Zoological Journal of the Linnean Society

Species-level delimitation within the genus Trichomycterus remains one of the main systematic challenges within the Trichomycterinae, particularly in lineages characterised by conservative external morphology, high apparent intraspecific variation, and historical diagnostic criteria based primarily on body proportions, colouration and a few meristic characters. In central Chile, Trichomycterus areolatus has traditionally been interpreted as a widely distributed and morphologically variable species, whilst T. maculatus, originally described from "Santiago du Chili", has remained subordinate to this broad conception without a modern phylogenetic reassessment. Here we reassess the specific boundaries of T. areolatus sensu lato using an integrative approach that combines complete mitogenomes, estimates of genetic divergence and comparative morphology of the cephalic laterosensory system associated with the neurocranium. Phylogenetic analyses reveal T. areolatus sensu lato to be non-monophyletic and identify a deeply divergent lineage, geographically coherent and attributable to T. maculatus. This lineage differs from restricted T. areolatus by extensive mitochondrial divergence, comparable to that observed between recognised species of Trichomycterinae, and by discrete characters of the cephalic lateral line system, primarily related to the continuity of the supraorbital canal and the arrangement of the associated pores. The congruence between mitogenomic, nuclear and morphological evidence supports the revalidation of Trichomycterus maculatus Valenciennes, 1846, and calls for a more restricted geographic circumscription of T. areolatus. These results demonstrate that the diversity of Trichomycterinae in central Chile has been underestimated, modify previous interpretation of the distribution of the species involved, and highlight the value of integrating mitogenomics and neurocranial/laterosensory characters into the taxonomy of morphologically conservative siluriform lineages.

Polytypic species with large ranges may harbor unrecognized diversity because taxonomy ranks populations differing subtly in plumage as subspecies. The Ruddy Foliage-gleaner (Clibanornis rubiginosus) exemplifies this problem. It ranges from Mexico to Brazil, with 15 subspecies, and forms a non-monophyletic complex with two congeners, yet its songs had not been compared. We measured ten spectral and temporal variables on 104 recordings covering 14 of 15 subspecies. Bayesian linear mixed models showed three song groups: eight subspecies west of the Andes share a single-note song, whereas Amazonian and Guianan populations add a short introductory note and sing longer, lower-pitched songs. Within this group, watkinsorum sings the lowest-pitched and longest song and is phylogenetically closer to C. cinnamomeigula than to its Amazonian neighbors. The third group is C. cinnamomeigula alone, a white-eyed taxon in an otherwise dark-eyed group. Its high-pitched, vibrato song resembles none other in the genus. One-note and two-note songs differ in kind without intermediates, and every two-note taxon sequenced to date falls in one clade, so C. rubiginosus is paraphyletic. We recognize four species, C. rubiginosus sensu stricto, C. cinnamomeigula, C. watkinsorum, and C. obscurus. This raises Clibanornis from five species to eight and divides its only polytypic species.

Phylogeny and taxonomy are related but not identical: phylogeny estimates common ancestry, whereas taxonomy must also provide diagnosable, stable, informative, and useful classifications. We examine this distinction using turtle and whale barnacles of the superfamily Coronuloidea, a group with an extensive fossil record, marked ecological specialization, a long history of morphology-based classification, and a recent topology-driven reclassification that merged recognized higher taxa to preserve monophyly. These changes reduced diagnosability and biological coherence, weakened historical continuity, and relied on placements insufficiently tested against morphological and phylogenetic evidence. Our reanalysis of published molecular datasets shows that critical relationships are sensitive to marker composition and taxon sampling. Together with morphological considerations on extant and fossil forms, these results indicate that topology alone is insufficient for coherent higher classification. We argue that higher taxa should be evaluated using complementary criteria: diagnosability, stability, utility, and monophyly, rather than by mechanically translating a preferred tree into Linnaean ranks.

Catenulids are free-living flatworms, common in eutrophic freshwaters such as ponds, ditches, or peatbogs, with most of the diversity described from tropical regions to date. Although the majority of the species have been described from warmer climates, most molecular studies have been done on specimens from temperate zones in Europe. We addressed this gap by sampling for exotic species in localities available in a temperate climate. In this study, we investigated catenulid diversity in the greenhouses at the University of Warsaw Botanic Garden and recorded two species known only from tropical areas (Stenostomum paraguayense and Suomina evelinae) and one exotic species recorded previously from a greenhouse in Poland (Stenostomum corderoi). Additionally, in the latter species, we provide evidence for environmentally induced coloration of sensory pits, which has not been reported thus far. We placed the collected species on a phylogeny using barcoding of 18S, 28S, and COI genes and retrieved paraphyly of the family Catenulidae, with S. evelinae forming a sister group to the genus Paracatenula, and hence we propose a revision of its systematic position. In total, we recorded six species, including three with a wide cosmopolitan distribution (C. turgida, S. grande and S. tuberculosum), and provided sequences for five of them, three of which had no previous molecular records (S. paraguayense, S. evelinae and S. corderoi). Thus, we confirm that greenhouses represent an important source of exotic species for taxonomic work on microscopic invertebrates.

The fieldwork was carried out between December 2023 and June 2025, with the aim, among others, of confirming the possible presence of the North African Gerbil (Gerbillus campestris) in Melilla. To this end, 56 camera trap stations were installed, each equipped with a camera trap. Of the total number of camera trap stations installed, the presence of Gerbillus campestris was detected in six of them, representing a capture success rate of 10.71%. Following the detection of the species, a specimen was captured using cage traps. For identification purposes, samples of epithelial tissue from the ear and faeces were collected. These samples were sent to the Institute of Vertebrate Biology of the Czech Academy of Sciences, Brno, Czech Republic, for analysis. There, DNA was extracted from the samples using the Innu PREP DNA Mini Kit 2.0 (IST Innuscreen GmbH, Germany) according to the manufacturer's protocol. The results obtained confirm, for the first time through records of the species, the presence of Gerbillus campestris in Melilla and, therefore, in Spain and Europe.

Tessellated calcified cartilage (TCC) is a hallmark of the chondrichthyan skeleton, yet its development early in ontogeny across the four major groups (batoids, galeomorphs, squalomorphs, and holocephalans) remains poorly understood. Specialised traits of TCC, such as multi-layered TCC and internal mineralised trabeculae, typically develop in response to feeding mechanics. In this study, we evaluated TCC morphology in the jaws of 12 representative taxa to observe its structure at an early ontogenetic stage to determine whether these specialised features had yet developed. Batoids consistently exhibited well-developed, homogeneous, polygonal tesserae early in ontogeny regardless of jaw morphology or feeding habit. In contrast, galeomorphs displayed high morphological heterogeneity. Notably, we document the first report of an extensive internal trabecular network in a non-batoid elasmobranch, observed in Ginglymostoma cirratum, which may serve to resist the mechanical pressures of specialised suction feeding. Furthermore, we identified voussoir tesserae in galeomorphs for the first time, extending their documented presence across all elasmobranch groups, where they display an inverted aspect ratio (wider than tall) compared to mature forms. The durophagous Mustelus mustelus exhibited surprisingly poor TCC development despite being a durophagous feeder, pointing to a pronounced ontogenetic lag. In Squatina oculata, TCC was characterised by large and thick tesserae and extensive fused tesseral regions which may relate to its explosive ambush predation mode, whereas the holocephalan Chimaera exhibited a poorly mineralized, mesh-like structure without resolvable discrete tesserae or trabeculae-matching findings from previous studies. Across all specimens, multi-layered TCC was absent, confirming that multi-layering develops later in ontogeny. These results demonstrate that generalised models of TCC development based on one group or a few taxa fail to capture the broader diversity of TCC morphology. It also opens up many exciting avenues for further study, and forms the basis for comparisons with fossil chondrichthyans, to investigate the evolution of TCC.

Metabolic rate governs the pace of life through the allocation and expenditure of energy, yet metabolic rate data are unavailable for most species. External morphological traits, such as gill slits, have been used to infer respiratory physiology and metabolism in sharks, yet, whether that pattern is generalizable across rays is unknown. In rays (Batoidea), spiracle length seems adaptive to oxygen uptake capacity, environmental oxygen availability, and activity level. Thus, we evaluate the allometry of external respiratory morphology (spiracles and gill slits) in rays using 550 museum specimens representing 88 species. We test whether spiracle length and gill slit height grow isometrically with body size (total length or disc width) within and across species. Overall, we find spiracles and gill slits are morphologically diverse, varying widely in shape within and between families. Spiracle length and gill slit height scale isometrically and are a consistent proportion of body size, despite such high morphological variability. While that proportion is consistent through ontogeny, each species proportion differs (e.g. gill slit height ranges from 0.93% of total length in Spotted Legskate to 2.93% in Pacific Electric Ray). Of the three swimming styles, pectoral oscillating species (Mobulid and Eagle Rays) may have steeper scaling of spiracle length with gill slit height - consistent with greater respiratory capacity in species with more active lifestyles. Our findings are consistent with literature hypothesizing external respiratory morphology (spiracles and gill slits) is tightly related to metabolic rate and activity level, and demonstrate the value of natural history collections in ecological research. Importantly, the isometry of both respiratory traits means that a single measure of any individual, irrespective of size, will be representative of the species thus providing a basis for expanding our understanding of metabolic morphology, enabling future work on life history, extinction risk, and ecological function.

Arcellidae is a family of testate amoebae within Arcellinida (Amoebozoa), comprising three recognized genera: Arcella, Galeripora, and Antarcella. Although species in the family have been studied for nearly two centuries, many historically described taxa and major morphological groups remain unsampled at the molecular level. Here, we provide a comprehensive review of Arcellidae and generate new cytochrome c oxidase subunit I (COI) sequences for arcellid species from Canadian peatlands, focusing on tall-shelled Arcella historically classified in section Altae sensu Deflandre. COI phylogenetic analyses recover a strongly supported monophyletic clade corresponding to North American representatives of Altae, providing the first molecular corroboration of this morphologically defined group. Within this clade, we redescribe Arcella leidyana based on modern material from Eeyou Istchee (Quebec). We further describe Galeripora purdoni sp. nov. from a calcareous fen in eastern Ontario, representing a novel terrestrial lineage within the genus, and redescribe Galeripora artocrea, which we transfer to Arcella based on congruent molecular and morphological evidence. Phylogenomic analyses of Arcellidae isolates from the Protist 10,000 Genomes Project reveal an additional deep lineage basal to Arcella and Galeripora. Together, these results highlight hidden diversity and demonstrate the importance of integrative approaches for resolving arcellid systematics and refining its classification.

Rheiformes are a South American lineage of large, flightless palaeognathous birds, currently represented by the genus Rhea and the two extant species R. americana and R. pennata. The fossil record of the group extends back to the Eocene, with its greatest diversification occurring during the Neogene. During the Quaternary, Rheiformes were widely distributed across southern and central South America. The Lagoa Santa region is particularly notable for its rich paleontological and archaeological record. Quaternary records of Rheiformes from caves in this region provide important evidence for understanding the distribution of the group in Brazil, although their chronological context remains uncertain owing to the absence of direct dating. Here, we describe and illustrate two rheiform bones recovered from a cave in the Lagoa Santa karst region. The material comprises a partially complete tibiotarsus and a fragmented tarsometatarsus, interpreted as belonging to a single individual. Comparative anatomical and morphometric analyses support their attribution to Rhea americana, based on morphological features and dimensions consistent with adult specimens of the species. This occurrence extends the Quaternary record of Rheiformes in the Lagoa Santa region and contributes to the fossil and subfossil record of Rhea in Brazil. The absence of stratigraphic and geochronological control precludes a precise age determination. Although the association with an extinct dasypodid indicates the presence of Late Pleistocene or Early Holocene faunal elements at the locality, a Holocene or even recent age for the rheiform remains cannot be excluded.

The marine sponge Hymeniacidon perlevis is a globally-distributed species, likely because it has been extensively spread through human means. The status of this species in the Western Atlantic has been difficult to discern due to the presence of a morphologically similar species Hymeniacidon heliophila. Here, we have collected Hymeniacidon specimens from the H. heliophila type location and compared their morphology and genotypes to material from Europe, North and South America, and publicly available datasets. Specimens from the type location closely matched the original description of H. heliophila, and were morphologically indistinguishable from H. perlevis. Multilocus sequencing data confirm that these samples are indistinguishable from H. perlevis, and that the name H. heliophila should therefore be considered a junior synonym of H. perlevis. Analysis of a global cox1 dataset further revealed significantly higher genetic diversity in Europe than in the remainder of the world combined, supporting a European origin for the species and suggesting that all other genotyped populations are introductions. In the Western Atlantic, we confirm the presence of H. perlevis in the Gulf of Mexico, but contrary to some past reports, are unable to confirm its presence in the Caribbean. Our results clarify the taxonomy, biogeography, and recent history of one of the worlds most widespread marine sponges.

Among Madagascar primates, the sportive lemurs (family Lepilemuridae) have seen their species diversity increase from eight in 2005 to 26 in 2009 mostly by applying the phylogenetic species concept to DNA barcode data. Despite the genus being speciose, only one case of sympatry is known from northern Madagascar, where two sportive lemur species described based on low mtDNA divergence, Lepilemur ankaranensis and Lepilemur milanoii, were found to co-occur at the center of their joint distribution range. Here, to clarify the taxonomy of these two species and examine their sympatry, we apply an integrative taxonomic framework to genomic and morphological data from 84 individuals of L. ankaranensis and L. milanoii, encompassing their entire distribution range and the forest of Analafiana, beyond their southernmost limit. Using clustering, multivariate, and isolation by distance analyses, we find no evidence of a sympatric zone and show that despite clear genetic differentiation between regions, the genomic and morphological diversity of the L. ankaranensis, L. milanoii-Analafiana group is clinal and explained by geographic distance. These results clarify that L. milanoii is a junior synonym of L. ankaranensis and that the Analafiana forest population belongs to L. ankaranensis, extending its distribution. It further implies that the 'sympatric' zone, the Andrafiamena forest, hosts conspecific individuals with slightly differentiated mtDNA backgrounds, rather than slightly differentiated sympatric species. Lastly, we re-evaluate the IUCN conservation metrics of L. ankaranensis, which continue to qualify as Endangered (EN) under the B1ab(i-v) criteria.

Genetic sex-determination mechanisms evolve rapidly in teleost fishes and are diverse within Beloniformes. In the present study, the sex-determination system of Pacific saury (Cololabis saira) was confirmed using double-digest, restriction-site-associated DNA sequencing (ddRAD-seq) and pooled whole-genome sequencing (Pool-seq), and a DNA marker was developed for inferring genetic sex. DNA sequence analysis detected numerous markers occurring predominantly or exclusively in phenotypic females suggesting a ZZ/ZW sex-determination system. Mapping of ddRAD-seq loci showed that these markers were concentrated within a 3-19 Mb region of chromosome 17 (Chr17). Pool-seq analyses also revealed elevated FST, sex-specific differences in sequencing depth, and nucleotide diversity within this region, supporting a ZZ/ZW sex-determination system involving Chr17. A PCR marker designated female-specific 1 (FS1), developed from a female-associated ddRAD sequence unmapped from the male-derived reference genome, was detected in all 43 phenotypic females and none of the 50 phenotypic males collected in 2025. In an independent sample collected in 2026, FS1 was detected in all 19 phenotypic females and one of the 29 phenotypic males, resulting in 97.9% concordance with phenotypic sex. The FS1-positive male may represent a genotypic-phenotypic sex mismatch or reflect recombination between FS1 and the causal sex-determining region; the linkage of FS1 to the W chromosome remains unconfirmed. Chromosome-scale synteny analysis showed that Pacific saury Chr17 and the sex chromosomes of Hyporhamphus sajori and Oryzias latipes are nonhomologous to one another. These findings reveal substantial sex-chromosome diversity among beloniform fishes and establish a basis for investigating genetic sex determination and genotypic-phenotypic sex concordance in wild Pacific saury.

Despite their currently scarce taxonomic diversity, rhinocerotoids were among the most evolutionarily successful clades of large herbivorous mammals throughout the Caenozoic. Within an array of morphological adaptations, the cranial horn represents the clades most remarkable and name-giving feature, imposing significant morpho-functional demands on the skull. In this paper, patterns and drivers of the rhinos cranial evolution were investigated for the first time by compiling a three-dimensional geometric morphometric dataset of living and extinct species. Multiple aspects of morphological evolution were explored using phylogenetic comparative methods, including craniofacial evolutionary allometry (CREA - tendency of larger species to have longer faces), phylogenetic constraints, and tempo of evolution. Cranial shape variation linked to horn presence and size evolved under a phylogenetically constrained framework, with a notable transition from hornless to horned species. Rhinos significantly deviated from CREA, likely due to diversity of cranial forms and proportions that evolved independently across the clade in response to varying horn morphologies and dietary habits. Hornless and horned rhinos exhibited similar rates of cranial evolution, and shape variation was obtained through multiple episodes of accelerated evolution. This highlights the role of morphological innovations and Caenozoic global cooling events in the emergence of phenotypic diversity.

Sexual dimorphism is a well-studied phenomenon in which males and females of a species exhibit different phenotypic characteristics. However, the degree of sexual dimorphism within a species may vary among populations and may be influenced by geographic factors such as seasonality, prey availability, and population density. In this study, we examined the effects of sexual dimorphism and geographic region on the craniomandibular morphology of bobcats (Lynx rufus) in Washington State. Using digital calipers, we measured 13 craniomandibular traits from 35 males and 42 females. We found that the degree of skull dimorphism does not differ between the eastern and western side of the Cascade Mountains. However, we found significant effects of sex and geographic region on craniomandibular morphology. Across the geographic regions, males were significantly larger than females in most craniomandibular traits, excluding postorbital constriction breadth (POC). After correcting for expected size differences, only condylobasal length (CBL) was disproportionately longer in males, indicating subtle shape dimorphism within this trait, that is largely unexplained by allometry. Mechanical advantage (MA) of the temporalis and masseter muscles did not differ between the sexes at the canine or the molar bite points. Comparisons between populations east and west of the Cascade Range revealed that while sex remains the primary indicator of overall size, there is a small regional effect on size as western bobcats are slightly larger than eastern bobcats. These regional differences highlight how environmental pressures including local prey availability and severe winter conditions may influence functional skull design. Future ecological and behavioral studies are needed to further clarify how these localized environmental drivers shape phenotypic variation across populations.

Kashmir voles (Hyperacrius) are poorly studied fossorial arvicolines of the southern Himalayan slopes, with two species recognized. We sequenced Cytb from voles collected in northern Pakistan and combined these data with existing GenBank sequences, yielding 194 tips spanning nearly all arvicoline genera. We analyzed five data sets with maximum likelihood and compared external measurements among species using principal component and linear discriminant analyses. We recovered five well-supported clades of Hyperacrius separated by ~12-14% p-distance, which we treat as distinct species. We recognize clade 1 as H. wynnei, and populations referred to H. w. traubi are genetically indistinguishable from H. w. wynnei. We recognize H. zygomaticus, new rank (clade 2), and tentatively allocate clade 5 to H. brachelix. Clades 3 and 4 likely represent undescribed species. Morphometric analyses of external measurements clearly distinguish H. wynnei but not clades 3 and 4 (overall classification accuracy 70.3%). Phylogenetic analyses including nuclear genes placed Hyperacrius as sister to a clade containing Microtini, Arvicola, Lagurini, Dinaromys, and Ellobiusini, whereas mitochondrial analyses weakly allied it with Ellobiusini. Its traditional placement in Clethrionomyini is thus unsupported, although its exact position remains unresolved. Alticola is nonmonophyletic, with A. macrotis sequences nested within Clethrionomys, likely reflecting mitochondrial capture.

Hydrozoa is a group of relatively simple animals with a well-developed nervous system. The nervous system in all hydrozoan medusae is highly conserved and includes outer and inner nerve rings at the bell margin, a neural network in the manubrium, and radial neural pathways that connect them. However, one element of the nervous system shows substantial variability among species: the subumbrella neural network. We examined the structure of the nervous and muscular systems in the subumbrella of 14 species of hydrozoan medusae. The main conclusion of this study is that the distribution of neural networks in the subumbrella strongly correlates with the distribution of smooth radial muscles. This correlation suggests that smooth radial muscles are the primary target of the subumbrella nervous system. Most species in the order Anthoathecata show a trend toward secondary loss of the neural networks and radial smooth muscle fibers in the subumbrella region, concentrating neural elements and smooth muscles only in the radial pathways along the radial canals. By contrast, all studied species in the order Leptothecata have neural networks in the subumbrella area, as well as radial smooth muscle fibers spread throughout the entire subumbrella region. The correlation between radial smooth muscles and the nervous system is also observed in the radial pathways along the radial canals. All species with thick bundles of smooth radial muscles along the radial canals have clearly defined, dense neural pathways running along or even embedded within the smooth muscle bundles.

Understanding patterns of evolution and divergence in populations is important for defining conservation units and informing taxonomy. However, when populations have low genetic diversity, or are closely related, genetic differentiation can be difficult to detect, particularly when relying on small numbers of genetic markers. Whole-genome sequencing allows for genome-wide identification of both neutral and outlier variation, improving the resolution of subtle population structure and providing insight into evolutionary processes. Here, we investigated genomic differentiation in a recently diverged and highly threatened albatross complex, the Antipodean (D. antipodensis antipodensis) and Gibson's albatross (D. a. gibsoni), using genome-wide neutral and outlier datasets. Whole-genome resequencing of 86 individuals sampled across Antipodes Island (D. a. antipodensis) and the Auckland Islands (D. a. gibsoni) identified 381,176 neutrally evolving and 57 independently segregating outlier (putatively adaptive) SNPs. Analyses of both datasets revealed significant genetic differentiation between the Antipodean and Gibson's albatross, no evidence of contemporary gene flow and evidence of selective sweeps suggesting local adaptation. Within-population structure was also identified for the Gibson's albatross, with genetic differentiation among sample sites from different islands (Adams Island and Disappointment Island). Patterns of heterogenous differentiation across the genome suggest the taxa are on different evolutionary trajectories. Together with existing morphological and behavioural evidence, these genomic results support reassessment of their conservation and taxonomic status. More broadly, this study demonstrates the value of combining neutral and putatively adaptive genomic variation to resolve subtle population structure in recently diverged taxa.

The genus Phakellia comprises predominantly fan-shaped deep-sea sponges, but its species diversity and boundaries remain unresolved. Here, we use an integrative approach combining morphological and molecular data to investigate the Phakellia robusta species complex and describe two new species, Phakellia pallida sp. nov. and Phakellia pusilla sp. nov. Overlap in external and spicular morphology makes these taxa challenging to identify. Molecular analyses based on mitochondrial (cox1) and nuclear (28S) markers support the recognition of the three taxa as distinct species, and reveal their close evolutionary relationship. The two new species co-occur in the NE Atlantic Ocean and the Mediterranean Sea, with overlapping depth ranges, although P. pallida sp. nov. is generally found at greater depths. Our results also suggest that several previous records of P. robusta from the Mediterranean and southern parts of the NE Atlantic may instead correspond to either P. pallida sp. nov. or P. pusilla sp. nov., indicating that its distribution requires reassessment. In addition, a group I intron previously reported in the cox1 gene of P. robusta was polymorphically detected at the same insertion site in some, but not all, individuals of P. pusilla sp. nov., but it was absent from P. pallida sp. nov. individuals. Overall, our results reveal pseudocryptic diversity within Phakellia and highlight the value of integrative approaches for resolving species boundaries and reassessing deep-sea sponge diversity and distributions.

Understanding when species diversification and phenotypic evolution are coupled remains a central challenge in macroevolution. Such coupling may depend on the traits considered, phenotypic integration, or the geographic context in which diversification occurs. Here, we investigate these relationships in Ithomiini, a diverse Neotropical butterfly tribe whose diversification is strongly associated with the Andes-Amazonia system. We quantified forewing and hindwing size and shape for 1,618 individuals representing 291 species using geometric morphometrics, and combined these data with phylogenetic comparative methods, diversification-rate estimates, and spatial analyses of species distributions. Evolutionary rate shifts were largely trait- and wing-specific, indicating limited coupling between forewing and hindwing evolution. Across the phylogeny, wing size evolution and extant wing size were not associated with species diversification, nor was hindwing shape evolution. By contrast, forewing shape evolutionary rates showed a positive association with diversification. Spatial analyses revealed that this coupling was geographically concentrated in the Andes, where high diversification rates coincided with elevated forewing size and shape diversity and increased forewing evolutionary rates. Hindwing diversity, by contrast, more closely tracked phylogenetic diversity and showed weaker or opposing relationships with diversification. Our results show that diversification and phenotypic evolution in Ithomiini are neither generally coupled nor fully independent, but linked in trait-specific and geographically structured ways. More broadly, they highlight the need to decompose complex phenotypes and integrate spatial context when testing macroevolutionary coupling.

Reliable morphological criteria for sexing birds without the need for invasive procedures are scientifically and ethically valuable. Smith et al (2005) reported that iris colour assigns the genetic sex of Starlings Sturnus vulgaris with 98% accuracy, and bill base colour with 100% accuracy when birds are in breeding condition, but these figures have not been tested independently. We applied their criteria to 31 captive Starlings in breeding condition, using three observers blind to genetic sex, two of them new to Starling sexing. Bill base colour correctly predicted genetic sex for all 29 birds scored, with complete observer agreement. Mean iris score was correct for 27 of 31 (87.1%), fewer than the 94 of 96 reported by Smith et al (2005) for birds in breeding condition (Fisher exact test, P = 0.031), although with so few errors in either study this comparison is imprecise. Observer agreement was high, so these errors reflect iris colouration conflicting with genetic sex rather than inconsistent scoring; bill base colour was correct for all four misassigned birds. Body mass and tarsus length classified 77% and 58% of birds correctly, their published thresholds missing in opposite directions. We conclude that bill base colour should be the primary criterion for sexing Starlings in breeding condition; the accuracy of iris colour should not be assumed to be 98%.