Integrating morphology with phylogenomics to describe four island endemic species of Temnothorax from Sicily and Malta (Hymenoptera, Formicidae)

. Temnothorax (Myrmicinae, Crematogastrini) is one of the most diverse Holarctic ant genera, and new taxonomic advancements are still frequent worldwide. The Mediterranean region, a global biodiversity hotspot characterized by a complex geographic history, is home to a substantial portion of its described diversity. Sicily is the region’s largest island and, as ongoing investigations are revealing, it is inhabited by a long-overlooked but highly diverse ant fauna that combines multiple biogeographic influences. We combined qualitative and quantitative morphology of multiple castes with phylogenomic analysis based on ultra-conserved elements (UCEs) to describe four species of Temnothorax endemic to Sicily and the neighboring Maltese Islands (Sicilian Channel). Three of these species, T. marae Alicata, Schifani & Prebus sp. nov., T. poldii Alicata, Schifani & Prebus sp. nov. and T. vivianoi Schifani, Alicata & Prebus sp. nov., are new to science, while a redescription clarifies the identity of T. lagrecai (Baroni Urbani, 1964). These descriptions highlight the current difficulties of delimiting monophyletic Temnothorax species groups based on morphological characters. The intra-insular endemicity patterns we revealed highlight the importance of Mediterranean paleogeography to contemporary ant diversity and distribution in the region.


Introduction
The hyperdiverse genus Temnothorax Mayr, 1861 is mainly distributed across the Holarctic and northern Neotropical regions and is only marginally present in the northern Afrotropics (Prebus 2015;Janicki et al. 2016;Guénard et al. 2017). It contains more than 450 valid species, including a notable number of species with small geographic ranges (Bolton 2021). Part of the tribe Crematogastrini Forel, 1893 (see Ward et al. 2015), Temnothorax are mostly diminutive, inconspicuous, timid and cryptically colored ants, which form small colonies with only a few notable exceptions (Prebus 2017;Seifert 2018). Due to these characteristics, most species are easily overlooked during field surveys, yet many may play important ecological roles (e.g., Prebus 2017;Seifert 2018;Giannetti et al. 2022). In addition, while the vast majority of the species are free-living, several are social parasites, the correct naming of which has become a debated issue among myrmecologists (Seifert et al. 2016;Ward et al. 2016). Here, we adopt the nomenclatural system proposed by Ward et al. (2015).
During the last decades, efforts to describe the taxonomic diversity of Temnothorax have multiplied globally: out of the about 490 valid species and subspecies, some 150 new species were described during the last 20 years, mostly from Asia and North America (Bolton 2021). Moreover, a major advancement in the understanding of the genus' phylogenetic diversity was recently achieved thanks to the analysis of more than 100 described and undescribed morphospecies belonging to 37 species-groups, resulting in the discovery of 7 major clades (Prebus 2017(Prebus , 2021b. In Europe, where three of these clades occur (Prebus 2017), the taxonomy of the genus has long been plagued by a lack of reliable diagnostic keys and descriptions of many so-called 'transitional forms' of uncertain taxonomic status, but has improved substantially during the last decades. The better investigated fauna is that of mainland Europe, where many of the recently conducted taxonomic investigations have uncovered cryptic diversity within the large geographic ranges of some species (e.g., Csősz et al. 2015Csősz et al. , 2018Seifert & Csősz 2015). For instance, the fauna of Central and Northern Europe consists of 24 species with relatively broad geographic distributions, and keys for their workers and queens are available (Seifert 2018). However, Europe and Asia Minor are expected to host some 150 species in total (Seifert 2018), and over 200 taxa are described from the Mediterranean region, which acts as a global biodiversity hotspot for the genus (Borowiec 2014). In this context, descriptions of non-cryptic species, usually with narrower geographic ranges, are not uncommon (e.g., Galkowski & Cagniant 2017;Salata et al. 2018;Salata & Borowiec 2019;Tinaut & Reyes-López 2020;Arcos 2021). Large Mediterranean islands may host a particularly rich and unique fauna. For instance, Crete is inhabited by 17 species, half of which are considered endemic (Salata et al. , 2020. Such diversity and high levels of endemicity are unparalleled by any other ant genus of the region, but much of the local fauna of Temnothorax still remains undescribed or little-known, and some serious efforts in this direction are required in the face of the threats the regional biodiversity is facing (Cuttelod et al. 2009).
Here, we present the descriptions of three new non-cryptic species of Temnothorax that we have found exclusively in Sicily or in the neighboring Aegadian and Maltese islands, as well as a redescription of T. lagrecai. We integrate qualitative and quantitative morphology of multiple castes with phylogenomic analysis of molecular data from ultra-conserved elements (UCEs), comment on the ecological and biogeographical features of these species, and illustrate their position within the global phylogeny of the genus. These descriptions allow us to present a complete key to the Sicilian light-colored species of Temnothorax with concolorous antennal clubs, and to comment on the current difficulties in speciesgroup classification of Temnothorax based on morphology.

Species delimitation criteria
We focus on identifying non-cryptic species, here defined as species that differ one from another by multiple qualitative morphological characters in a consistent way (i.e., in > 95% of the examined individuals), and we consider such differences to represent a reliable proxy indicating "separable clusters that have passed a threshold of evolutionary divergence" sensu Seifert (2020). To achieve a satisfactory species delimitation and a fully informative species description, we integrate the evidence provided by qualitative morphological characters with quantitative morphology as well as phylogenomic evidence in the form of an analysis of ultra-conserved element (UCE) locus data, which we integrate into the global phylogeny of the genus Temnothorax presented in Prebus (2017). Finally, we carefully interpret the results considering the available biogeographic data. Support from these different sources of information are then integrated and discussed to produce final species hypotheses.

Examined material
Sicilian specimens of Temnothorax were primarily obtained through field-collecting across the island by the authors and occasional collaborators as described in Schifani et al. (2021a) (Fig. 1), extending to the Italian Peninsula and the circum-Sicilian islands (including the Maltese islands). Sampling techniques consisted of direct sampling, soil sifting, pitfall traps, and Milieu Souterrain Superficiel (MSS) traps (Agosti et al. 2000;Mammola et al. 2016). These samples were compared either through pictures or direct investigation of other specimens of Temnothorax from our collections, specimens from museum collections (investigated directly or through photographs and belonging to: Stephen Schembri personal collection) and specimens from museum and private collections whose high-quality images are available on the AntWeb.org archive (Bolton 2021).
Concerning the four taxa treated in this study, we examined 1180 workers, 8 males and 28 queens from 60 collecting sites, whose full list is given in Supp. file 1: Table S1. Holotype specimens of the species described in this study were deposited at the Museo Civico di Storia Naturale "G. Doria" (MSNG -Genoa, Italy).

Qualitative morphological characters
Directly available specimens were examined under stereoscopic microscopes at 60-180 × magnification. Qualitative morphological characters were defined as discrete characters (presence or absence of certain traits) easily observable by trained myrmecologists without the need of detailed numerical recording, and were investigated on all specimens examined during this study. The following characters were used in this study: Antennal club pigmentation Antennal clubs may be concolorous with the rest of the antenna, or, in light-colored individuals, they can also exhibit a darker pigmentation (Figs 2-3). This is considered a steady character in several (albeit not all) European species of Temnothorax (Seifert 2018).

Antennal segments
The standard number of segments in the Euro-Mediterranean species of Temnothorax is 12 in females (workers and queens) and 13 in males, but in at least one European free-living species, T. flavicornis, as well as in most members of the gordiagini group (i.e., of the former genus Myrmoxenus Ruzsky, 1902), this number is reduced to 11 in females and 12 in males (Emery 1870(Emery , 1895Figs 2-3).

Eye size
The relative size of compound eyes is quite variable in Temnothorax, and is evaluated as a quantitative character in this study (see eye length [EL], eye width [EW] and their arithmetic mean [EYE]), but in the reference area there is a group of 'large-eyed' species distributed from the Maghreb to Iberia that represents a clear outlier. Outside this condition, eyes are defined 'normal', while finer differentiation is achieved by means of quantitative morphology .

Metanotal groove of workers
The metanotal groove (or depression) is generally either present or absent in a single species of Temnothorax (Figs 6-7), sometimes becoming a species group-defining character (Seifert 2018;Prebus 2021b). In dubious cases, to facilitate its observation the standard lateral view of the mesosoma may be angled slightly upwards.  (Santschi, 1922), type worker from Italy (CASENT0912964), photo by Will Ericson, antenna of 12 segments and darkened club. (Emery, 1870), type worker from Italy (CASENT0904761), photo by Will Ericson, antenna of 11 segments and concolorous club.

Propodeal spines
The length of propodeal spines is a quantitative character in this study (see the distance between the center of the propodeal spiracle and the spine tip [SPST]), but at the same time we also rely on simpler categorical definition for practical reasons, since the species treated in this study show a rather dichotomic division in long-spined and short-spined species. These categories refer to workers, and are based on the length of the propodeal spine visually compared with that of the propodeal declivity as in Prebus (2021b): short spines are those that are long about ⅓ or less of the propodeal declivity; long spines are those that are roughly long at least ½ of the propodeal declivity (Figs 6-7).
Concerning males, propodeal spines are entirely absent in most of the described W-Palearctic Temnothorax, but may be present in some cases, sometimes as normally-developed spines similar to those of workers and queens, sometimes as small dentiform protuberances (Figs 8-10).

Petiole shape
In species of Temnothorax, the petiole shape exhibits a remarkable interspecific but in some cases also intraspecific variation, making it at the same time a very useful character and one that requires extra vigilance from the investigator. Two parts of the petiole are important in our revision and should be observed in lateral view : i) its dorsal profile, characterized by the way the anterior and posterior margins meet may form a single edge with no horizontal component or two edges delimiting a clear horizontal section; ii) the subpetiolar process, which may develop downwards in correspondence to the anterior part of the petiole, forming a tooth-like protuberance, or may be simply represented by a ventral carina to the petiole.

Surface sculpturing
Only two types of surface sculpturing play an important role in this paper, and our terminology is here a strong simplification of what is described by Harris (1979): we use the term alveolate sculpture whenever a light sculpture divides the surface in numerous small, relatively regular spaces; we use the term striae to describe parallel or subparallel lineations, more or less markedly raised, that may be linked by anastomoses. Finally, the term 'shiny' is used to indicate the apparent lack of sculpturing at the given magnifications, which produces a shiny and glossy appearance. Rugae may occur above a weaker background of alveolate or shiny sculpture. See Figs 13-15.

Quantitative morphological characters
Morphometric measurements were taken using high-quality pictures of specimens photographed with a Canon 1300D reflex and Canon MP-E 65mm f/2.8 1-5 × Macro Photo lens and the software ImageJ (Schneider et al. 2012). To analyze differences between the species, Principal Component Analysis (PCA) was employed using the software R and R Studio and the function princomp from the package stats (R Core Team 2021;Rstudio Team 2021). In these analyses, each species was represented by a set of workers about equally divided between multiple nests and sites, thus resulting in a similarly distributed number of pseudoreplicates. A total of 648 morphometries were recorded from 52 workers, 10 males and 10 queens by recording the following nine morphometric characters on each specimen = scape length, measured as the maximum length of the scapus but excluding the basal constriction or neck that occurs just distal of the condylar bulb SPST = maximum distance between the center of the propodeal spiracle and the distalmost tip of the spine. Measured in profile view of the specimen (not measured in spineless male specimens) All measurements are given in µm as mean ± standard deviation (minimum, maximum). Based on these nine characters, we computed CS (cephalic size) as the arithmetic mean between CL and CW and EYE as the arithmetic mean between EL and EW (Seifert 2018). Furthermore, we calculated indices to weight each of the nine characters based on the relative size of the specimen. To achieve this, we divided them by CS (Seifert 2018) with the following exceptions: SL and PoOC were divided by CL, while CL was divided by CW. Concerning the SL and PoOC indices, the aim was to provide a ratio more associated with the visual comparison of antennal scapes and post-ocular distance with cephalic length in frontal view. Concerning CL, dividing it by CW allowed us to describe of the shape of the cephalic capsule more clearly. All morphometric data are provided in Supp. file 1: Table S3.

Sequencing and analysis of Ultra-Conserved Elements (UCEs)
The sequence dataset used in this study was compiled from previously published datasets (Branstetter et al. 2017;Prebus 2017Prebus , 2021aBlaimer et al. 2018) as well as newly generated data (NCBI BioProject PRJNA770978). See Supp. file 1: Table S2 for NCBI accession numbers.
The sequenced taxa for this study were selected based on the sampling used in Prebus (2017), with the addition of populations of the four focal taxa.
For the newly generated data, adult worker ants were prepared for nondestructive DNA extraction by using a flame-sterilized size 2 stainless steel insect pin to pierce the cuticle of the head, mesosoma, and gaster on the right side of the specimens. These prepared specimens were then extracted using the DNeasy Blood & Tissue Kit (Qiagen, Inc.) following the manufacturer's protocols, using a 12-hour digestion in proteinase-K solution on a shaking heat block. The extracted specimens were stored in 95% ethanol prior to being mounted as molecular vouchers. Up to 50 ng of DNA was used as input, sheared to a target fragment size of 400-600 bp into a genomic DNA library preparation protocol for targeted enrichment of ultraconserved elements (UCEs) following Faircloth et al. (2015) as modified by Branstetter et al. (2017) using a unique combination of iTru barcoding adapters for each sample (Glenn et al. 2019; see Supp. file 1: Table S2 for a list of the adapters used). Enrichments were performed on pooled libraries using the custom version of the Hym 2.5Kv2A ant-specific RNA probes (Branstetter et al. 2017; ArborBiosciences, Ann Arbor, MI), which target 2524 UCE loci in the Formicidae Latreille, 1809. The library enrichment procedures for the probe kit were followed, except that the RNA probe concentration was reduced to 0.1X (note that this step is only necessary for the custom kit; the currently available catalog kit is already diluted to 0.1X concentration), custom adapter blockers were used instead of the standard blockers, and enriched DNA was left bound to the streptavidin beads during PCR, as described in Faircloth et al. (2015). Following post-enrichment PCR, the resulting pools were purified using SpeedBead magnetic carboxylate beads (Rohland & Reich 2012; Sigma-Aldrich) and adjusted their volume to 22 μL.
Enrichment success was verified and measured size adjusted DNA concentrations of each pool with qPCR using a SYBR ® FAST qPCR kit (Kapa Biosystems) and a Bio-Rad CFX96 RT-PCR thermal cycler (Bio-Rad Laboratories) and combined all pools into an equimolar final pool. The final pool was sequenced as a single lane at the High Throughput Genomics Facility at the University of Utah on an Illumina HiSeq 2500 (125 cycle paired end sequencing v4).
Following sequencing, raw reads were trimmed of adapter contamination, Illumina sequencing artefacts, and low-quality bases using the program illumiprocessor, which is included in PHYLUCE ver. 1.7.1 (Faircloth 2016). Cleaned reads were assembled denovo with PHYLUCE using SPAdes ver. 3.12.0 (Bankevich et al. 2012). All newly generated raw sequence reads have been submitted to the National Center for Biotechnology Information (NCBI) Sequence Reads Archive (BioProject PRJNA770978).
The standard PHYLUCE protocol was followed for processing UCEs in preparation for phylogenomic analysis, aligning the monolithic unaligned fasta file with the phyluce_align_seqcap_align command, using MAFFT (Katoh & Standley 2013) as the aligner (-aligner mafft) and opting not to edge-trim the alignment (-no-trim). The resulting alignments were trimmed with the phyluce_align_get_gblocks_ trimmed_alignments_from_untrimmed command in PHYLUCE, which uses GBlocks ver. 0.91b (Castresana 2000), using the following settings: b1 0.5, b2 0.5, b3 12, b4 7. After removing UCE locus information from taxon labels using the command phyluce_align_remove_locus_name_from_nexus_ lines, the alignment statistics were examined using the command phyluce_align_get_align_summary_ data, and a dataset was generated in which each locus contains a minimum of 85% of all taxa using the command phyluce_align_get_only_loci_with_min_taxa.
Because the assumption that the evolutionary rates of sequence data are homogenous is often violated in empirical data (Buckley et al. 2001), we partitioned our UCE loci into sets of similarly evolving sites. To achieve this, we used the command phyluce_align_format_nexus_files_for_raxml which concatenates loci into a single alignment, and generates a partition file for input into the SWSC-EN method (Tagliacollo & Lanfear 2018). The resulting datablocks were used as input for partitioning in IQTREE ver. 2.1.2 (Nguyen et al. 2015), using the command -m TESTNEWMERGEONLY. The substitution model was set to 'general time reversible' (-mset GTR), and the rate heterogeneity models were set to a subset of rate heterogeneity models that includes everything except the combination of gamma and proportion of invariable sites (-mrate E, I, G). The combination of gamma and proportion of invariable sites (+I+G) has been demonstrated to result in anomalies in likelihood estimation (Sullivan & Swofford 2001;Yang 2006). The search algorithm was set to -rclusterf 10. The resulting partitioned dataset was used as input for maximum likelihood tree inference in IQ-TREE, using 1000 ultrafast bootstrap replicates (-bb 1000).

Diagnostic character combination
Antennal clubs concolorous yellowish, antennae 12-segmented in females and 13 in males, eyes normal, metanotal groove very weak or absent, worker propodeal spines long, male propodeal spines dentiform, petiole upper profile usually with a short horizontal component, subpetiolar process toothlike, sculpturing mostly areolate and weak.

Etymology
Baroni Urbani (1964) dedicated this species to the Italian entomologist Marcello La Greca .

Material examined
We investigated 56 colony samples from 23 localities, consisting in a total of 212 ☿☿, 8 ♀♀, 2 ♂♂ from our collections, one colony under rearing in our possession, holotype queen and paratype workers from the MSNV and NHMW collections, and additional non-type workers from the personal collections of David Misfud and Stephen Schembri and from the MSNM. A detailed list is provided in the Supp. file 1: Table S1.

Redescription
Worker ( (120-175) coloration. Entirely yellowish with the exception of a variably large and often not clearly demarcated black transverse band on the gaster.
MesosoMa. May present a slight metanotal depression, its dorsal profile being from almost straight to definitely rounded in lateral view. Propodeal spines are relatively long (SPST/CS: 0.30 ± 0.02), usually proportionally thin in minor worker but sometimes considerably thick in large ones.
MetasoMa. The petiole in profile view usually presents a short but visible horizontal section. A subpetiolar process is normally visible, consisting of a small tooth angled down at up to 90°. Postpetiole ordinarily roundish in lateral profile, subrectangular in dorsal view.
surface sculpturing. Most of the body is characterized by a very fine areolate-rugose sculpture, the longitudinal component of which tends to be notably more marked on the frontal side of the head only in larger specimens. The gaster and appendages are smooth, as well as the clypeus and a variable area extending around the frons. A central longitudinal stria visible in the lower portion of the clypeus. Very sparse, occasionally suberect but usually erect setae all over the body; dense, fine, and mostly adpressed pilosity on all appendages, especially abundant on the antennal flagelli. coloration. Entirely yellowish with the main exception of parts of the gaster: at least the first tergite characterized by a blackish transverse band. Moreover, the mesoscutellar disk is darkened caudally.
MetasoMa. Dorsum of petiole in profile view without a horizontal section. Subpetiolar process usually visible, consisting of a small tooth. Postpetiole ordinarily roundish in profile, subrectangular in dorsal view.
surface sculpturing. Most of the body is characterized by a variably fine areolate-rugose sculpture, strong longitudinal rugae on the head. Clypeus. gaster and appendages smooth; a variable area extending around the frons, most of the pronotum, anepisterna, katepisterna, mesoscutellar disk and mesoscutum. A central longitudinal stria visible in the lower portion of the clypeus. Very sparse, occasionally suberect but usually erect setae all over the body; dense, fine, and mostly adpressed pilosity on all appendages, especially abundant on the antennal flagelli. coloration. Whole body yellowish, head slightly darker, appendages whitish, gaster dark and mesoscutellar disk caudally darkened.
surface sculpturing. Sculpture relatively strong on the head, with well-developed longitudinal striae on its frontal side, very light all over the propodeum, petiole, postpetiole and in some areas near the mesosoma lateral sutures, all the remaining parts smooth. A central longitudinal stria visible in the lower portion of the clypeus. Sparse, erect setae all over the body; fine and mostly adpressed pilosity over the appendages.

Distribution and biogeography (Figs 80-81)
Occurs almost all over Sicily and is also found in the Maltese Islands. In the past, Sicily and the Maltese Islands have been connected via an extensive land bridge covering a large area of currently shallow waters, and the last time such a connection existed was during the Last Glacial Maximum (Foglini et al. 2016).

Ecology and conservation
Relatively thermophilous, collected from 5 to 840 m a.s.l. (see Fig. 82). Temnothorax lagrecai inhabits Mediterranean shrublands and relatively open forests like native and artificial forests of Pinus halepensis Mill., sometimes found under trees of Quercus ilex L., but was also collected in artificial gardens in the leaf litter of trees of Citrus L. Due to the wide ecological niche and distribution it may face a relatively positive situation in conservationist terms, but further assessment would be interesting.

Nesting
Nests are found in the soil, probably opportunistically exploiting several kinds of microhabitats when available. On Monte Etna entire nests were found several times under moss.

Biology
Monogynous in all documented cases.

Social parasites
One colony was found to be hosting T. muellerianus (Finzi, 1922) in the R.N.O. Pino d'Aleppo (Vittoria).

Phenology
Flying queens and males (often attracted by artificial lights) and mating were observed in Mondello (Palermo) from the early days of July to late August at least.

Notes
Baroni Urbani (1964) assembled the type series of this species from an unspecified number of worker specimens and a single queen collected in Bosco di S. Pietro (Hyblaean Plateau, SE Sicily) by Giovanni Sichel. No additional information on this taxon was published after its description, and its name was mentioned only on very few occasions ( Baroni Urbani 1971;Salata & Borowiec 2019). Baroni Urbani decided to define the only queen he had as the holotype, despite the specimen being damaged (missing both antennae) and with the taxonomy of Temnothorax being mostly built around workers. He based the worker caste description on a specimen he defined as the 'ergatotype', but this definition has no legal value anymore according to the ICZN code, therefore that specimen must be considered as a simple paratype. We examined the holotype along with several paratypes preserved at the Natural History Museums of Verona and Vienna, but our efforts to find the ergatotype failed. Over 20 years ago, it was briefly observed at the home of Bruno Poldi (1920Poldi ( -2002 by one of us (AA), but it appears to be absent from the MNHM where Poldi's collection is kept, as well as from the MSNV, NHMB and NHMW where other material from Baroni Urbani and specimens of T. lagrecai are stored. According to its description, the ergatotype shows several characters deviating from all type and non-type workers of T. lagrecai that we have found; however, it is unclear whether this is due to the actual features of this lost specimen or an imprecise description. Temnothorax marae sp. nov. cooccurs with T. lagrecai in the Bosco di S. Pietro, yet it is not characterized by the sharp petiole and mesoepinotal furrow of the drawing of the ergatotype of T. lagrecai found in Baroni Urbani's original description. In any case, the holotype of T. lagrecai, although partly damaged, is fully coherent with the morphology of the queens collected alongside workers with the same characters of T. lagrecai that we present here, so that we consider the issue over the identity of T. lagrecai to be resolved.
Maltese records under Leptothorax rabaudi Bondroit, 1918 by Schembri & Collingwood (1981) are partly based on misidentified T. lagrecai according to an investigation of their voucher specimens.

Diagnostic character combination
Antennal clubs concolorous yellowish, antennae 12-segmented in females and 13 in males, eyes normal, metanotal groove absent, worker propodeal spines short, male propodeal spines absent, petiole upper profile usually with a short horizontal component but no well-defined edges, subpetiolar process carinalike, sculpturing mostly areolate and weak.

Etymology
The species is dedicated to Mara La Rocca, wife of Antonio Alicata.

Material examined
We investigated 17 colony samples from 11 localities, consisting in a total of 41 ☿☿, 2 ♀♀, 4 ♂♂ from our collections, one colony under rearing in our possession, and additional workers from the personal collections of David Misfud and Stephen Schembri. A detailed list is provided in the Supp. file 1: Table S1.  coloration. Entirely yellowish with the exception of the gaster, which is often entirely blackish (but in some colonies the black part forms a transverse band).
MetasoMa. The petiole in profile view rather blunt dorsally, lacking well-defined edges; on its anteroventral part, a small carina weakly emerges in profile view. Postpetiole ordinarily roundish in profile, subrectangular in dorsal view.
surface sculpturing. Most of the body characterized by a variably fine areolate-rugose sculpture, the longitudinal component of which tends to be more marked on the frontal side of the head. Clypeus, gaster, and appendages smooth; a variable area extending around the frons. Very sparse, occasionally suberect but usually erect setae all over the body; dense, fine, mostly adpressed pilosity on all appendages, especially abundant on the antennal flagelli. surface sculpturing. Most of the body is characterized by a variably fine areolate-rugose sculpture, the longitudinal component of which tends to be more marked on the frontal side of the head. Clypeus, gaster, appendages, a variable area extending around the frons, anepisterna, katepisterna, mesoscutellar disk, and mesoscutum smooth. A central longitudinal stria visible in the lower portion of the clypeus. Very sparse, occasionally suberect but usually erect setae all over the body; dense, fine, and mostly adpressed pilosity on all appendages, especially abundant on the antennal flagelli. coloration. Whole body yellowish, appendages whitish, gaster dark.
MesosoMa. Propodeum spineless and rounded, most of the length of its lateral profile composed by the declivious posterior margin.
MetasoMa. Petiole ordinarily low and blunt. Postpetiole ordinarily roundish in lateral profile, subrectangular in dorsal view.
surface sculpturing. Sculpture relatively strong on the head, with well-developed longitudinal striae on its frontal side, very light all over the propodeum, petiole, postpetiole and in some areas near the Phylogeny (Fig. 79) Sister species of T. lagrecai, which together are closely related to T. flavicornis.

Distribution and biogeography (Figs 80-81)
Apparently restricted to SE Sicily (Hyblaean Plateau) and the Maltese islands. The Hyblean Pleateau may be the earliest sector of Sicily to have emerged from the Mediterranean Sea and has a strong biogeographic characterization (Guarino & Pasta 2018).

Ecology and conservation
More thermophilous than any other species treated in this study, it was found at altitudes between 5 and 175 m a.s.l. (see Fig. 82). Mainly found in the leaf litter of phryganas or shrublands, but also within a native P. halepensis forest and under riparian vegetation. Its habitat was likely severely reduced by strong anthropization of coastal areas and agricultural activities.

Biology
Small and monogynous colonies in all documented cases.

Social parasites
One colony was found to be hosting T. muellerianus (Finzi, 1922) in the R.N.O. Vendicari.

Nesting
Nests were always found on dead twigs, either on bushes or laying on the ground, although nesting directly on the soil or in other microhabitats on the ground may also occur.

Phenology
Males were produced under rearing conditions during July.

Diagnostic character combination
Antennal clubs concolorous and ferruginous, 12-segmented in females and 13 in males, eyes normal, metanotal groove absent, worker propodeal spines long, male propodeal spines absent, petiole upper profile usually with a clear horizontal component, subpetiolar process tooth-like, sculpturing characterized by distinct rugae, especially in larger workers, and a shiny area at the center of the frons.

Etymology
This species is dedicated to the Italian physician and amateur myrmecologist Bruno Poldi , which has also been a mentor and friend to Antonio Alicata in his early approach to the study of ants.

Material examined
We investigated 62 colony samples from 21 localities, consisting in a total of 820 ☿☿, 6 ♀♀, 2 ♂♂ from our collections. A detailed list is provided in the Supp. file 1: Table S1.  coloration. Entirely ferruginous with the possible exception of the gaster, which varies from slightly darker than the rest of the ant to substantially black in a minority of examined colonies.
MesosoMa. Without a metanotal depression, its dorsal profile being from usually rounded in lateral view. Propodeal spines relatively long and moderately thick (SPST/CS: 0.29 ± 0.02).
MetasoMa. Petiole in profile view relatively high, usually presenting a clear dorsal horizontal component in profile view. On its antero-ventral part, the subpetiolar process is usually visible, consisting of a small tooth. Postpetiole ordinarily roundish in lateral profile, subrectangular in dorsal view. surface sculpturing. Body sculpture relatively strong, with well-marked longitudinal rugae on the mesosoma, head sides, more variably on the dorsal surfaces of the waist segments. Development of anastomoses and variably finer areolate-rugose areas between the strong longitudinal rugae is quite variable in many areas, but usually absent in the lateral sides of the pronotum. Completely smooth areas are often observed between the larger rugae. The waist segments and some areas of the mesosoma may be entirely characterized by a finer areolate-rugose sculpture not interrupted by strong rugae. The gaster, appendages, and a long area from the clypeus to the occiput through the frons are smooth. Very sparse, occasionally suberect but usually erect setae all over the body; dense, fine, and mostly adpressed pilosity on all appendages, especially abundant on the antennal flagelli. coloration. Whole body dark reddish-brownish, appendages whitish.
surface sculpturing. Rugulose-areolate sculpture on head and propodeum, everything else smooth. Sparse erect setae all over the body; fine and mostly adpressed pilosity over the appendages. Phylogeny (Fig. 79) Appearing as the sister taxon of Temnothorax ibericus (Menozzi, 1922) among the sequenced species. Temnothorax ibericus is a mountain species like T. poldii sp. nov. and is endemic to Iberia (Espadaler et al. 2017), but the two species are not particularly similar in broad morphological terms, and close relatedness appears unlikely. (Figs 80-81) Occurs almost all over Sicily (although its ecological requirements clearly make it less widespread than T. lagrecai, e.g., absence from the westernmost section may be due to aridity and lack of suitable forests). An apparently similar niche is occupied by T. alienus in the southern Italian Peninsula (Aspromonte, Calabria). (Fig. 82) Collected between 340 and 1612 m a.s.l., mostly in hilly to mountainous conditions, always under tree coverage, usually consisting of different oak species. Massive historic deforestation of Sicily likely resulted in a huge habitat loss and fragmentation into isolated populations at least outside the mountain chains along the Tyrrhenian coast (Sicilian Apennines).

Nesting
Nests are found in the soil, probably opportunistically exploiting several kinds of microhabitats when available.

Biology
Monogynous in all documented cases.

Diagnostic character combination
Antennal clubs concolorous yellowish, antennae 12-segmented in females and 13 in males, eyes normal, metanotal groove absent, worker propodeal spines short, male propodeal spines absent, petiole upper profile usually without a horizontal component, subpetiolar process carina-like, sculpturing mostly areolate and weak.

Etymology
The species is dedicated to our friend Roberto Viviano, who donated to us most of the material of this species we had the opportunity to examine, collected by him during malacological surveys.

Material examined
We investigated 20 colony samples from 11 localities, consisting in a total of 111 ☿☿, 14 ♀♀, 1 ♂ from our collections. A detailed list is provided in the Supp. file 1: Table S1. coloration. Entirely yellowish with the exception of a black transverse band on the first gaster tergite.
MetasoMa. The petiole in profile view usually presents no dorsal horizontal component. Subpetiolar process carina-like. Postpetiole ordinarily roundish in profile, subrectangular in dorsal view. surface sculpturing. The body is almost entirely meticulously covered with a relatively fine areolaterugose sculpture, the longitudinal component of which tends to slightly more marked on the frontal side of the head. Clypeus, gaster, and appendages smooth; a variable area extending around the frons. Very sparse, occasionally suberect but usually erect setae all over the body; dense, fine, and mostly adpressed pilosity on all appendages, especially abundant on the antennal flagelli.
MetasoMa. The petiole in profile view sharp dorsally and rather high; on its antero-ventral part, a small carina weakly emerges in profile view. Postpetiole ordinarily roundish in lateral profile, subrectangular in dorsal view. surface sculpturing. Most of the body is covered with moderately marked longitudinal rugae, which are stronger on head. Very fine areolate sculpture is also present on propodeum and waist segments. Clypeus, gaster, and appendages smooth; a variable area extending around the frons, anepisterna, katepisterna, mesoscutellar disk and mesoscutum. Central longitudinal stria visible in the lower portion of the clypeus. Very sparse, occasionally suberect but usually erect setae all over the body; dense, fine, and mostly adpressed pilosity on all appendages, especially abundant on the antennal flagelli.
Male  MeasureMents and indices (1 individual -unfortunately, only a single damaged specimen of this species was found so far, solely consisting of the mesosoma, wings and legs). ML: 765; MW: 385.
MesosoMa. Propodeum spineless and rounded, in profile view propodeal declivity clearly shorter than the propodeal dorsum.

Ecology and conservation
Found from 75 to 1080 m a.s.l. (see Fig. 82). Most of our sites are characterized by forest or shrubs of Quercus ilex/Fraxinus ornus L. growing on rocky landslides or very rocky substrates, usually at the base of hundred meters-high north-facing cliffs hosting relatively wet and cool conditions compared to the surroundings. At least twice it was found in more open habitats characterized by rocky outcrops with sparse shrubs, in one case surrounded by agricultural areas. Populations may be fragmented due to anthropogenic fragmentation of forested areas and shrublands, but further assessment are required.

Biology
Small and monogynous colonies in all documented cases.

Nesting
Nests are found in the soil, probably opportunistically exploiting several kinds of microhabitats when available. The large number of the samples we examined consisted of colonies living entirely within abandoned shells of gastropods. In Monte Pellegrino, on several occasions entire colonies were found inside the abandoned shells of Marmorana (Murella) sicana (Férussac, 1822), a short-range endemic species which is locally very abundant. In Monte Pecoraro, a colony was found under a stone inside an abandoned shell of another short-range endemic of Palermo mountains, Chilostoma macrostoma (Rossmässler, 1837). It has also been found within shells of Rumina decollata (Linnaeus, 1758). However, these repeated findings may not indicate a true specialization in the use of abandoned gastropod shells, but a reflection of the nesting opportunities offered by many of the collecting sites, as well as the malacological nature of the sampling efforts during which it was found.

Phenology
Nuptial flights observed in late August in Mondello (Palermo), where queens were attracted by artificial lights.

Fig. 82.
Spatial characterization of the sites where the four species treated in this study were found.

Worker-based key to the Sicilian species of Temnothorax with light-colored pigmentation and concolorous antennal clubs
Note that recently emerged workers and queens of dark species (e.g., T. exilis) may temporarily present a yellowish pigmentation. The key is meant to be used for species in which the final pigmentation of females is naturally light-colored. High-quality images of all the species are available on AntWeb.org. Quantitative and qualitative morphological characters mentioned in the key are defined in the Material and methods section of this paper.

UCE sequence processing and phylogenomic inference
Following assembly and UCE extraction, the mean number of loci per specimen was 2242, with a mean contig length of 885 bp, and a mean coverage score of 44.3x (see Supp. file 1: Table S2). Following alignment, trimming and filtering of the full UCE dataset to loci with ≥ 85% taxon presence, the dataset had 2039 loci, with a mean locus alignment length of 793 bp. The concatenated matrix was 1 617 690 bp in length, in which 819 040 sites (50.6%) were variable, 533 245 sites (33%) were parsimony informative, with 19% missing data.
The partitioning analysis of the dataset resulted in a 260-partition scheme (see Dryad data repository; https://doi.org/10.25338/B8K63Z). The tree resulting from the IQTREE analysis (Fig. 80) had strong overall maximum likelihood bootstrap support. The phylogenetic placements of the species described in the current study, all of which fell within the subclade 'Palearctic clade IV' (sensu Prebus 2017), were unambiguous with respect to their closest relatives. As a side note, 'Palearctic clade II' (sensu Prebus 2017), which was composed of taxa from eastern and southern Asia, was recovered as paraphyletic in the current study. It now appears to consist of two clades, which are renamed 'Palearctic clade IIa' and 'Palearctic clade IIb' (see Fig. 79).

Discussion
The ideal context for descriptive alpha-taxonomic studies is often that of revisions carried out at the species group or species complex level. If the species of a genus clearly cluster into multiple species groups and complexes of easily definable boundaries, describing new taxa within the context of welldelimited groups is a safe way to build a stable taxonomic framework and avoid missing synonymies. However, while a clear morphological definition of monophyletic species groups may be easily achieved in some ant genera, for the time being this is rarely the case within the highly diverse W-Palearctic fauna of Temnothorax. This impediment clearly conditioned our investigation, whose results further suggest caution in future studies. We could not detect any described species closely resembling T. poldii sp. nov. and we were unable to define a species group hosting this species even after obtaining phylogenetic data on its account. Its (relative) phylogenetic proximity with T. ibericus is interesting. The latter was lately proposed to be a member of the sordidulus group (Arcos 2021), yet T. sordidulus (Müller, 1923) and the species complex named after it were defined as a part of the nylanderi group , which is unrelated to either T. ibericus or T. poldii according to our phylogenomic analysis. Molecular data on T. sordidulus to settle this issue are not yet available. The situation was even more complex for the other three species treated in this study. For instance, T. lagrecai was previously thought to belong to the luteus complex (Lebas et al. 2016), and T. flavicornis was thought to belong to the nylanderi group . Our phylogenomic evidence shows that the luteus complex belongs to the rottenbergii clade and is close to the exilis group, while T. lagrecai and T. flavicornis cluster together within the Palearctic clade, but far from the nylanderi group. On the other hand, some authors claimed the existence of a Ibero-Maghrebian species complex ('superspecies') composed by taxa such as T. atomus (Cagniant & Espadaler, 1997), T. curtulus, T. gentilis, T. monjauezi (Cagniant, 1968) and T. tebessae spp. and belonging to the tuberum group (Cagniant & Espadaler 1997; also see Reyes-López & Carpintero-Ortega 2013). Following the key of Cagniant & Espadaler (1997), T. lagrecai, T. marae sp. nov. and T. vivianoi sp. nov. could also belong to the same group, which would also be biogeographically credible. While we are inclined to think that the closest relatives of these three species treated in this study may be found within the tebessae complex sensu Cagniant & Espadaler (1997), we doubt it can represent a monophyletic entity anyway: not only even T. luteus would key out as a member of it, but T. vivianoi is not closely related to T. lagrecai and T. marae. Also, none of them belongs to the tuberum group anyway. And while T. lagrecai and T. marae clustered closely together, there was no easily identifiable morphological character that could have led us to believe, prior to the phylogenomic results, that T. marae was closer to T. lagrecai compared to T. vivianoi. In other words, the taxonomy of Palearctic Temnothorax currently suffers from an absence of clearly defined and reliable morphological characters to delimit the boundaries of species groups at least for a large proportion of species. It is possible to speculate on whether such characters may be hidden in the often undescribed male or even queen castes of Temnothorax (although queens often share many characters with workers). In fact, male morphology may, at least occasionally, be a powerful source of information on species group delimitation in some other ant genera (e.g., Alicata & Schifani 2019). Moreover, dispersing sexuals can occasionally prove very important to detect the presence of certain ant species (Espadaler & López-Soria 1991;Schifani et al. 2021b;Menchetti et al. 2022). However, less than 20% of the descriptions of Temnothorax of the last 20 years included the male caste (Bolton 2021), and even for well-studied regions like Central Europe no keys based on males are available yet (Seifert 2018). Integrating morphological descriptions with molecular data proves especially important to understand the diversification patterns and evolutionary relationships within Mediterranean Temnothorax.
Further investigation should also focus on the ecology and possible conservation issues of the four species treated in this study. Certain morphological traits here described for their workers may offer some first speculative hypotheses over their lifestyle based on possible functional implications: greater absolute size variation and relatively wider heads of T. poldii sp. nov. compared to the other species may indicate slightly more developed task subdivision and stronger mandible force (e.g., Retana & Cerdá 1994;Gronenberg et al. 1997), while relatively larger eyes in T. lagrecai may be linked to different light conditions associated with foraging activities (e.g., Weiser & Kaspari 2006). These and several other biological aspects of these four species remain to be explored.