of the galliform birds (Aves, Galliformes)
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Cellular scaling rules for brains of the galliform birds (Aves, Galliformes)
Martin Kocourek1*, Yicheng Zhang1, Seweryn Olkowicz1, Lucie Marhounová1, Barbora Straková1, Zuzana Pavelková1, Patrik Stehlík1, Tomáš Kušta2, Radek K. Lučan1, Tomáš Hájek1, Kristina Kverková1 and Pavel Němec1
1 Department of Zoology, Faculty of Science, Charles University in Prague, Viničná 7, CZ-128 44
Praha 2, Czech Republic
2 Department of Forest Protection and Wildlife Management, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, CZ-165 21 Praha 6, Czech Republic
Address for correspondence:
Pavel Němec
Department of Zoology Charles University Viničná 7
CZ-128 44 Praha 2 Czech Republic
Phone: ++420 2 2195 1855 Fax: ++420 2 2195 1841
e-mail: [email protected]
Songbirds, especially corvids, and parrots are remarkably intelligent. Their cognitive skills are on par with primates and their brains harbour 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 linages.
Here we use the isotropic fractionator to determine numbers of neurons in specific brain regions of galliform birds, which have small brain relative to body size and a proportionally small telencephalon and are often perceived as cognitively inferior to most other birds. We demonstrate that brains of galliforms contain on average half the number of neurons found in parrot and songbird brains of the same mass. Additionally, in contrast to these birds, galliforms resemble mammals in having small telencephalic and dominant cerebellar neuronal fractions. Consequently, their brains and especially their forebrains harbor much smaller absolute numbers of neurons than those of equivalently sized songbird and parrots, the fact that undoubtedly constrains cognitive abilities of galliforms. However, their neuronal densities exceed those in most non-primate mammals and their brains contain about equal numbers of neurons as brains of equivalently sized rodents and marsupials with larger brains.
Moreover, galliforms have higher telencephalic neuronal densities and higher proportion of brain neurons located in the pallial telencephalon compared to these mammals. Thus, galliform birds have forebrain neuron counts greater than rodents or marsupials of the same body mass. Therefore, it is not surprising that cognitive abilities of galliforms are on par with mammals in many domains.
Keywords: Intelligence, cognition, evolution, brain size, number of neurons, birds, mammals.
Introduction
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 (Emery 2006; Güntürkün and Bugnyar 2016; Kabadayi et al. 2016; Lambert et al. 2018; Rössler and Auersperg 2022). 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.(Bugnyar et al. 2016;
Kabadayi and Osvath 2017; Pika et al. 2020), to mention just a few complex abilities. They have relatively large brains for their bodies (e.g. Ksepka et al. 2020), characterized by a large telencephalon, which is dominated by the associative nidopallium and mesopallium, and the striatopallidal complex (Iwaniuk and Hurd 2005). Recently, it has been shown that brains of corvids and parrots contain huge numbers of neurons, packed in high densities in the telencephalic pallium (Olkowicz et al. 2016), particularly in the associative areas (Ströckens et al. 2022). Moreover, pallial neuron numbers scale with body size with a steeper slope compared to other bird clades (Sol et al. 2022). The nidopallium caudolaterale (NCL), which is involved in executive control and is generally considered to be a functional equivalent of the mammalian prefrontal cortex (e.g.
(Güntürkün 2005; Nieder 2017) has been shown to be enlarged and compartmentalized in songbirds, and especially in corvids (von Eugen et al. 2020).
In contrast, basally diverging birds, namely ratites and galliforms, feature a lower degree of encephalization (e.g.
(Ksepka et al. 2020) and a proportionally smaller telencephalon (Iwaniuk and Hurd 2005). Recent studies suggest that compared to songbirds and parrots, they have a smaller telencephalic and dominant cerebellar neuronal fraction and generally lower neuronal densities (Olkowicz et al. 2016; Kverková et al. 2022), with rather modest numbers of neurons in associative telencephalic regions (Ströckens et al. 2022). However, they included only a handful of species from these clades or divided the brain into crude compartments, not allowing for a thorough statistical comparison of cellular scaling rules and allocation of neurons to different brain parts. Here, we present such 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 (Gill et al., 2022). Together with anseriform birds, they form the clade Galloanserae, a sister group to all living birds except for tinamous and ratites (Jarvis et al. 2014; Prum et al. 2015; Kimball et al. 2019, Kuhl et al. 2021). Therefore, they are typically considered baseline for avian encephalization (Portman 1947; Boire and Baron 1994) and others consider small volumes of different pallial regions, typical for galliforms, as a predictor of low level of innovativeness (Lefebvre et al., 1997; Timmermans et al., 2000). Their brains are characterized by a relatively large optic tectum, diencephalon, and brainstem and a relatively small Wulst and mesopallium (Iwaniuk and Hurd 2005) and a small, simply organized NCL (von Eugen et al. 2020).
Galliforms are traditionally viewed as birds with rather low cognitive abilities. Chicken (Gallus gallus) is the predominant model species for testing fundamental cognitive processes such as associative learning, imprinting, or visual perception (Vallortigara 2012; Marino, 2017). Chickens also display some aspects of physical reasoning (e.g. amodal completion, Regolin and Vallortigara, 1995), self-control (Abeyeshinghe et al. 2005; Meier et al. 2017), temporal cognition (e.g. perception of time duration, Taylor et al. 2002) or numerical competencies, Rugani et al. 2008; Rugani et al. 2009; Rugani et al. 2016; Kobylkov et al. 2022). Because the body of research on sophisticated cognitive processes is biased mostly towards corvids and parrots, future studies may reveal a larger repertoire of cognitive skills in gallinaceous birds, especially in the social domain (Marino, 2017). In fact, rather sophisticated capacities such as perspective-taking (Smith et al. 2011) and intentional or tactical deception have been reported in chicken (Gyger and Marler 1988).
In this study, we quantify neurons and non-neuronal cells in six brain divisions in 15 species of gallinaceous birds and compare their numbers and distributions with those of parrots and songbirds. These data provide new insights into the evolution of avian brain-neuron scaling and information processing capacity.
Materials and Methods
Animals. Altogether 36 birds belonging to 14 species of galliform birds were used in this study (Figure 1, Table 1). Previously, small part of the data were used in another studies (Massen et al., 2021, Sol et al., 2022, Kverková et al., 2022) and published data for the red junglefowl Gallus gallus were also included (Olkowicz et al., 2016). Three individuals per species were collected with exception of the Yellow-knobbed curassow, the California quail, the Black francolin and the Black grouse, in which only one or two birds were examined. The common quail and the common pheasant were wild-caught in Czech Republic (Permission No. 00212/CS/2013 and 446/2013); the Grey partridge, the Red-legged partridge and the Reeves's pheasant were pen-reared birds obtained from semi-natural populations; all other species were purchased from local breeders. Only adult birds were used in this study and their sex was determined upon dissection.
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. Skulls were partially opened and postfixed for 30-60 minutes, after which brains were dissected and weighed. Brains were postfixed for additional 7-21 days and then dissected. All procedures were approved by Institutional Animal Care and Use Committee at Charles University in Prague, Ministry of Culture (Permission No. 47987/2013) and Ministry of the Environment of the Czech Republic (Permission No. 53404/ENV/13-2299/630/13).
Dissection. Brains were dissected into distinct components using the Olympus SZX 16 stereomicroscope as described earlier (Olkowicz et al., 2016). 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 left and right tectum were processed together.
The cerebellum was cut off at the surface of the brainstem. Finally, the remaining structures were dissected into diencephalon (rostral part) and brainstem (caudal part) along the plane connecting the posterior commissure dorsally and hypothalamus- mesencephalon boundary ventrally. Because we detected negligible differences between left and right hemisphere mass and cell numbers in our pilot studies, only 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 size of a 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 subpallium on each section by cutting along the pallial-subpallial boundary, as defined by Puelles et al. (2007). The dissected structures were dried with paper towel, weighed, incubated in 30% sucrose solution until they sank, then transferred into antifreeze (30% glycerol, 30%
etylene glycol, 40% phosphate buffer) and stored at -25°C until further processing.
Isotropic fractionator. We estimated total numbers of cells, neurons and nonneuronal cells following the procedure of isotropic fractionation, described earlier (Herculano-Houzel and Lent, 2005). 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 turned into an isotropic suspension of isolated cell nuclei, homogenates were stained with the florescent DNA marker DAPI, adjusted to a defined volume, and kept homogenous by agitation. The total number of nuclei in suspension, and therefore the total number of cells in original tissue, was estimated by determining density of nuclei in small fractions drawn from a 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 (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 reach the coefficient of variation (CV) among counts ≤ 0.15 (usually, CV ≤ 0.10 was achieved).
Once the total cell number was known, the proportion of neurons was determined by immunocytochemical detection of neuronal nuclear marker NeuN (Mullen et al., 1992). This neuron-specific protein was detected by the mouse monoclonal antibody anti-NeuN (clone A60, Chemicon, Temecula, CA, USA; dilution 1:800), which was recently characterized by Western blotting with chick brain samples and shown to react with a protein of the same molecular weight as in mammals (Mezey et al.,2012), 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-labelled and NeuN-immunopositive nuclei in the Neubauer chamber. A minimum of 500 nuclei was counted to estimate percentage of double labelled neuronal nuclei. Numbers of nonneuronal cells were derived by subtraction.
Data analysis. All analyses were performed using average values for each species; variables were log-transformed before the subsequent statistical analyses. Correlations between variables were assessed using nonparametric Spearman rank test. If a significance criterion of p < 0.05 was reached, the reduced major axis regressions were calculated to describe how structure mass, numbers of cells and densities are interrelated across species using the Smatr package for R (Warton et al., 2012). The same package was used to compare scaling among groups (taxonomic orders or brain regions). We first tested whether the regression lines differ in slopes. If so, the group(s) with significantly different slope(s) were excluded from the model and, subsequently, the effect of categorical predictor was tested across groups with statistically homogenous slopes and their differences were compared based on differences in the intercepts. To compare relative brain size between cracids (Aves, Cracidae) and other galliform birds, we computed t-test on the residuals of a log-log regression of brain mass against body mass (residual brain mass, hereafter).
For the comparison with cellular scaling rules reported previously for mammals, the reduced major axis regressions were calculated from quantitative data published for primates (Herculano-Houzel et al., 2007; Gabi et al., 2010; Azevedo et al. 2009), rodents excluding the naked mole-rat (Herculano-Houzel et al., 2006, 2011), Eulipothyphla (Sarko et al.,2009) and marsupials (Dos Santos et al., 2017).
All statistical analyses were performed in R 3.3.2. (R Core Team, 2016) the graphs were plotted in JMP 10.0 (SAS Institute, Cary, NC, USA).
Results
Total numbers of neurons. Among fifteen galliform species studied, weighing between 44 and 3,600 g, brain mass ranges from 0.52 to 9.02 g, and total numbers of neurons in the brain from 80 to 653 million (Figure 2, Table 1). The relationship between brain mass and the number of brain neurons can be described by the power function MBR = 1.583 × 10-11 × NBR1.331
(Table 2). Because the scaling exponent is significantly higher than 1.0 (95% confidence interval (CI) = 1.168–1.516), any gain in 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 21.4 larger brain. Figure 3a compares brain mass scaling with total number of brain neurons between galliform birds, songbirds and parrots. Allometric lines for these three groups do not differ in slope (Likelihood ratio statistic (LRS)2,37 = 3.384, p = 0.184), but allometric line for galliform birds have a significantly higher intercept (galliforms versus songbirds: Wald statistic (WS)1,27 = 107.31, p < 10-15; galliforms versus parrots: WS1,25 = 84.36, p < 10-15), clearly indicating taxonomic 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 (Figures 3c, 4a). The relationship between brain volume and body mass among 15 species examined in this study can be described by the power function MBR = 0.047 x MBo0.616 (r2 = 0.964, p < 10-10; Figure 3c), among 71 species of galliforms collated from the literature by the power function MBR = 0.082 × MBo0.558 (r2 = 0.905, p < 10-15; Figure 4a). The scaling coefficient is not only significantly smaller than 1.0 (CI = 0.518–0.600), but also significantly smaller than that for parrots and songbirds (galliforms versus songbirds: LRS1,887 = 38.21, p < 10-9; galliforms versus parrots: LRS1,221 = 33.39, p < 10-8). 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, brains of galliform birds harbor much smaller absolute number of neurons than brains of equivalently sized songbirds or parrots. For instance, the common pheasant is somewhat heavier than the blue-and-yellow macaw, but its brain has ~10-fold less neurons. Likewise, the red junglefowl is ~50-fold heavier than the great tit, 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 (Figure 2, Table 1) a representative of a basal clade Cracidae (including the chachalacas, guans and curassows). Indeed, cracid brains tend to be larger than brains of other galliform birds. Technically, residual brain mass calculated from regressions for all galliforms is significantly larger in cracids than in non-cracid galliforms (t1,70 = 4.745, p = 0.0006, Figure 4a).
Neuronal densities. In galliform birds, neuronal density varies greatly among principal brain divisions examined and decreases significantly with increasing brain mass in all these divisions (scaling exponent ranges between -0.271 and -0.548; r2 between 0.470 and 0.905, p ≤ 0.005 in all cases; Figure 5b). Just as in other avian groups, the highest neuronal densities are in the cerebellum (310–660 × 103 N/mg), the lowest in the brainstem (4–28 × 103 N/mg). Curiously, the rate of decrease in neuronal density with increasing brain mass tend to be negatively correlated with overall neuronal density in a division (scaling exponents: cerebellum, - 0.271; tectum, -0.457; telencephalon, -0.454; diencephalon, -0.463; brainstem, -0.547). Neuronal densities in all divisions are significantly lower than those observed in songbirds and parrots (Figure 5d). Slopes of allometric lines for all divisions but the telencephalon do not differ among avian groups examined (LRS: p > 0.224 in all cases), but allometric lines for galliform birds have significantly lower intercepts than those for the other two avian groups (WS: p < 10-5 for all pairwise comparisons). In these divisions, galliforms have 1.3–2.2-fold lower neuronal densities than have songbirds and parrots. In case of the telencephalon, this difference is even more pronounced because allometric line for galliforms not
only have a lower intercept but also a lower slope (slope: LRS2,37 = 6.31, p = 0.043; intercept: WS2,37 = 189.9, p = < 10-15), the fact that has important consequences for relative distribution of neurons among major brain compartments (see below).
Density of telencephalic neurons is 2.3–4.9-fold lower in galliforms than that in songbirds and parrots.
Relative distribution of mass and neurons. The telencephalon mass fraction increases with brain size at the expense of all other brain components but cerebellum, ranging from 52% to 64% (Figures 6a–c, Table 3). However, the relative proportion of the telencephalon is much smaller in galliforms than in songbirds and parrots (songbirds: 63% to 80%, parrots: 71 to 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 (Figure 6b). 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 (Table 4, Figures 6d–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, cerebellar neuronal fraction increases and telencephalic neuronal fraction tend to decrease as brain get larger; the reverse patterns are 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 larger the larger the brain. For instance, the telencephalon of the king quail has
~2.6-fold less neurons than that of the zebra finch, the telencephalon of the wild turkey has ~4-fold less neurons than that of the jackdaw and the yellow-knobbed curassow has almost 8-fold less neurons than that of the grey parrot.
Subpallium. The subpallium (comprising the striatum, pallidum and septum) constitute 13–18% of total telencephalon mass and houses 14–19% of all telencephalic neurons (Tables 3, 4; Figures 7a, b). Neither the relative mass of the subpallium nor the fraction of telencephalic neurons contained within it correlate with telencephalon size (mass fraction: ρ = 0.143, p = 0.612;
neuronal fraction: ρ = 0.04, p = 0.899). Number of subpallial neurons scales almost linearly with number of pallial neurones (scaling exponent = 1.004 ± 0.1; NSUBPALL = 0.184 × NPALL1.004; Figure 6c), indicating concerted gain of neurons that maintains a ratio of ~ 5.1 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 = 1.19 ± 0.13), while an opposite trend is observed in songbirds (scaling exponent = 0.91 ± 0.1).
Nonneuronal scaling rules. Although statistically different in many cases (statistics not shown), nonneuronal scaling rules are, in contrast to neuronal scaling rules, remarkably similar across brain divisions and avian lineages (Figure 3b, Table 2). 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 (galliform birds: t2,13 = 11.132, p < 10-13; songbirds: t2,11 = 24.199, p < 10-15; parrots: t2,9 = 13.777, p < 10-14; Figure 5c, e). Because the lower nonneuronal density in the telencephalon was observed in all three avian groups studied but never in mammals (Herculano-Houzel et al., 2014; Dos Santos et al., 2017), it seems to be a specific avian feature.
In galliform birds, nonneuronal cell density decreases significantly with increasing brain size in all brain division examined (Figure 5c, p < 0.014 in all cases), the rate of this decrease is the highest in the cerebellum (scaling exponent for the cerebellum
= – 0.462, for other structures it ranges between –0.166 and – 0.211).
Glia/neuron ratio. In contrast to songbirds and parrots, in which neurons clearly predominates, nonneuronal cells are about equally numerous or slightly outnumber neurons in galliform birds (Figure 8a). 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 analysed, 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 (Figure 8b). 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 low proportion of neurons in all brain divisions but the cerebellum.
Comparison with mammals. Brains of galliform birds tend to have less neurons than equivalently sized brains of primates and more neurons than equivalently sized brains of rodents and marsupials (Figure 9a, for statistics see the figure legend).
However, brains of galliform birds are small for their body mass compared to mammals (Figures 4b, 9c). Consequently, brains