Abstract

Cuticular microsculpture is an important morphological feature in insects, but it has not been systematically investigated in psyllids (Hemiptera: Psylloidea). Here, we examine the microsculpture of the vertex and thoracic dorsum in two subfamilies of Aphalaridae, focusing mainly on European taxa and using scanning electron microscopy to evaluate its taxonomic and phylogenetic significance. In Aphalarinae, imbricate microsculpture was observed in Aphalara, Eumetoecus and Rhodochlanis; rugose microsculpture in most species of Colposcenia; and several microsculptural types in Craspedolepta, including imbricate, smooth–imbricate, areolate–rugose, and alveolate patterns. A unique tuberculate microsculpture was found in Rhinocola aceris (Linnaeus, 1758), whereas other Rhinocolinae exhibited an imbricate pattern. Ancestral state reconstruction indicates that imbricate microsculpture represents the plesiomorphic condition in Aphalaridae and remains expressed to varying degrees in most extant representatives of the family. Multiple transitions among sculptural types within Craspedolepta and partly also Colposcenia indicate that microsculpture can evolve relatively rapidly, although the distribution of character states across the phylogeny shows that it still retains phylogenetic signal at shallow to intermediate taxonomic levels, despite a moderate level of homoplasy. Overall, these results demonstrate that head and dorsal thoracic microsculpture constitute diagnostic and phylogenetically informative characters that can be more widely applied in taxonomy, systematics and evolutionary research on psyllids.

ancestral character reconstruction, cuticle, integument, morphology, psyllids, scanning electron microscopy, surface sculpturing

The arthropod cuticle is a complex, multifunctional structure composed of distinct layers (epicuticle, exocuticle and endocuticle) that form the interface between the organism and its environment (Moussian 2013; Campli et al. 2024). While functioning as a mechanically protective barrier, the cuticle also exhibits intricate surface ornamentation collectively termed cuticular micro- or nanosculpture (Watson et al. 2017; Buxton et al. 2021; Hellenbrand and Penick 2023; Finet 2024). These structures, arising from physical rather than chemical differentiation, include ridges, pits, meshes, granules, spines, tubercles and other relief elements (Harris 1979; Richards and Richards 1979). Their diverse functions are ranging from mechanical reinforcement and improved grip to modulation of light reflection, aero- or hydrodynamics, thermoregulation, camouflage, grooming, food processing, filtration, secretion dispersal, sound production, oxygen retention, and reduction of water loss or microbial colonization (Gorb 2001; Kment and Vilímová 2010; Sun et al. 2012; Politi et al. 2021).

Because of this diversity, surface sculpture has long been used as an important taxonomic character in arthropods at both species and higher taxonomic levels, particularly since the advent of electron microscopy. However, despite several terminological systems and classification schemes for cuticular structures (Harris 1979; Richards and Richards 1979), detailed comparative studies across arthropod groups remain relatively scarce (e.g. Shear and Gruber 1983; Mitov 2011; Scarparo et al. 2017; Li et al. 2023). Terminological overlap is common, which complicates making broad comparisons and highlights the need to apply sculptural terminology within a taxon-specific context (Hellenbrand and Penick 2023).

Within Hemiptera, studies of the morphology and function of the integument remain limited (e.g. Sun et al. 2012; Hartung et al. 2016; Hemala et al. 2021). Nevertheless, scanning electron microscopy (SEM) has increasingly been used in recent years to document cuticular surfaces and sensilla, particularly in aphids (Kanturski et al. 2015; Kaszyca-Taszakowska et al. 2022; Kanturski and Lee 2023). In psyllids, earlier works (e.g. Pflugfelder 1941) addressed certain aspects of the integument, while Vondráček (1957) and Dobrеanu and Manolache (1962) were among the first to explicitly mention variation in microsculpture among taxa and body parts, including pitting, wrinkling, grooving, ridging and scale-like ornamentation. More recent taxonomic studies have incorporated cuticular patterns into species and genus diagnoses, especially those of the vertex, which is readily observable (e.g. Burckhardt 1988; Brown and Hodkinson 1988; Malenovský and Burckhardt 2009; Burckhardt et al. 2024; Serbina et al. 2025). However, apart from the study by Drohojowska (2015), which examined thoracic microsculpture in 59 psyllid species as a source of morphological characters for phylogenetic analysis, cuticular microsculpture in psyllids has not been investigated within a broader comparative or phylogenetic framework.

Psyllids, or jumping plant lice (Hemiptera: Psylloidea), are small sternorrhynchan insects (body length 1–10 mm, including folded wings) that feed on plant sap. They are typically highly host-specific, most often associated with eudicots and magnoliids, and only rarely developing on monocots or conifers (Burckhardt et al. 2014). Several species are economically important as crop pests or as biological control agents of invasive plants (Moreno et al. 2021; Camargo et al. 2022). Approximately 4,200 species have been described worldwide in seven families, with at least a comparable number still undescribed (Burckhardt et al. 2021; Mauck et al. 2024; Malenovský et al. 2025).

Within Psylloidea, the family Aphalaridae is the third largest, comprising about 770 described species (Ouvrard 2020). As in many psyllid groups, species identification is often difficult due to subtle morphological differences and overlapping host ranges (Burckhardt and Lauterer 1997; Halbert and Burckhardt 2020). These challenges highlight the need for additional diagnostic characters, including molecular, bioacoustic and morphological traits, to improve taxonomy and our understanding of aphalarid biology and phylogeny (Pramatarova et al. 2024; Tischechkin 2025).

In this study, we investigate variation in head and thorax microsculpture in two aphalarid subfamilies, Aphalarinae and Rhinocolinae, using SEM in a phylogenetic framework to evaluate its taxonomic and phylogenetic significance. Our analysis is based primarily on 31 species (Aphalarinae 26, Rhinocolinae 5) recorded from Bulgaria (Pramatarova et al. 2025), supplemented by additional taxa. We test the hypotheses that (I) cuticular microsculpture is species- or genus-specific and (II) reflects phylogenetic relationships within Aphalaridae.

This study provides the first comparative examination of vertex and dorsal thoracic microsculpture in jumping plant lice, focusing on mostly European representatives of the Aphalarinae and Rhinocolinae. Aphalaridae has been recovered as a basal assemblage of Psylloidea in recent phylogenetic analyses (Percy et al. 2018; Cho et al. 2019; Wang et al. 2023).

We have identified six main surface microsculpture types in the examined taxa. Sometimes the assignment of a particular structure to one of these categories may prove problematic. This difficulty could arise from continuous variation between certain types—for instance, the distinction between imbricate and alveolate microsculpture—or from the potential existence of additional types not captured in our study. Consequently, our proposed classification constitutes a preliminary framework that needs further testing including a broader range of psyllid taxa.

Our ancestral state reconstruction under an equal-rates Mk1 model suggests that imbricate microsculpture represents the most likely ancestral condition within Aphalaridae, in contrast to patterns observed in some other insects such as ants (Hymenoptera: Formicidae), where smooth cuticle appears plesiomorphic (Hellenbrand and Penick 2023). This inference, however, should also be tested with broader taxon sampling including other subfamilies of Aphalaridae and families of Psylloidea. Several microsculptural types evolved subsequently in different lineages of Aphalarinae and Rhinocolinae. Some of these, such as the rugose microsculpture in Colposcenia, appear to represent genus-level synapomorphies, whereas alveolate, areolate–rugose, smooth–imbricate and tuberculate patterns are restricted to relatively few species. Multiple transitions among sculptural types within Craspedolepta and partly also Colposcenia indicate that microsculpture can evolve relatively rapidly, although the distribution of character states across the phylogeny shows that it still retains phylogenetic signal at shallow to intermediate taxonomic levels, despite a moderate level of homoplasy. While the imbricate state is ancestral, the phylogenetic signal largely arises from rare derived states shared within smaller clades.

Although the microsculpture on the head and thoracic dorsum does not consistently differ among most examined genera, it provides useful diagnostic characters at the species level. In particular, fine details of the scale-like elements of the imbricate microsculpture can be used to distinguish between some species of Aphalara, for example A. exilis and A. nigrimaculosa, which are morphologically similar and belong to the same species group (Burckhardt and Lauterer 1997). In Craspedolepta, the long macrosetae, usually covered with wax, together with imbricate pattern with multiple granules at the bases of cuticular “scales”, distinguish C. araneosa and C. conspersa from the other species. Craspedolepta malachitica can be distinguished by its alveolar microsculpture from the otherwise similar C. artemisiae and C. latior displaying imbricate microsculpture, and areolate–rugose microsculpture can be used to distinguish C. nervosa and C. pontica from C. bulgarica. Another potentially useful character for species or genus diagnosis might be the presence of granules on the scale-like structures of the imbricate microsculpture, which are well-distinct in Agonoscena pistaciae and Megagonoscena viridis.

Taken together, this study highlights the potential of cuticular microsculpture as an additional morphological character set for both phylogenetic inference and species-level taxonomy in Psylloidea. The taxonomic value of cuticular microsculpture has also been demonstrated in other insect groups. In Hymenoptera, particularly within Chrysididae and Formicidae, variation in surface structures has been used in classification and comparative studies (Martynova 2017; Hellenbrand and Penick 2023). Similarly, in Coleoptera, microsculptural characters have been successfully applied in taxonomic studies (e.g. Khalaf 1980; Liu 2010).

Further research on the functional significance of the microsculptural traits should clarify their ecological role and test if the transformations between microsculptural types are adaptive in Psylloidea, where similar diversity may be expected beyond Aphalaridae. Potential functional roles include modification of cuticular wettability, facilitation of wax retention, mechanical reinforcement of the cuticle, or optical effects such as light scattering and camouflage (Parker 2000; Kuitunen and Gorb 2011; Sun et al. 2012; Baio et al. 2019; Urca et al. 2024); these hypotheses are testable in a comparative framework.

Author Contributions. Conceptualization (MP, AL-G), Methodology (MP, IM, IG, AL-G), Validation (IG), Formal analysis (MP, IM, AL-G), Investigation (MP, DB, IM, IG, AL-G), Resources (MP, DB, IM, IG), Data Curation (MP, IG), Writing - Original draft (MP, DB, IM, AL-G), Writing – Review and Editing (MP, DB, IM, IG, AL-G), Visualization (MP), Supervision (AL-G), Project administration (MP, AL-G), Funding acquisition (MP, AL-G).

Conflict of interests. The authors declare that no competing interests exist.

Funding. The authors have no funding to report.

We would like to express our sincere thanks to Dr Diana Percy (Department of Botany and Biodiversity Research Centre, University of British Columbia, Canada), Dr hab. Jowita Drohojowska (Institute of Biology, Biotechnology and Environmental Protection, University of Silesia, Poland), and an anonymous reviewer for their positive feedback and constructive evaluation of our manuscript. We also thank Dr Atanas Tzonev (Faculty of Physics, Sofia University) for assistance with the operation of the SEM device. MP, IG, and AL-G acknowledge support from the National Science Fund, Bulgaria, under Grant No. KP-06-N-91/11 (awarded on 4 December 2025). This study was supported by the grant BG-RRP-2.004-0008 SUMMIT-3.3 (I. Gjonov).