Cellular Scaling Rules for Brains of the Galliform Birds (Aves, Galliformes) Compared to Those of Songbirds and Parrots: Distantly Related Avian Lineages Have Starkly Different Neuronal Cerebrotypes Open Access

Introduction: Songbirds, especially corvids, and parrots are remarkably intelligent. Their cognitive skills are on par with primates and their brains contain primate-like numbers of neurons concentrated in high densities in the telencephalon. Much less is known about cognition and neuron counts in more basal bird lineages. Here, we focus on brain cellular composition of galliform birds, which have small brains relative to body size and a proportionally small telencephalon and are often perceived as cognitively inferior to most other birds. Methods: We use the isotropic fractionator to assess quantitatively the numbers and distributions of neurons and nonneuronal cells in 15 species of galliform birds and compare their cellular scaling rules with those of songbirds, parrots, marsupials, insectivores, rodents, and primates. Results: On average, the brains of galliforms contain about half the number of neurons found in parrot and songbird brains of the same mass. Moreover, in contrast to these birds, galliforms resemble mammals in having small telencephalic and dominant cerebellar neuronal fractions. Consequently, galliforms have much smaller absolute numbers of neurons in their forebrains than equivalently sized songbirds and parrots, which may limit their cognitive abilities. However, galliforms have similar neuronal densities and neuron counts in the brain and forebrain as equally sized non-primate mammals. Therefore, it is not surprising that cognitive abilities of galliforms are on par with non-primate mammals in many domains. Conclusion: Comparisons performed in this study demonstrate that birds representing distantly related clades markedly differ in neuronal densities, neuron numbers, and the allocation of brain neurons to major brain divisions. In analogy with the concept of volumetric composition of the brain, known as the cerebrotype, we conclude that distantly related birds have distinct neuronal cerebrotypes.

Birds can be remarkably smart, even though they have small brains compared to mammals. This is especially true for parrots and corvids who are on par with primates in many cognitive domains [1‒5]. They plan for the future, make inferences about causality, understand the minds of others, and have high levels of self-control and behavioral flexibility (e.g., [6‒8]), to mention just a few complex abilities. They have relatively large brains for their bodies (e.g., [9]), characterized by a large telencephalon, which is dominated by the associative nidopallium and mesopallium, and the striatopallidal complex [10]. Recently, it has been shown that brains of corvids and parrots contain huge numbers of neurons, packed in high densities in the telencephalic pallium [11], particularly in the associative areas [12]. Moreover, their pallial neuron numbers scale with body size with a steeper slope compared to other bird clades [13]. The nidopallium caudolaterale, which is involved in executive control and is generally considered to be a functional equivalent of the mammalian prefrontal cortex (e.g., [14‒17]), has been shown to be enlarged and compartmentalized in songbirds, and especially in corvids [18].

In contrast, basally diverging birds, namely ratites and galliforms, feature a lower degree of encephalization (e.g., [9]) and a proportionally smaller telencephalon [10]. Recent studies indicate that compared to songbirds and parrots, they have a smaller telencephalic and dominant cerebellar neuronal fraction and generally lower neuronal densities [11, 19], with rather modest numbers of neurons in associative telencephalic regions [12]. However, these studies included only a handful of species from these clades or divided the brain into broad compartments, restricting comprehensive statistical comparison of cellular scaling rules and allocation of neurons to different brain parts. Here, we address this gap and present a detailed comparison of brain cellular composition between gallinaceous birds and songbirds and parrots.

Gallinaceous birds or galliforms are ground-feeding precocial birds with a huge variation in body size. The order contains about 300 species, inhabiting every continent except Antarctica [20]. Together with anseriform birds, they form the clade Galloanserae, a sister group to all living birds except for tinamous and ratites [21‒23]. Therefore, they are typically considered baseline for avian encephalization [24, 25] and some authors maintain that small volumes of the pallium or its parts (namely, the mesopallium and nidopallium), typical for galliforms, are an indicator of a low level of innovativeness [26, 27]. Galliform brains are characterized by a relatively large optic tectum, diencephalon, and brainstem and a relatively small Wulst and mesopallium [10] and a small, simply organized nidopallium caudolaterale [18].

Galliforms are traditionally viewed as birds with rather low cognitive abilities. The chicken (Gallus gallus) is the predominant model species for testing fundamental cognitive processes such as associative learning, imprinting, or visual perception [28, 29]. Chickens also display some aspects of physical reasoning, e.g., amodal completion [30], self-control [31, 32], temporal cognition, such as perception of time duration [33], or numerical competencies [34‒37]. Because the body of research on higher cognitive processes is skewed mostly toward corvids and parrots, future studies may reveal a larger repertoire of cognitive skills in gallinaceous birds, especially in the social domain [29]. In fact, rather sophisticated capacities such as perspective-taking [38] and intentional or tactical deception have been reported in the chicken [39].

In this study, we quantify neurons and nonneuronal cells in 6 brain divisions in 15 species of gallinaceous birds and compare their numbers and distributions with those of parrots, songbirds, and four mammalian orders. These data provide new insights into the evolution of avian brain-neuron scaling and information processing capacity.

A total of 39 birds belonging to 15 species of galliform birds were used in this study (online suppl. Fig. 1; online suppl. Table S1; for all online suppl. material, see https://doi.org/10.1159/000545417). Some of the data have been previously used in other studies [11, 13, 19, 40]. Three individuals per species were collected with the exception of the yellow-knobbed curassow (Crax daubentoni), the California quail (Callipepla californica), the Black francolin (Francolinus francolinus), and the Black grouse (Tetrao tetrix), where only 1 or 2 birds were examined. The common quail (Coturnix coturnix) and the common pheasant (Phasianus colchicus) were wild-caught in the Czech Republic (Permission Nos. 00212/CS/2013 and 446/2013); the Grey partridge (Perdix perdix), the Red-legged partridge (Alectoris rufa), and the Reeves’s pheasant (Syrmaticus reevesii) were pen-reared birds obtained from seminatural populations; all other species were purchased from local breeders. Only adult birds were included in this study and their sex was confirmed upon dissection.

The animals were killed by an overdose of halothane. They were weighed and immediately perfused transcardially with warmed phosphate-buffered saline containing 0.1% heparin followed by cold phosphate-buffered 4% paraformaldehyde solution. The skulls were partially opened and postfixed for 30–60 min, after which the brains were dissected and weighed. Brains were postfixed for additional 7–21 days and then dissected.

Brains were dissected into distinct compartments using the Olympus SZX 16 stereomicroscope as previously described [11]. Briefly, the cerebral hemispheres were detached from the diencephalon by a straight cut separating the subpallium from the thalamus. The tectum (comprising most of the tectal gray, optic tectum, and torus semicircularis) was bilaterally excised from the surface of the brainstem. Both the left and right tecta were processed together. The cerebellum was cut off at the surface of the brainstem. Finally, the remaining structures were dissected into the diencephalon (rostral part) and the brainstem (caudal part) along the plane connecting the posterior commissure dorsally and the hypothalamus-mesencephalon boundary ventrally. Because we detected negligible differences between the left and right hemisphere mass and cell numbers in our pilot studies, only one hemisphere per individual was processed. In one individual per species, the second hemisphere was dissected into the pallium and the subpallium. These hemispheres were embedded in agarose and sectioned on the Leica VT1200 S vibratome at 300–500 μm (depending on the size of the hemisphere) in the coronal plane. Under oblique transmitted light at the stereomicroscope and with the use of a microsurgical knife (Stab Knife Straight, 5.5 mm, REF 7516, Surgical Specialties Corporation, Reading, PA, USA), we manually dissected the pallium from the subpallium on each section by cutting along the pallial-subpallial boundary, as defined by Puelles et al. [41]. The dissected structures were dried with paper towels, weighed, and then incubated in a 30% sucrose solution until they sank. The samples were subsequently transferred into antifreeze (30% glycerol, 30% ethylene glycol, 40% phosphate buffer) and stored at −25°C until further processing.

We estimated the total numbers of cells, neurons, and nonneuronal cells using the isotropic fractionator, as described earlier [42]. Briefly, each dissected brain division was homogenized in 40 mm sodium citrate with 1% Triton X-100, using Tenbroeck tissue grinders (Wheaton, Millville, NY, USA). When the sample turned into an isotropic suspension of isolated cell nuclei, the homogenate was stained with the fluorescent DNA marker DAPI, adjusted to a defined volume, and kept homogeneous by agitation. The total number of nuclei in the suspension, and therefore the total number of cells in the original tissue, was estimated by determining the density of nuclei in small fractions drawn from the homogenate. At least four 10 μL aliquots were sampled and counted using a Neubauer improved counting chamber (BDH, Dagenham, Essex, UK) with an Olympus BX51 microscope equipped with epifluorescence and appropriate filter settings (specifically, Olympus filters U-MWU2 for DAPI and U-MWG2 for Alexa Fluor 546-conjugated secondary antibodies); additional aliquots (typically 2–5) were assessed when needed to achieve a coefficient of variation among counts ≤0.15 (usually, a coefficient of variation ≤0.10 was achieved).

Once the total cell number was known, the proportion of neurons was determined by immunocytochemical detection of the neuronal nuclear marker NeuN [43]. This neuron-specific protein was detected by the mouse monoclonal antibody anti-NeuN (clone A60, Chemicon, Temecula, CA, USA; dilution 1:800), which has been characterized by Western blotting with chick brain samples and shown to react with a protein of the same molecular weight as in mammals [44], indicating that it does not cross-react with other proteins in birds. The binding sites of the primary antibody were revealed by Alexa Fluor 546-conjugated goat anti-mouse IgG (Life Technologies, Carlsbad, CA, USA; dilution 1:500). An electronic hematologic counter (Alchem Grupa, Torun, Poland) was used to count simultaneously DAPI-labeled and NeuN-immunopositive nuclei in the Neubauer chamber. A minimum of 500 nuclei were counted to accurately estimate the percentage of double-labeled neuronal nuclei. Numbers of nonneuronal cells were derived by subtraction.

All analyses were performed using the average values for each species; all variables were log-transformed. Ordinary least squares regressions were calculated to describe how structure mass, numbers of cells, and densities are interrelated across species. To compare scaling among groups and account for phylogenetic dependence, we used phylogenetic least squares (PGLS) with simultaneous phylogenetic signal estimation, as implemented in the nlme package [45]. The phylogenetic trees were created from published species-level time-calibrated phylogenies (birds [9]: mammals [46]) pruned to match the datasets (online suppl. Fig. S2).

For the comparison with cellular scaling rules reported previously for mammals, we used previously published data for primates [47‒49], rodents excluding the naked mole-rat [50, 51], Eulipotyphla [52], and marsupials [53]. Marsupials were selected as early-diverged mammals, rodents as typical representatives of non-primate mammals, Eulipotyphla as mammals exhibiting high absolute neuronal densities, and primates as mammals with the highest relative neuronal densities.

All statistical analyses were performed in R 4.1.2 (R Core Team, 2021). Graphs were plotted in JMP 10.0 (SAS Institute, Cary, NC, USA).

All quantitative data gathered in this study are summarized in online supplementary Dataset S1.

Among the 15 galliform species studied, weighing between 44 and 3,600 g, brain mass ranges from 0.52 to 9.02 g and the total number of neurons in the brain from 80 to 653 million (Fig. 1; online suppl. Table S1). The relationship between brain mass and the number of brain neurons can be described by the power function $MBR=2.939×10−11×NBR1.299$ (online suppl. Table S5). Because the scaling exponent is significantly higher than 1.0 (95% confidence interval = 1.125–1.472), any gain in the number of brain neurons is accompanied by an even more pronounced gain of mass: a 10-fold increase in the number of neurons results in a 19.9 larger brain. Figure 2a compares brain mass scaling with the total number of brain neurons between galliform birds, songbirds, and parrots. Allometric lines for these three groups do not differ in slope (PGLS: p > 0.08), but the allometric line for galliform birds has a significantly lower intercept (galliforms vs. songbirds: t₍₃₅₎ = −11.12, p < 0.001; galliforms versus parrots: t₍₃₅₎ = −8.8, p < 0.001; λ = 0), clearly indicating a phylogenetic difference (grade shift) in total numbers of neurons between galliform birds and the other two avian groups. Brains of galliforms accommodate about half the number of neurons found in parrot and songbird brains of the same mass. Moreover, galliform birds show small brain mass for their body mass compared to songbirds and parrots (Fig. 2c, 3a). The relationship between brain volume and body mass among the 15 species examined in this study can be described by the power function $MBR=0.051×MBo0.605$ (r² = 0.965, p < 0.001; Fig. 2c), among the 71 species of galliforms collated from the literature by the power function $MBR=0.098×MBo0.531$ (r² = 0.905, p < 0.001; Fig. 3a). The scaling coefficient is not only significantly smaller than 1.0 (confidence interval = 0.490–0.572) but also significantly smaller than that for parrots and songbirds (PGLS, galliforms vs. songbirds: t₍₇₂₀₎ = 5.66, p < 0.001; galliforms versus parrots: t₍₇₂₀₎ = 4.68, p < 0.001; λ = 0.842). Thus, the difference in relative brain size between galliform birds and the other two avian groups increases with body size. Because they have smaller brains and lower average neuronal densities, the brains of galliform birds harbor a much smaller absolute number of neurons than the brains of equivalently sized songbirds or parrots (galliforms vs. songbirds: t₍₃₃₎ = 3.82, p < 0.001; λ = 1; galliforms versus parrots: t₍₃₃₎ = 4.17, p < 0.001; λ = 1) (Fig. 2d). For instance, the common pheasant is somewhat heavier than the blue-and-yellow macaw (Ara ararauna), but its brain has ∼10-fold fewer neurons. Likewise, the red junglefowl is ∼50-fold heavier than the great tit (Parus major), but both birds have approximately the same number of brain neurons.

Interestingly, the largest brain and the highest absolute number of neurons were observed in the yellow-knobbed curassow (Fig. 1; online suppl. Table S1), a representative of the clade Cracidae (including the chachalacas, guans, and curassows). Indeed, cracid brains tend to be large when compared to the brains of other galliform birds (Fig. 3a).

In galliform birds, neuronal density varies greatly among the principal brain divisions examined and decreases significantly with increasing brain mass in all these divisions (scaling exponent ranges between −0.186 and −0.502; r² between 0.470 and 0.905, p ≤ 0.005 in all cases; Fig. 4b). Just as in other avian groups, neuronal densities are highest in the cerebellum (310–660 × 10³ N/mg) and lowest in the brainstem (4–28 × 10³ N/mg). Neuronal densities in all divisions are significantly lower than those observed in songbirds and parrots (Fig. 4d, statistics not shown). Typically, galliforms have 1.3–2.2-fold lower neuronal densities than songbirds and parrots. In case of the telencephalon, this difference is even more pronounced because the allometric line for galliforms has not only a lower intercept but also a lower slope (slope, galliforms vs. songbirds: t₍₃₃₎ = 2.35, p = 0.025; galliforms versus parrots: t₍₃₃₎ = 2.75, p = 0.01; intercept, galliforms versus songbirds: t₍₃₃₎ = 11.47, p < 0.001; galliforms versus parrots: t₍₃₃₎ = 8.65, p < 0.001; λ = 0.842). The density of telencephalic neurons is 2.3–4.9-fold lower in galliforms than that in songbirds and parrots. This has important consequences for the relative distribution of neurons among major brain compartments (see below).

The telencephalon mass fraction increases with brain size at the expense of all other brain components but the cerebellum, ranging from 52% to 64% (Fig. 5a–c; online suppl. Table S2). However, the relative proportion of the telencephalon is much smaller in galliforms than in songbirds and parrots (songbirds: 63–80%, parrots: 71–85%). The mass fractions of other brain divisions are larger in galliforms. Cerebellum, tectum, diencephalon, and brainstem constitute 12–16%, 10–14%, 7–10%, and 9–12% of the total brain mass, respectively. Moreover, in contrast with that of songbirds and parrots, the cerebellar mass fraction does not decrease with brain size (Fig. 5b). Thus, the relative size of major brain divisions differs starkly between the three avian groups analyzed. The difference in the relative distribution of neurons is even more striking (online suppl. Table S3; Fig. 5d–f). Just like mammalian brains (see below) and in marked contrast to brains of songbirds and parrots, brains of galliform birds are characterized by small telencephalic and dominant cerebellar neuronal fractions. Moreover, the cerebellar neuronal fraction increases and telencephalic neuronal fraction tends to decrease as brains get larger; the reverse pattern is observed in songbirds and parrots. Therefore, the cerebellum of galliforms houses as much as 52–72%, whereas their telencephalon houses only 23–36% of all brain neurons. Taken together, the telencephala of galliform birds accommodate much smaller absolute numbers of neurons than the telencephala of equivalently sized brains of songbirds or parrots; this difference is more pronounced the larger the brain (Fig. 6). For instance, the telencephalon of the king quail (Coturnix chinensis) has ∼2.6-fold less neurons than that of the zebra finch (Taeniopygia guttata), the telencephalon of the turkey (Meleagris gallopavo) has ∼4-fold less neurons than that of the jackdaw (Coloeus monedula), and the telencephalon of the yellow-knobbed curassow has almost 8-fold less neurons than that of the gray parrot (Psittacus erithacus).

The subpallium (comprising the striatum, pallidum, and septum) constitutes 13–18% of total telencephalon mass and houses 14–19% of all telencephalic neurons (online suppl. Tables S3; Fig. 7c,d). Neither the relative mass of the subpallium nor the fraction of telencephalic neurons contained within it correlates with telencephalon size (mass fraction: ρ = 0.143, p = 0.612; neuronal fraction: ρ = 0.04, p = 0.899). The number of subpallial neurons scales almost linearly with the number of pallial neurons (⁠$scalingexponent=0.989±0.101;Nsubpall=0.239×Npall0.989$⁠; Fig. 7b), indicating a concerted gain of neurons that maintains a ratio of ∼4.2 neurons in the pallium to every neuron in the subpallium. This feature seems to be taxon-specific, because in parrots, the number of neurons in the subpallium increases faster than in the pallium (scaling exponent = 1.185 ± 0.15), while an opposite trend is observed in songbirds (scaling exponent = 0.891 ± 0.09).

Nonneuronal scaling rules are remarkably similar across brain divisions and avian lineages (Fig. 2b, online suppl. Tables S4, S5). In contrast to neuronal scaling rules, they do not differ significantly between avian groups (PGLS, galliforms vs. songbirds p = 0.76, galliforms vs. parrots p = 0.76, λ = 0.94). The densities of nonneuronal (glial and endothelial) cells remain similar in all brain structures, except for the telencephalon, where nonneuronal cell density is distinctively lower (galliforms: −0.283 ± 0.037, t_{3, 27} = 7.695, p < 0.001; songbirds: −0.132 ± 0.031, t_{3, 19} = 4.303, p < 0.001; parrots: −0.296 ± 0.013, t_{3, 23} = 23.486, p < 0.001; Fig. 4c, d). Because the lower nonneuronal density in the telencephalon was observed in all three avian groups studied but never in mammals [53, 54], it seems to be a specific avian feature.

In galliform birds, nonneuronal cell density decreases significantly with increasing brain size in all brain divisions examined (Fig. 4c, p < 0.014 in all cases), and the rate of this decrease is the highest in the cerebellum (scaling exponent for the cerebellum = –0.303; for other structures, it ranges between −0.124 and −0.150).

In contrast to songbirds and parrots, in which neurons clearly predominate, nonneuronal cells are about equally numerous or slightly outnumber neurons in galliform birds (online suppl. Fig. S3a). The proportion of nonneuronal cells to neurons in the brain ranges between 48% and 58%. Hence, the maximal glia/neuron ratio (if all nonneuronal cells were glial cells) for the whole brain ranges from 0.92 to 1.38. Nonneuronal cells constitute a minor cellular fraction (20–35%) in the cerebellum, but predominate in the remaining brain regions analyzed, representing 48–71% of all cells in the telencephalon, 84–92% of all cells in the diencephalon, 54–77% of all cells in the tectum, and 86–95% of all cells in the brainstem (online suppl. Fig. S3b). When compared to songbirds and parrots, the proportion of nonneuronal cells is distinctly higher in the cerebellum and the telencephalon. This difference is particularly conspicuous in the telencephalon, which is dominated by nonneuronal cells in galliform birds (see above) but by neurons in songbirds and parrots (nonneuronal cells represent 21–40% and 31–43%, respectively). Thus, the numeric preponderance of nonneuronal cells over neurons in the brain of galliform birds is caused by the low proportion of neurons in all brain divisions but the cerebellum.

Brains of galliform birds tend to have fewer neurons than equivalently sized brains of primates and about the same number of neurons as equivalently sized brains of rodents, marsupials, and insectivores (Fig. 8a; for statistics, see the figure legend). However, brains of galliform birds are small for their body mass compared to mammals (Fig. 3b, 8c). Consequently, brains of galliform birds harbor a much smaller absolute number of neurons than the brains of equivalently sized primates but about equal numbers as equivalently sized rodents and marsupials (Fig. 8d). For example, the king quail has similar body mass and total number of brain neurons as the mouse (Mus musculus). By contrast, the yellow-knobbed curassow is almost 7-fold larger than the common marmoset (Callithrix jacchus), but both species have approximately the same number of brain neurons.

Neuronal densities in the pallium of galliform birds are similar to those observed in the pallium of mammals, although there is a nonsignificant trend for higher neuronal densities in primates (Fig. 9a; online suppl. S4a; for statistics, see the figure legend). The pallium of galliform birds houses 19–30% of all brain neurons (online suppl. Fig. S4d). This percentage is comparable to that of primates and insectivores (9–44% and 13–28%, respectively) but is higher than that of rodents and marsupials (11–19% and 11–20%, respectively). The telencephala of galliform birds accommodate smaller absolute numbers of neurons than the telencephala of equivalently sized primates but about the same number of neurons as telencephala of equivalently sized non-primate mammals (Fig. 9b; for statistics, see the figure legend).

Cerebellar neuronal densities of galliform birds are comparable to those of other mammals but primates, in which cerebellar neuronal densities are higher (online suppl. Fig. S4b; for statistics, see the figure legend). Like in mammals, the cerebellum of galliform birds houses the majority of brain neurons (online suppl. Fig. S4e). However, despite the fact that the numerical preponderance of cerebellar neurons significantly increases with brain size (which is not the case in mammals), it never reaches as extreme values as in many mammals. Neuronal densities as well as neuronal fractions contained in the rest of brain (comprising the subpallium, diencephalon, tectum, and brainstem) are higher in galliforms than in mammals (online suppl. Fig. S4c, f; for statistics, see the figure legend). Average densities of nonneuronal cells are comparable in galliform birds and mammals (PGLS: F_4,44 = 0.677, p = 0.611, λ = 0.94; Fig. 8b).

We examined the number and distribution of neurons and nonneuronal cells in brains of galliform birds and compared these features with those of songbirds and parrots. In line with earlier findings based on a smaller number of species [11, 19], we clearly show that galliform birds have significantly lower neuronal densities and their brains accommodate about half the number of neurons of songbird or parrot brains of the same mass. The difference in densities is most pronounced (up to 5 times lower) in the telencephalon. Because the telencephalon is also proportionally smaller in galliforms [10, 24, 25], it harbors a much smaller absolute number of neurons than the telencephalon of songbirds and parrots with equivalent brain size. Consequently, the relative distribution of neurons among the major brain components also differs markedly between these avian clades. Similar to mammals [55], in galliform birds the cerebellum houses the majority of brain neurons, while the telencephalon typically contains less than 35% of brain neurons. Moreover, the cerebellar neuronal fraction increases with brain size. The opposite pattern was observed in songbirds and parrots [11, 19]. Analogous to the concept of volumetric composition of the brain, termed the cerebrotype [10, 56], we suggest the term “neuronal cerebrotype” for the allocation of brain neurons to major brain parts. We conclude that galliforms, representing early diverging birds, and songbirds and parrots, representing the core landbirds (Telluraves), have distinct neuronal cerebrotypes.

The most striking and functionally important feature of galliform brains is the lower number of neurons in the telencephalon compared with core landbirds. They have significantly fewer neurons in the associative parts, especially in the nidopallium and mesopallium [12]. These traits are presumably ancestral to birds, as they are shared with paleognaths [12, 19] and likely constrain the cognitive abilities of galliform birds. Indeed, the size of these associative areas [27, 57] and the number of neurons in the telencephalon [13] are good predictors of the propensity to innovate. Innovativeness is a major feature of intelligence, in which galliforms do not excel [58]. Likewise, experimental studies suggest that galliforms do not perform on par with songbirds and parrots in cognitive tasks, although their abilities may be underestimated because they are rarely the subject of research focused on higher forms of cognition ([29], for details see also Introduction). In this context, it should be emphasized that although galliforms, together with palaeognaths, have the lowest relative neuronal densities (for a given brain size) of all birds studied to date, these densities are still several times higher than those of non-avian reptiles [19, 59, 60] and about the same as in non-primate mammals ([55]; this study). Thus, galliform birds have much higher numbers of brain neurons than equivalently sized reptiles and about equal numbers of brain neurons as similarly sized rodents and marsupials.

Differences in the densities and numbers of cerebellar neurons in the studied avian taxa are not nearly as pronounced as differences in the telencephalon. However, galliforms have significantly lower cerebellar neuron densities than songbirds and parrots. This has been independently demonstrated by stereological methods [61, 62]. In this context, however, it should be noted that the estimates of cerebellar neurons for some species have been higher than in our study [61, 62]. These numbers are based on stereological approaches and use conventional histological staining; i.e., neurons were not specifically labeled. Thus, it is possible that the counts were overestimated because glial cells were included in granule cell counts. However, glial cells make up only about 30% of the cells in the cerebellum, so even their potential inclusion in granule cell counts cannot explain the large differences reported in some species (e.g., 2.1-fold higher number of cerebellar neurons in the common pheasant and 2.8-fold higher in the turkey [61]). These marked interobserver differences fall within the range of individual variation reported for other vertebrate groups, which can be surprisingly large (individual variability – [60, 63‒65]; interpopulation variability – [60]; age-related variability – [66]). Moreover, individual variability in the number of neurons in the cerebellum appears to be higher than in other parts of the brain [63]. It also has to be noted that the turkeys examined in this study were wild type but captive-bred and captive breeding may have affected their neuroanatomy, although probably much less than domestication [67]. Nevertheless, the cerebellar neuronal fraction might be even more dominant in some galliform birds than reported in this study.

The extent to which the avian cerebellum is involved in cognitive functions remains uncertain. Its role in cognition is supported by the correlation between avian innovativeness and the number of cerebellar neurons [13], as well as by the observation that the medial spiriform nucleus, which relays information from the pallium to the cerebellum, is enlarged in large-brained birds, particularly in parrots [68]. However, the most intelligent birds, such as corvids and parrots, have evolved an enlarged telencephalon that contains the majority of brain neurons, without a corresponding proportional increase in the size of the cerebellum or the number of cerebellar neurons [11, 19, 62]. Therefore, the role of the cerebellum in cognition may be less significant in birds than in mammals.

Recent studies comparing red junglefowl and domestic chicken have demonstrated that the absolute size of the telencephalon and cerebellum and the total number of cerebellar neurons increased due to domestication [69‒71]. In contrast, the wild junglefowl and its domesticated counterpart have very similar numbers of telencephalic neurons [11, 12, 65]. As shown in this study, it is the low neuronal densities in the cerebellum that distinguish the red junglefowl from other galliforms. However, all the studied specimens of red junglefowl were captive-bred [11, 69‒71] and there is evidence for gene flow between its wild and domestic populations [72]. Therefore, the degree of conservation of its ancestral neuroanatomical features is uncertain. It also remains unclear to what extent the above findings can be generalized, as brain composition differences have been reported between chicken breeds [73, 74]. However, the fact that the relative cerebellar size increased in several species of domesticated birds suggests that cerebellar enlargement may be a byproduct of domestication [69].

Individual variability in brain size and cellular composition was recently studied in an intercross between junglefowl and domestic chicken (white leghorns) [65]. These data allow us to compare within-species (static) and among-species (evolutionary) allometries for galliform birds. The brain-body mass scaling is much steeper in galliform birds (the scaling exponent ∼0.6) than intraspecific brain-body scaling in G. gallus (b ∼ 0.25). This result is consistent with shallow intraspecific brain-body allometries typically observed in birds and mammals [75]. The same comparison can also be made for the brain structure mass-neuron number allometries. Within-species, scaling of the telencephalon mass with the number of telencephalic neurons is linear (b ∼ 1), strongly suggesting that neither neuron size nor neuron densities change with the size of the telencephalon. Among-species (intraordinal) scaling for galliforms is much shallower (b ∼ 0.65), because the increase in the telencephalon size is associated with increasing neuron size and decreasing neuron density. Surprisingly, different scaling rules apply to the cerebellum, where intraspecific mass-neuron number scaling is shallower (b ∼ 0.73) than a rather steep intraordinal scaling (b ∼ 0.81). Mechanisms underlying brain region-specific regulation of neuron size and number at within- and among-species levels remain to be determined.

To summarize, in this paper, we have studied neuron numbers and distributions in galliform birds in detail and compared the cellular scaling rules for their brains with those for songbirds and parrots. We show that galliforms have generally lower neuronal densities, small telencephalic and dominant cerebellar neuronal fractions, larger glia/neuron ratios, and much smaller absolute numbers of neurons than brains of equivalently sized songbirds or parrots. On the other hand, galliforms have similar neuron counts in the brain and forebrain as equally sized non-primate mammals. These comparisons (i) demonstrate that distantly related birds can have distinct neuronal cerebrotypes and (ii) provide supporting evidence for the notion that the number of neurons in the pallium is a reliable neural correlate of cognitive and behavioral capabilities [13, 76, 77].

We thank V. Miller for logistic support and P. Benda and J. Matějů for helping us acquire the animal experiment approvals.

All procedures were approved by the Institutional Animal Care and Use Committee at Charles University in Prague (Permission No. UKPRF/28830/2021), the Ministry of Culture (Permission No. 47987/2013), and the Ministry of the Environment of the Czech Republic (Permission No. 53404/ENV/13-2299/630/13).

The authors declare no conflict of interest.

This research was funded by the Czech Science Foundation (22-35153S, to P.N.), the Charles University Research Centre Program (UNCE/24/SCI/006, to K.K.), and the Charles University Grant SVV (260685/2024, to Y.Z., P.S., and A.P.).

M.K., K.K., and P.N. designed research; M.K., Y.Z., L.S., P.S., A.P., S.O., and P.N. performed experimental investigations; B.S. and Z.P. collated data on brain mass and body mass from the literature; M.K., Y.Z., and K.K. analyzed data; T.H., T.K., and R.K.L. collected experimental animals; M.K., K.K., and P.N. wrote the original draft of the paper; M.K., Y.Z., and L.M. made figures; all authors reviewed and edited the manuscript and gave final approval for publication.

Martin Kocourek and Yicheng Zhang contributed equally to this work.

All quantitative data gathered in this study are provided in Supplementary Dataset S1 associated with this paper. Further inquiries can be directed to the corresponding author.