1. Introduction
The genus Euathlus Ausserer, 1875 comprises a monophyletic group of medium-sized tarantulas (Perafán and Pérez-Miles 2014; Ferretti 2015; Allegue and Ferretti 2025) distributed in South America (Argentina, Chile and Peru). Its distribution extends almost exclusively along the Andean highlands and lowlands, although some species are present in flat regions of the Argentinean Patagonia (Allegue and Ferretti 2025). These tarantulas show a high diversity in Chile and also one species has been recorded for Peru. During recent years, in Argentina, the number of species described has been increasing (Allegue and Ferretti 2025). However, there is still a huge gap regarding the knowledge of the systematics, evolution and diversification of the species of this genus in South America. For example, some preliminary molecular evidence was reported for the genus by Allegue and Ferretti (2025), who included mitochondrial data for five Euathlus species. However, because most taxa were not represented in that phylogeny, the molecular framework for the genus remains incomplete.
Tarantulas and most mygalomorph spiders usually represent a challenge to taxonomists due to their extreme morphological homogeneity (Wilson et al. 2023). Fortunately, the robustness of tarantula systematics has also increased considerably in recent years due to numerous studies incorporating molecular data. DNA-based methods are usually used in systematics as a tool for species delimitation, discovery, and identification. By analyzing discrete mitochondrial genetic regions, it is often possible to achieve reliable species-level identifications and detect cryptic diversity in animals (Ferretti et al. 2024; Hebert et al. 2003; Montes de Oca et al. 2015; Vasconcelos et al. 2016; Zhou et al. 2019). The use of a universal DNA ‘barcode’ fragment of the cytochrome c oxidase I (COI) gene has been established as a useful tool for generating the initial species hypotheses (Korba et al. 2022). Mitochondrial genes have been preferred for studies at species-level systematics because: i) they are strongly conserved across animal groups; and ii) they are highly variable within natural populations because of their relatively elevated mutation rates. Additionally, they are easy to amplify at a relatively low cost (Galtier et al. 2009). Despite some limitations, COI sequences have proven to be useful to provide a framework for the taxonomy of some poorly studied groups, such as tarantulas (Candia-Ramírez and Francke 2021) and specifically, in this case, the genus Euathlus (Allegue and Ferretti 2025).
Beyond molecular information, morphology-based cladistics is essential in generating phylogenetic hypotheses, not only when we do not have access to fresh material or tissues from all species but also to achieve an integrative approach of total evidence. Identifying and testing patterns of shared derived characters allow the reconstruction of evolutionary relationships independently from molecular data. This independence makes them particularly valuable for assessing the congruence between morphological and molecular hypotheses, strengthening the evidence for species boundaries and lineage diversification. In taxa such as tarantulas, where morphological differentiation can be subtle or convergent, cladistic analyses based on morphology remain essential for revealing underlying phylogenetic structure and guiding an integrative taxonomy approach (Bertani 2001; Fukushima and Bertani 2017; Rios-Tamayo 2024)
Consequently, from the integration of many lines of evidence (molecular, morphology and behavior), we describe, diagnose and illustrate three new species of the genus Euathlus from central-south Argentina. In addition, the female paratype previously assigned to E. tenebrarum is herein reassigned to E. basalticussp. nov., based on morphological and geographic information. Thus, we formally describe for the first time a conspecific female of E. tenebrarum from Junín de los Andes, Neuquén, Argentina. Moreover, we provide an updated molecular phylogeny for some representatives of the genus and performed a cladistic analysis based on morphology including the new species. Finally, we describe for the first time the sexual behavior of E. susanaesp. nov.
2. Materials and Methods
2.1. Morphological work
Specimens. Specimens of Euathlus basalticussp. nov. were obtained both from field surveys and from preserved material. Euathlus susanaesp. nov. was collected during fieldworks. Male and female of Euathlus kupalsp. nov. and females of E. tenebrarum are described from preserved specimens. The material examined is deposited in the collection of the Museo de Ciencias Naturales “Bernardino Rivadavia” (MACN-Ar), Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina; Centro de Recursos Naturales Renovables de la Zona Semiárida (CERZOS-CONICET, UNS), Universidad Nacional del Sur, Bahía Blanca, Buenos Aires, Argentina, and the Entomological Collection of the IPEEC-CONICET (CNP-CE), Puerto Madryn, Chubut, Argentina.
Methods. Specimens were examined using a Leica S APO stereoscopic microscope equipped with a MShot digital camera, and a Leica S9 with MC170 integrated camera. The structures were photographed in different focal plains and then stacked using Helicon Focus v.7. The spermathecae were dissected and then cleaned with ©Naclens enzymatic pills in distilled water. All measurements are given in millimeters and were made with digital dial calipers with an error of 0.01 mm. Appendage measurements were obtained from left appendages in dorsal view. Total lengths do not include chelicerae or spinnerets.
Terminologies. Spine notation follows Petrunkevitch (1925). Male palpal organ keel terminology follows Bertani (2000). Urticating setae terminology follows Cooke et al. (1972) and Bertani (2002). The spermathecae terminology follows Perafán and Pérez-Miles (2014), Ferretti (2015) and Ríos-Tamayo (2020).
Abbreviations. A = apical keel, ALE = anterior lateral eyes, AME = anterior median eyes, D = dorsal, OQ = ocular quadrangle, P = prolateral, PB = prolateral branch of tibial apophysis, PI = prolateral inferior keel, PLE = posterior lateral eyes, PLS = posterior lateral spinnerets, PME = posterior median eyes, PMS = posterior median spinnerets, PS = prolateral superior keel, R = retrolateral, RB = retrolateral branch of tibial apophysis, V = ventral.
2.2. Molecular work
Specimens. DNA sequences were obtained from the following specimens (GenBank accession numbers in parentheses for sequences downloaded from there): Bistriopelma matuskai — Perú, Abancay, Nevado de Ampay (OR178612); Bistriopelma sp. — Perú, Cusco, La Raya; Euathlus ameghinoi — Argentina, Chubut, Villa Dique Florentino Ameghino; Euathlus basalticussp. nov. — Argentina, Neuquén, Ñorquín Department, Caviahue; Argentina, Neuquén, Ñorquín Department, Caviahue near Salto del Agrio waterfall; Euathlus sagei — Argentina, Neuquén, Zapala; Euathlus susanaesp. nov. — Argentina, Mendoza, Agua Escondida; Argentina, La Pampa, Cerro Negro; Euathlus ventus — Argentina, Chubut, Sarmiento; Eupalaestrus sp. — Argentina, Santiago del Estero, Parque Nacional Copo; Hapalotremus sp. — Perú, Cusco, Ollantaytambo (OR178636); Grammostola inermis — Argentina, Catamarca, Belén; Grammostola vachoni — Argentina, Buenos Aires, Tandil.
Methods. Specimens used for molecular analysis were sacrificed by putting them in the freezer, assuring a relatively quick death without causing them excessive suffering and tissue degradation. Extractions and isolation of DNA were made at the Genomic Services Laboratory (GENeTyC) from CERZOS-UNS. Muscle tissue was extracted from legs III and IV and stored in absolute ethanol at –80°C. The DNA was quantified or visualized through agarose gel electrophoresis. DNA amplification was performed for a 710-bp region of the mitochondrial gene cytochrome c oxidase subunit I (COI). We use a single set of primers to amplify the barcode region of mitochondrial COI gene: LCO-1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO-2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994). Polymerase chain reaction (PCR) amplification was performed under the following conditions for COI: 94°C for 9 min, then 34 cycles of 94°C for 45 s; annealing 48°C for 45 s; 72°C for 60 s; with a final elongation step of 72°C for 6 min. PCR products were purified following manufacturer’s protocol and sequenced in Macrogen (Korea). Chromatograms were edited, and sequences managed with the help of the software Geneious R11 (https://www.geneious.com).
2.3. Phylogenetic analyses
2.3.1. Maximum parsimony
A cladistic analysis based on morphology included the newly described species (Euathlus basalticussp. nov., Euathlus kupalsp. nov., and Euathlus susanaesp. nov.), scored for 41 characters obtained by Perafán and Pérez-Miles (2014) and Ferretti (2015), with the addition of four new characters: (7) presence of teeth on the embolus keel PI, (8) number of teeth on the embolus keel PI, (40) retrolateral teeth on female chelicerae, and (41) retrolateral teeth on male chelicerae. The ingroup comprised sixteen taxa: Euathlus ameghinoi Allegue and Ferretti, 2025, E. antai Perafán and Pérez-Miles, 2014, E. atacama Perafán and Pérez-Miles, 2014, E. basalticussp. nov., E. condorito Perafán and Pérez-Miles, 2014, E. diamante Ferretti, 2015, E. grismadoi Ríos-Tamayo, 2020, E. kupalsp. nov., E. manicatus Simon, 1892, E. mauryi Ríos-Tamayo, 2020, E. pampa Ríos-Tamayo, 2020, E. parvulus (Pocock, 1903), E. sagei Ferretti, 2015, E. susanaesp. nov., E. tenebrarum Ferretti, 2015, E. truculentus L. Koch, 1875, E. vanessae Quispe-Colca and Ferretti, 2021, E. ventus Allegue and Ferretti, 2025, and E. walteri Taucare-Ríos, Plaza and Pérez-Miles, 2025. The outgroup included Homoeomma uruguayense (Mello-Leitão, 1946), Grammostola vachoni (Schiapelli and Gerschman, 1961), Phrixotrichus jara Perafán and Pérez-Miles, 2014, Phrixotrichus scrofa (Molina, 1782), Phrixotrichus vulpinus (Karsch, 1880), and Plesiopelma longisternale (Schiapelli and Gerschman 1942). The tree was rooted using H. uruguayense. The data matrix was constructed using Mesquite v3.7 (Maddison and Maddison 2021). The cladistic analysis was carried out with TNT v1.5 (Goloboff and Catalano 2016) and to decide upon appropriate k-values, we followed the proposal by Mirande (2009) which uses a heuristic search. This method divides the values of fit/distortion into regular intervals, obtained under different k-values. This was accomplished by employing the “aaa.run” script (Mirande 2009) implemented in TNT under the commands 3 10 70 95 7. A consensus tree was also calculated with Majority rule (Cut-off = 50). Character optimization were performed with the computer software WINCLADA-ASADO 1.61 (Nixon 2004). The editing of the trees was carried out with Corel Photo Paint X7 v17.0.0.491.
Morphological data set. Multistate characters were coded as non-additive. The data matrix comprised 25 species scored for 41 characters (Table S1). Characters:
(1) Embolus direction: directed ventrolaterally = 0; directed retrolaterally = 1. (2) Relative width of bulb sclerites II + III: wide = 0; narrow (less than 10% of length) = 1. (3) Position of distal prolateral inferior keel (PI): prolateral = 0; prolateroventral = 1. (4) Apical keel: absent = 0; present = 1. (5) Ventral crest on PI: absent = 0; present = 1. (6) Subapical tooth on PI: absent = 0; present = 1. (7) Teeth on embolus keel PI: absent = 0; present =1. (8) Number of teeth on embolus keel PI: low (≤ 2) = 0; median (3–5) = 1; high (≥ 6) = 2. (9) Tegular apophysis on bulb: absent = 0; present = 1. (10) Position of male tibial apophysis: ventral = 0; prolateroventral = 1. (11) Male tibial apophysis: branches with fused bases = 0, branches with non-fused bases = 1. (12) Male tibial apophysis: with one retrolateral spine = 0; with two retrolateral spines = 1; without retrolateral spines = 2. (13)PB: with basal spine = 0; without basal spine = 1. (14) Spines of RB: without spines = 0; with 1 spine = 1; with more than one spine. (15) Position of distal spine on RB: subapical = 0; apical = 1. (16) Ventral spine on RB: absent = 0; present = 1. (17) Flexion of male metatarsus I: between the branches of tibial apophysis = 0; on the apex to the RB = 1; retrolateral to the tibial apophysis = 2. (18) Male metatarsus I: strongly curved = 0; straight = 1. (19) Spermathecal morphology: spheroid shape = 0; not spheroid shape = 1. (20) Spermathecae with a lateral spheroid chamber: absent = 0; present = 1. (21) Spermathecal receptacles: single = 0; bifurcated = 1. (22) Spermathecal neck: straight = 0; spiralled = 1. (23) Digitiform projections on spermathecae: absent = 0; present = 1. (24) Female palpal tibia spination: with apical spines only = 0; with apical and others ventral spines = 1; with apical and prolateral spines = 2. (25) Labial cuspules: numerous (> 20) = 2; moderate amount (10–20) = 1; few (< 10) = 0. (26) Sternum: as long as wide = 0; longer than wide = 1. (27) Extension of scopula on metatarsus I: complete = 0; more than a half (distal 2/3) = 1; distal half = 2. (28) Extension of scopula on metatarsus II: more than half (distal 2/3) = 0; distal half = 1. (29) Extension of scopula on metatarsus III: distal half = 0; less than half (1/3) = 1; only apical (1/4, 1/5) = 2. (30) Extension of scopula on metatarsus IV: less than half (1/3) = 0; only apical (1/4, 1/5) = 1; absent scopula = 2. (31) Scopulae on tarsi I: entire = 0; widely divided = 1. (32) Scopulae on tarsi II: entire = 0; narrowly divided = 1; widely divided = 2. (33) Scopulae on tarsi III: entire = 0; narrowly divided = 1; widely divided = 2. (34) Scopulae on tarsi IV: entire = 0; narrowly divided = 1; widely divided = 2. (35) Tarsal claws: with teeth = 0; without teeth = 1. (36) Length of urticating setae type III: short (< 0.75 of optical field diameter of microscope; 40×) = 0; medium sized (0.75–1.5 of optical field diameter; 40×) = 1; long (> 1.5 of optical field diameter; 40×) = 2. (37) Tibial apophysis: with two branches = 0; with one branch = 1. (38) Tibial apophysis with two branches: both with equal development = 0; both with subequal development = 1; RB more developed than PB = 2. (39) Number of patches of urticating setae: one central dorsal = 0; two lateral patches = 1. (40) Retrolateral teeth on female chelicerae: absent = 0; present = 1. (41) Retrolateral teeth on male chelicerae: absent = 0; present = 1.
2.3.2. Maximum Likelihood
Sequence alignment of the COI fragment was conducted with the MAFFT v7.017 (Katoh et al. 2002) plug-in implemented in Geneious R11 using default parameters. Maximum Likelihood phylogenetic inference was estimated with the software IQ-TREE v1.6.8 (Nguyen et al. 2015). We used IQ-TREE to first select the best-fit partitioning scheme and corresponding evolutionary models of the matrix through ModelFinder (Kalyaanamoorthy et al. 2017), and then to infer the best tree and estimate clade support by means of 1000 replicates of ultrafast bootstrapping (Hoang et al. 2018). The tree was rooted with Hapalotremus sp. and edited using FigTree v1.4.3 (Rambaut 2009) (http://tree.bio.ed.ac.uk/software/figtree) and Corel Photo Paint X7 v17.0.0.491.
2.4. Map construction
The map (Fig. 18) was made using the software QGIS (https://qgis.org). The distribution points of Euathlus species were obtained from geographic coordinates from published taxonomic works available in the World Spider Catalog (2025) (https://wsc.nmbe.ch). The records of the species described in this study were georeferenced directly at the field or the data from museum labels.
2.5. Sexual behavior
For the description of the sexual behavior of Euathlus susanaesp. nov. we performed two male-female pairings. We obtained one mating. Durations of behaviors are expressed in seconds. The courtship event was estimated before the male-female contact, from the first behavioral unit observed, in our case leg tapping with leg I. Copulation started when the male performs the first palpal insertion.
4. Discussion
The genus Euathlus seems to be more diverse than previously thought because although most species described share a common morphology regarding their genitalic features, a considerable variation among them actually exists. For example, females have a spermatheca that typically consist of wide seminal receptacles with lateral chambers; however, these structures vary widely in shape, position, and orientation. In addition, males possess palpal bulbs with a consistent architecture, yet exhibiting marked interspecific differences. The embolus varies from thinner to thicker forms, and may be either slightly or strongly curved, always directed retrolaterally. The distal portion of the embolus bears a PI keel, which may present teeth or serrated edges; an apical keel can be present or absent, and accessory keels may vary in their presence and development. Secondary genitalic structures, such as the tibial apophysis, also show notable variation, ranging from one to two branches, with differences in thickness and degree of development. In addition, the number and morphology of the spines associated with the tibial apophysis vary significantly among species (Allegue and Ferretti 2025; Ferretti 2015; Perafán and Pérez-Miles 2014; Ríos-Tamayo 2020; Taucare-Ríos et al. 2025).
Compounding the taxonomic challenges in Euathlus is the ontogenetic development of the spermatheca. The structures are often homogeneous in immature females, making species diagnosis difficult until the specimen reaches full maturity and the spermathecae acquire their definitive morphology. Consequently, these structures may vary significantly throughout the individual’s development, preventing accurate descriptions, identifications, and comparisons among species (Schwerdt et al. 2021).
Regarding the cladistic analysis, although some clades exhibit high support values in the majority-rule consensus tree (e.g., 100%), they are supported exclusively by homoplasies. This indicates that, despite their stability across the trees, these groupings are not underpinned by unique morphological synapomorphies. Such results underscore the limitations of the morphological dataset, including the scarcity of clear synapomorphies, the recurrence of certain character states, and possible convergences. Nevertheless, the analysis recovered an interesting clade within Euathlus, supported by character 16 (the presence of a ventral spine on the retrolateral branch of the tibial apophysis) as a synapomorphy that distinguishes a clearly cordilleran clade (including all Chilean species, except for E. diamante, which is found in Argentina) from another group distributed across the cordillera, precordillera, and steppe (with Argentinean and Peruvian species). The latter group (E. tenebrarum, E. kupalsp. nov., E. basalticussp. nov., E. sagei, E. vanessae, E. susanaesp. nov., E. ameghinoi, E. ventus) is further supported by two additional synapomorphies: the presence of an apical keel (character 4) and the presence of teeth on the prolateral inferior (PI) keel of the embolus (character 7).
In this analysis, the genus Euathlus was recovered as paraphyletic, including representatives of the genus Phrixotrichus. Although these two genera were clearly diagnostic and recovered as two different lineages in previous morphological cladistics analyses (Perafán and Pérez-Miles 2014; Ferretti 2015; Allegue and Ferretti 2025), we must take into account the complex and long-standing taxonomic history which includes multiple transfers between these genera. For example, Phrixotrichus was originally considered as a junior synonym of Euathlus by Schmidt (1996), but was later revalidated by Perafán and Pérez-Miles (2014). Taxonomic confusion between these groups has persisted over time. For instance, Strand (1908) described the genus Ashantia, which Gallon (2005) later considered a junior synonym of Euathlus. However, it is now recognized that Ashantia latithorax corresponds to Phrixotrichus vulpinus, and Ashantia is currently treated as a junior synonym of Phrixotrichus. Then, why did our study not recover both genera as distinct clades? Arguably, both the cladistic analyses from Perafán and Pérez-Miles (2014) and Ferretti (2015) included just less than half of the Euathlus species that we know to date, thus the full picture of the variation of the genitalic features and secondary sexual characters were underrepresented. Indeed, the morphologies observed from many taxa seem to indicate that some characters are more informative for their grouping, for example, the shape of the tibial apophysis or the embolus from the palpal organ, instead of the presence of one/two urticating setae patches, which has been used traditionally to discriminate between these two genera. Then, we could have taken the action of synonymizing Phrixotrichus with Euathlus from the cladistics analysis, but for now, we prefer to be cautious. One reason is because to date, there are still no mitochondrial fragment sequences available to test the monophyly or distinctiveness of Phrixotrichus in relation to Euathlus and testing the monophyly of these genera would require analyses based on multiple independent loci.
A striking result from the molecular phylogenetic analysis was the non-grouping of the sequences of Euathlus basalticussp. nov. in a clade, as one of the sequences was grouped closely related with E. sagei, while the other remained outside that clade. This clade ((E. sagei + E. basalticus sp. nov) E. basalticus sp. nov) is well supported. This unexpected pattern of intraspecific polyphyly matches another findings in other mygalomorph taxa. Indeed, Hamilton et al. (2024) documented a similar case in Aphonopelma jacobii Hamilton and Hendrixson, 2024, where mitochondrial haplotypes from different populations were split across divergent clades, with some nested within A. marxi (Simon, 1891), rendering it paraphyletic. This phenomenon, known as gene-tree/species-tree incongruence, occurs when gene trees reveal that alleles from one species are more closely related to those from another species than to conspecific alleles. Such patterns are often attributed to ancient admixture and asymmetric mitochondrial introgression (Horoiwa et al. 2021) and have been reported in other theraphosid genera (Hamilton et al. 2016). This incongruence may also result from incomplete lineage sorting or an underestimation of the extent of gene flow among populations (Funk and Omland 2003).
Despite the mitochondrial incongruence observed here, we believe that the clear morphological differences between E. basalticussp. nov. and E. sagei support their recognition as distinct species. Nevertheless, the presence of divergent mitochondrial lineages within a single morphologically diagnosable species may reflect similar evolutionary processes. Past climatic fluctuations and habitat shifts in the Andean and Patagonian regions could have facilitated secondary contact and introgression between lineages now occupying disjunct habitats (Sérsic et al. 2011). Further sampling across the distribution range of E. basalticussp. nov., combined with multi-locus genomic analyses, are required to unveil the extent of mitochondrial introgression, test for potential cryptic diversity, and clarify the species’ evolutionary history.
It is important to note that sequences for many described species are still lacking. Future efforts will focus on incorporating additional molecular markers, particularly nuclear genes, which are essential for a more robust phylogenetic framework. Despite the current limitations in taxon and marker sampling, we consider it valuable to present the available molecular data, as the results demonstrate that the studied lineages are distinct from previously known Euathlus sequences and also from outgroup taxa.
Euathlus susanaesp. nov. is distributed in two different localities: Agua Escondida, Mendoza, and the Cerro Negro Reserve, La Pampa. Individuals from both sites cluster together in the molecular analysis, supporting the hypothesis that they belong to the same species. Morphologically, females from both populations are similar, particularly in the shape of the spermatheca. Unfortunately, males from Agua Escondida are unknown, thus further morphological comparisons were not possible to conduct. Additionally, mating trials were performed to explore potential reproductive barriers. In these trials, males and females from different populations successfully performed courtship and copulation, indicating the absence of prezygotic isolation, at least at the level of courtship behavior, as similarly interpreted for other theraphosid spiders (Costa and Pérez-Miles 2002). However, some specific behavioral differences were observed e.g. during the intraspecific trial within Cerro Negro specimens, the female responded quickly and positively to the male’s courtship. In contrast, from the trial using individuals from different localities, the female initially showed a more reluctant behavior, taking more time to accept the male. Nevertheless, mating occurred, reinforcing the hypothesis that these populations are reproductively compatible. The combined molecular, morphological, and behavioral evidence suggests that E. susanaesp. nov. represents a single species composed of at least two geographically distinct but reproductively connected populations.
Morphologically, it is important to note that Ríos-Tamayo (2020) suggested the existence of morphological differences between males from Caviahue (now described as Euathlus basalticussp. nov.) and those of E. tenebrarum from Lago Curruhué Chico. Based on our comparisons of specimens of E. basalticussp. nov. and E. tenebrarum, we were able to clearly differentiate them. Furthermore, we assigned females to E. tenebrarum from other localities near the type locality, along with a male that exhibited morphological characteristics consistent with those already described for the species. In E. basalticussp. nov., males possess only type III urticating setae, whereas females have both types III and IV. The absence of type IV setae in males may be associated with the loss of structures related to passive defense. According to Ortiz-Villatoro et al. (2020) and Russi and Pérez-Miles (2023), type IV setae are mainly incorporated into the ootheca and moulting mats, suggesting a role in passive defense against arthropods or phorid larvae rather than in active defense against vertebrate predators. Males, which lead a more mobile lifestyle and do not construct oothecae, would not require this type of setae, relying instead on type III for active defense. This pattern may therefore reflect a sexually dimorphic adaptation linked to differences in ecology and behavior between sexes.
The distribution pattern of Euathlus also deserves further attention. Many species, including some described in this study, inhabit small rocky relicts, some located far from the Andean Cordillera. These isolated and spread populations led us to think about the historical and ecological processes shaping the current distribution of the genus. Although the present work focuses on taxonomic aspects, arguably, these biogeographical patterns may provide valuable context for future studies on diversification and endemism within Euathlus.
Overall, both the cladistic and molecular analyses highlight the complex evolutionary history of Euathlus, revealing patterns of morphological convergence, taxonomic uncertainty, and possible historical gene flow. While these approaches provide complementary evidence, the limited sampling and absence of molecular data for some key species preclude definitive taxonomic conclusions. Future studies integrating expanded molecular datasets, particularly multi-locus information, alongside with more comprehensive morphological analyses, will be essential to fully resolve the phylogenetic relationships and species boundaries within the genus.