; l.arc. faintly punctate</td><td>l.o.p. united to l.m.t. and with ramus; a.p. distinct, coarsely punctate; larc. single and strongly procurved</td><td>l.o.p. disjunct from l.m.t. and with an weak ramus in some specimens; a.p. reduced to a few small scattered punctate; larc. single and strongly procurved</td></tr><tr><td>Male Male dorsal shield setae ornamentation</td><td>Unknown</td><td>Known WI</td><td>Unknown</td><td>Unknown</td><td>Unknown</td><td>Unknown</td><td>Known Most smooth (j1, j4, z4, r2, r3, S4, S5 and Z5 distally pilose; J5 finely pilose)</td><td>Known Most smooth (j1, j4 and z4 distally pilose; J5 slightly serrate)</td></tr><tr><td>Male Z4</td><td>-</td><td>WI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>reaching with their tip the base of the Z5</td><td>not reaching half the distance between its base and the base of Z5</td></tr><tr><td>Shape of male ventranal shield</td><td>-</td><td>Subcordate: convex anterolateral margin adjacent to the insertions of coxe IV</td><td>-</td><td>-</td><td>-</td><td>-</td><td>Deeply concave anterolateral margin adjacent to the insertions of coxe IV</td><td>Peltate: slightly convex anterolateral margin adjacent to the insertions of coxe IV</td></tr><tr><td>Male legs</td><td>-</td><td>Femur II with a large spur. Femur IV with a spur and a tubercle carrying a seta.</td><td>-</td><td>-</td><td>-</td><td>-</td><td>Trochanter IV has a triangular spur. Femur IV with two spurs and a tubercle carrying a terminal thick-spiniform seta (v1). Genu IV with av 1 on a tubercle.</td><td>Trochanter IV without spurs. Femur IV with two spurs and a tubercle carrying a terminal small spiniform seta (v1). Genu IV with av 1 on a barely visible tubercle.</td></tr><tr><td>Distribution</td><td>South Africa</td><td>Asia (China, Java, Philippines, Taiwan, Vietnam)</td><td>Southern Africa</td><td>Northern Europe (Hamburg, Germany)</td><td>Southeast Africa (Potchefstroom, South Africa; Mozambique; East Africa; Zaire)</td><td>Southern Africa (Tanzania, Zimbabwe, South Africa)</td><td>Old World (Europe, North Africa, Israel, India, China, China, Philippines, Central Asia, New Zealand)</td><td>Israel</td></tr><tr><td>Known phoretic associations</td><td>Scarabaeus (Coleoptera: Scarabaeidae)</td><td>Catharsius molossus (Linnaeus, 1758), Catharsius sp., Enoplotrupes, and Paragymnopleurus (Coleoptera: Scarabaeidae, Geotrupidae)</td><td>Heliocopris (Coleoptera: Scarabaeidae)</td><td>Unknown</td><td>Heliocopris and Catharsius (Coleoptera: Scarabaeidae)</td><td>Catharsius, Copris, Onthophagus, Scarabaeus and Heliocopris (Coleoptera: Scarabaeidae)</td><td>Geotrupes, Onitis, Copris, Onthophagus (Coleoptera: Geotrupidae, Scarabaeidae), Rattus exulans (Peale, 1848) (Rodentia: Muridae)</td><td>Unknown</td></tr><tr><td>Reference</td><td>(Walter and Krantz 1992)</td><td>(Berlese 1910; Walter and Krantz 1992)</td><td>(Berlese 1918; Walter and Krantz 1992)</td><td>(Krantz 1972; Walter and Krantz 1992)</td><td>(Ryke and Meyer 1958; Walter 1985; Walter and Krantz 1992)</td><td>(Walter and Krantz 1992)</td><td>(Berlese 1904; Filipponi and Pegazzano 1962; Walter and Krantz 1992; Özbek, Bal and Doğan 2015)</td><td>This study</td></tr></tbody></table>

<footnote>* Noted on Özbek, Bal and Doğan (2015), but holotype has not been examined.</footnote>
<figure><img src="image_17.png" /><figcaption>**Figure 15** Morphological comparison between an Italian population of *Macrocheles scutatus* and *Macrocheles karkomensis n. sp.* (a) Phase-contrast image of the sternal shield of *M. karkomensis n. sp.*, (b) DIC image of the sternal shield of *M. karkomensis n. sp.*, (c) DIC image of sternal shield of *M. scutatus*, (d) DIC image middle portion of dorsal shield of *M. karkomensis n. sp.*, and (e) DIC image of middle part of dorsal shield of *M. scutatus*. *l.lang.*: linea angulata; *l.arc.*: linea arcuata; *l.m.t.*: linea media transversa; *l.o.a.*: linea obliqua anterior; *l.o.p.*: linea obliqua posterior; *rm*: ramus of *l.o.p*; *w*. *rm*: weak ramus of *l.o.p*; *p.l.*: procurved line; *w*. *p.l.*: weak procurved line.</figcaption></figure>

**Biological observations**

*Macrocheles karkomensis n. sp.* has been observed in the laboratory to consume nematodes of the following species: *Acrobeloides bodenheimeri* (Steiner, 1936) Thorne, 1937 (Rhabditida: Cephalobidae), *Oscheius tipulae* (Lam & Webster, 1971) (Rhabditida: Rhabditidae), *Pristionchus pacificus* Sommer, Letter, Kim & Sternberg, 1996 (Rhabditida: Diplogastridae), *Panagrellus nepenthicola* (Menzel, 1922) Goodey, 1945, *Panagrellus redivivus* (Linnaeus, 1767) Goodey, 1945, *Panagrellus silusioides* Thalolikhin, 1965 (Rhabditida: Panagrolaimidae), *Meloidogyne incognita* (Kofoid & White, 1919) Chitwood, 1949 (Rhabditida: Meloidogynidae) and two unidentified species of *Panagrolaimus* (Rhabditida: Panagrolaimidae).

Immature development in the laboratory was shortest for mites fed on *A. bodenheimeri* and the two *Panagrolaimus* species (approximately four days). Duration was slightly longer for mites fed with *M. incognita* (about five days). Mites failed to develop on *O. tipulae* beyond the protonymphal stage (Rueda-Ramírez et al. 2023). Mite fecundity was highest when feeding on *A. bodenheimeri* and was almost null (approximately 0.12 eggs/female) when feeding on *M. incognita* (Rueda-Ramírez et al. 2023). *Macrocheles karkomensis n. sp.* has also been observed to feed on the astigmatine oribatid *Carpoglyphus lactis* (Linnaeus, 1758) (Carpoglyphidae) (Rueda-Ramírez pers. obs).

Unlike most of the known species of the *scutatus* subgroup, no phoretic associations are
<figure><img src="image_18.png" /><figcaption>**Figure 16** Morphological comparison between an Italian population of *Macrocheles scutatus* and *Macrocheles karkomensis* n. sp. (a) DIC image of the ventrianal shield of *M. karkomensis* n. sp., (b) DIC image of the ventrianal shield of *M. scutatus*, (c) DIC image of leg IV of *M. karkomensis* n. sp., (d) DIC image of leg IV of *M. scutatus*.</figcaption></figure>

known for *M. karkomensis* n. sp. However, females in laboratory cultures readily grasped the hairs of a fine brush when offered, suggestive of a phoretic response.

**Discussion**

The *scutatus* complex was proposed by Walter and Krantz (1992) within the *scutatus* subgroup of the *glaber* group, to include seven species. This complex comprises species with reduced ornamentation on the ventral shields, but which retain the basic *glaber* ornamentation. Species were separated mainly by degree of pilosity of the female idiosoma and the pattern of ornamentation of their sternal shield (Walter and Krantz 1986; Walter and Krantz 1992) (Table
6).

Our observations of *M. karkomensis n. sp.* and of *M. scutatus* show that male characteristics may play an important role in the separation and identification of species in the *scutatus* complex. Unfortunately, few males of the *scutatus* complex have been described (*M. karkomensis n. sp.*, *M. scutatus*, and *M. dispar*). Unlike the phoretic females, most of which have been collected from scarabaeine beetle phoronts, males of *M. karkomensis n. sp.* and *M. scutatus* have been recovered only from laboratory-reared populations. The provenance of known *M. dispar* males (Berlese 1918) is unclear, but data in the Berlese Acaroteca in Florence suggests that at least some male dispar may have been collected along with females on the same individual beetle (*Copris* sp.) (Castagnoli and Pegazzano 1985).

The females of the species here described conform to the diagnosis for the *scutatus* complex proposed by Walter and Krantz (1992). However, knowledge of the males of the other species will allow better characterization of the diagnoses. Based on the present status of our knowledge of the males of this complex, a new characterization of recognized *scutatus* complex species based on females and known males is presented below (Table 6).

## Updated characterization of the *scutatus* complex

Sternal shield typically longer than wide and with reduced ornamentation; *l.arc.* not deeply punctate, sometimes weak, occasionally doubled; *l.m.t.* distinct, *l.o.p.* weak, *ramus* often lost or weak; *a.p.* reduced to scattered punctae or lost. Epigynal shield varying from smooth to reticulate, with reticules formed by punctate lines. Ventrianal shield rounded-subcordate, length equal to or greater than width, evenly reticulate; postcoxal pore (gv2) distinct and adjacent to parapodal plate. Dorsal shield reticulate, with procurved line present, sometimes weak; setae *j1* appressed and distally pilose, setae *j4* and *z4* similarly ornamented and often thickened, *J5* serrate. Known males with separate sternitigenital and ventrianal shields; ventrianal shield with deeply concave or convex (subcordate or peltate) anterolateral margins adjacent to insertions of coxa IV; femur II with one large spur; leg IV heavily ornamented with several spurs: femur IV with 1–2 spurs and a tubercle, genu IV with tiny or large protuberance, and tarsus IV with a distal rounded spur.

## Updated key to known *scutatus* complex species initially published by Walter and Krantz (1992)

<table><tr><td>1. Setae *j3* distally pilose. Southern Africa</td><td>2</td></tr><tr><td>— Setae *j3* smooth</td><td>3</td></tr></table>

<table><tr><td>2. Dorsal shield setae bipectinate (*z1*, *z5* and *j6* may be smooth in some specimens), postanal and opisthogastric setae similarly ornamented</td><td><i>M. agnosticus</i></td></tr><tr><td>— Most dorsal shield setae smooth (*j1*, *j3-4*, *s2*, *r2-3* bipectinate distally); postanal and opisthogastric setae without ornamentation</td><td><i>M. subscutatus</i></td></tr></table>

<table><tr><td>3. <i>L.o.p.</i> disjunct from <i>l.m.t.</i> and with a weak <i>ramus</i> or without <i>ramus</i>, <i>a.p.</i> reduced to a few punctae, or lost</td><td>4</td></tr><tr><td>— <i>L.o.p.</i> united to <i>l.m.t.</i> and with <i>ramus</i>; <i>a.p.</i> distinct</td><td>6</td></tr></table>

<table><tr><td>4. <i>L.arc.</i> lost; setae *j4* distally pectinate, other dorsal setae smooth. Southern Africa</td><td><i>M. neovernalis</i></td></tr><tr><td>— <i>L. arc.</i> present or weak; setae *j4* and *z4* distally pilose. Male ventrianal shield with convex anterolateral margin adjacent to the insertion of coxae IV</td><td>5</td></tr></table>

<table><tr><td>5. <i>L. arc.</i> weak; setae *r2* and *r3* usually distally pilose. Femur IV of male with a spurs and a set setiferous tubercle. Asia</td><td><i>M. dispar</i></td></tr></table>
— *L. arc.* strongly procurved; setae *r2* and *r3* always simple. Femur IV of male with two spurs and tubercles with a small terminal spiniform seta. Israel. *M. karkomensis* n. sp.

6. Sternal shield distinctly reticulate anterior to *l.m.t.*, *l.arc*. indistinct; *a.p.* finely punctate. Western Europe. *M. neoscutatus*
— Sternal shield with one or two *l.arc.*, without distinct reticulation as above; *a.p.* coarsely punctae. 7

7. With a single strongly procurved *l.arc*. Male ventrianal shield with deeply concave antero-lateral margin adjacent to the insertions of coxae IV. Presently considered cosmopolitan. *M. scutatus*
— With two transverse parallel *l.arc.* which are weakly procurved medially. Southern Africa. *M. medialis*

Additional observations

Based on Calugar (2008) and O. Makarova (pers. comm.), the *scutatus* complex appears to include additional species whose females display little or no significant morphological differences from females of *M. scutatus s. str.*, but which may be distinguished based on male characters alone. Clearly, characterization of the complex must be considered as ongoing, eventually to be supplemented with data from additional *scutatus* populations worldwide and hopefully supported by comparative genetic information.

An interesting observation in the immature stages of the new species described here is the absence of seta Zv1 in the deutonymph, which only appears in the adult stage. Evans &Till (1965) observed that, at least in certain dermanyssoid Gamasina, opistogastric setae Zv1 first appear in the deutonymphal stage. In the new species, Zv1 appears to be smaller and more anteriorly located than observed in other species of the same complex (Walter and Krantz 1992). However, we interpret its presence in adults as being associated with *gv2* and *ivo*, suggesting the possibility of its loss in the deutonymph stage and reappearance in the adult stage. At this point, it is not possible to conclude that loss and reappearance of Zv1 is characteristic of the *scutatus* complex or of its subgroup, given the limited information presently available on immatures.

Variation in opistogastric chaetotaxy of some female Macrochelidae was reported earlier by Hyatt & Emberson (1988). Similar variation was found in males of *M. karkomensis* n. sp. during this study, particularly on the sternogenital shield. Most of the males in our series had only one seta *st4* (Figure 8a, c), while in others both *st4* setae were either present or absent (Figure 8b).

Another characteristic seen in adults of *M. karkomensis* n. sp. is the presence of two
lyrifissures on the peritreme. These structures have previously been observed in *Macrocheles
embersoni* Azevedo, Castilho & Berto (2017) and *Macrocheles neotransversus* Quintero-
Gutierrez and Cómbita-Heredia (2020). Based on our observations, the lyrifissures referred to
as *gp*? and *gp* in the original description of *M. neotransversus* are homologous with the anterior
and posterior peritrematic lyrifissures depicted in *M. embersoni* by Azevedo *et al.* (2017).
These structures are identified in this paper as lyrifissures *ipc1* and *ipc2* (Figures 2a (*ipc1*
expressed anterodorsally) and 4 in the female, and in Figures 7a (also expressed anterodorsally)
and 8a in the male). Therefore, the pore and the lyrifissure on the peritrematic shield correspond
to *gd3* (anterior) and *ip* (posterior) (Figure 17).

Although genetic information on most species of the *scutatus* group is not currently
available, we were able to include in our genetic comparison with *M. karkomensis* two species
that appear to belong to this group or are close to it. In light of their potential usefulness as
biological control agents (Azevedo *et al.* 2015; Krantz 1983), further efforts should be made to
expand collection of *scutatus* group species so that reliable morphological identifications may
<figure><img src="image_19.png" /><figcaption>**Figure 17** Comparison of peritreme and peritrematic lyrifissures and pores of *a* – *Macrocheles embersoni* Azevedo, Castilho & Berto, 2017 (modified from Azevedo *et al.* 2017), *b* – *Macrocheles neotransversus* Quintero-Gutierrez and Cómbita-Heredia, 2020 (modified from Quintero-Gutierrez *et al.* 2020), and *c* – *Macrocheles karkomensis* *n. sp.*</figcaption></figure>

be correlated with genetic information extracted from collected specimens (Rueda-Ramírez *et al.* 2022; Young *et al.* 2019; Young and Hebert 2022). This in turn may lead to a better understanding of species differences and increased precision in phylogenetic, ecological, and practical application studies. We also highlight the importance of including more molecular markers to increase phylogenetic precision (Rokas and Carroll 2005) and confirm identity of specimens whose sequences are publicly available.

**Acknowledgements**

To Shira Gal, Newe Ya'ar Research Center, Agricultural Research Organization (ARO), Israel, for his outstanding assistance with colonies, photos and conveying biological material. To Jason Dunlop, Anja Friederichs, and Birger Neuhaus, Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, Berlin, Germany, for opening a space for the first author to look through the collection, use the microscopes and work with the material. To Bruno Cicolani for the loan of slides of *M. scutatus* from his personal collection. To Thomas Stach and Peer Martin, Institute of Biology, Molecular Parasitology group, Humboldt Universität zu Berlin, Germany, for granting access to their scanning electron microscope, advising and helping in preparing the samples, and taking photographs. To Gilberto J. de Moraes, Department of Entomology and Acarology, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Brazil, for answering questions, reviewing the manuscript, and providing valuable suggestions. To María L. Moraza, Universidad de Navarra, Instituto de Biodiversidad y Medioambiente, for her time and invaluable and accurate comments and suggestions on the adenotaxy and porotaxy of *M. karkomensis*. To Olga Makarova, Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, for important information shared, and for her enthusiasm for future cooperative research on the *scutatus* complex. To Wayne Knee, Agriculture and Agri-Food Canada, for kindly sending photos of his specimens and for allowing
us to base our phylogenetic analysis on his previously published analysis of some *Macrocheles* species. Finally, to the anonymous reviewers whose comments and suggestions significantly improved the manuscript. Funding: This work was supported by German Research Foundation (DFG) [grant number RU780/20-1] ('Harnessing the soil food web for the biological control of rootknot nematodes').

ORCID

Diana Rueda-Ramírez https://orcid.org/0000-0002-7642-2256

Gerald W. Krantz https://orcid.org/0000-0002-5241-1771

Eric Palevsky https://orcid.org/0000-0002-4958-8920

Liliane Ruess https://orcid.org/0000-0001-8061-6208

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