Evol Biol (2014) 41:229–239 DOI 10.1007/s11692-013-9262-3

RESEARCH ARTICLE

A Dysfunctional : The Irreversibility of Olfactory Evolution in Free-Living

Valeria Maselli • Gianluca Polese • Greger Larson • Pasquale Raia • Nicola Forte • Daniela Rippa • Roberto Ligrone • Rosario Vicidomini • Domenico Fulgione

Received: 11 March 2013 / Accepted: 4 November 2013 / Published online: 13 November 2013 Ó Springer Science+Business Media New York 2013

Abstract Artificial selection began to override natural significantly larger brain per unit mass than domestic pigs, selection in domesticated wild boar and other species about and FLPs’ brains are not significantly smaller than wild 10,000 years ago. The intentional selection of a desired boar’s. Similarly, both wild boars and FLPs have signifi- phenotypic trait is a complex process, and comes along cantly higher cell density than domestic pigs in the olfac- with unexpected or even unwanted changes in other traits, tory mucosa. Yet, at the functional level, olfactory marker because of epistatic effects, and ontogenetic con- and neuropeptide Y, both of which are important to straints. The loss of brain mass in domestic ungulates is the correct functioning of the sense of smell, are fully related to selection for reduced reaction to external stimuli. expressed only in wild boar. These results suggest that Evolutionary losses in body structures and were once FLPs reacquired structural, but not the biochemical capa- considered mostly irreversible, in keeping with Dollo’s bility in their olfactory system. law. Here we studied the biochemical and the histological functioning of the free-living pigs (FLPs) olfactory system, Keywords Smell Free-living Evolution to see if and to what extent does FLPs regain a full sense of Domestication Wild boar Brain smell, as compared to the domestic pigs and wild boar Sus scrofa. In our samples both wild boars and FLPs have Introduction

Valeria Maselli and Gianluca Polese have contributed equally to this Artificial selection started to override natural selection in study. domesticated breeds of wolves, wild boar and a few other species some 10,000 years ago (Bo¨ko¨nyi 1974; Hemmer V. Maselli R. Ligrone 1990; Giuffra et al. 2000; Larson et al. 2007). Humans took Department of Environmental Biological and Pharmaceutical Sciences and Technologies, Second University of Naples SUN, a leading role in these species evolution since, by Via Vivaldi 48, 81100 Caserta, Italy exploiting existing genetic variation in their and pets in order to fulfil their own economic interests (Roots V. Maselli G. Polese N. Forte D. Rippa R. Vicidomini 2007). To successfully manage domesticates, early farmers D. Fulgione (&) Department of Biology, University of Naples Federico II, selected animals with reduced wariness and aggressiveness Campus Monte S. Angelo, 80126 Naples, Italy (Price 1998, 2002; Albert et al. 2012; Zeder 2012). Asso- e-mail: [email protected] ciated with this selection was a strong reduction in brain size in the domesticates. The extent of brain mass loss goes G. Larson Durham Evolution and Ancient DNA, Department of from about 10 % in pet rodents, to 15–25 % in domestic Archaeology, University of Durham, Durham DH1 3LE, UK ungulates, to[30 % in pigs and carnivores (Kruska 1988). Not all brain structures were affected equally. The P. Raia greatest loss of the cerebral mass took place in the telen- Department of Earth Sciences, Resources and Environment, University of Naples Federico II, Largo San Marcellino 10, cephalon, and in the limbic system in particular. This is 80138 Naples, Italy unsurprising since the limbic system controls behavioral 123 230 Evol Biol (2014) 41:229–239 responses to external stimuli (reviewed in Zeder 2012). et al. 1995; Hutcheon et al. 2002; Safi and Dechmann Thus, reduction in limbic structures is directly linked to 2005). Here, in order to test the degree to which domestic selection for reduced wariness and aggressiveness. Despite pigs can regain a full sense of smell once freed from the generalized nature of these observations across species captivity, we studied the biochemical and histological as phylogenetically distant as cats and birds (Kruska 1988; functioning of suid olfactory system in wild boar, Ebinger and Ro¨hrs 1995; Price 2002), there are notable domestic pig, and Sardinian free-living pig (FLP) differences in brain functioning among domestic species. populations. For instance, brain parts involved in the senses of hearing According to the zoo-archaeological record Sus scrofa and smell in pigs are less reduced than visual centers, as presence in Sardinia traces back to the first human settle- compared to other domesticates (Plogmann and Kruska ments on the islands, during the early Neolithic (Wilkens 1990). 2003; Albarella et al. 2006). The Sardinian wild boar Evolutionary losses in body structures and genes were population is supposed to have originated when pigs once considered mostly irreversible. In evolutionary biol- escaped from human control and became wild during the ogy, this idea is known as Dollo’s law (Dollo 1893, 1922; Neolithic (Albarella et al. 2006; Scandura et al. 2011). Gould 1970; Porter and Crandall 2003). Although Dollo’s Sardinian populations of wild boar and pig breed have law is pervasive, exceptions are common. Evolutionary experienced a multi-millennial history of independent reversals have been shown to occur in the dentition of the evolution (geographic isolation) (Scandura et al. 2008, lynx (Kurte´n 1963), limb muscles in birds (Raikow et al. 2009; Larson et al. 2005; Randi 1995) followed by intro- 1975), life cycle in marsupial frogs (Wiens et al. 2007), gressive hybridisation, and by gene flow with continental sight in blind cavefish (Borowsky 2008), oviparity in sand wild boars transplanted on the island only in recent years boas (Lynch and Wagner 2010), and might be even more (Scandura et al. 2011). Nowadays FLPs live in open air common among invertebrates. The number of exceptions conditions since a number of generations, therefore they undermines the interpretation of Dollo’s idea as a ‘‘law’’ are domesticated pigs which are exposed to the same (Collin and Miglietta 2008). In particular, the most logi- selective pressures as wild boars. In summary, two genet- cally prominent explanation for Dollo’s law, i.e. the ically distinct free populations exist in Sardinia: wild boars, accumulation of deleterious mutations in genes coding for deriving from the mentioned ancient introduction, and no longer expressed phenotype, is regarded as potentially FLPs, of uncertain origin. ineffective. Given normal mutation rates, it may take up to Here, we compared the density of receptor cells of the 10–20 million years to inactivate these genes, leaving a olfactory epithelium in wild boar, domestic pig, and FLP large window of time for the lost phenotype to reappear. individuals. Receptor cells are the primary sensory neu- Genes linked to pleiotropic pathways may survive in a rons. In addition to structural differences among the three functionally unaltered manner even longer. Overall, this strains analysed, we assayed quantitatively expression suggests that evolutionary reversals are quite possible levels in both the olfactory marker protein (OMP), and (Collin and Miglietta 2008). neuropeptide Y (NPY). The OMP is a protein involved in Like the observations that gave rise to Dollo’s Law, , found in mature olfactory receptor phenotypic changes that result from the domestication neurons of all vertebrates. OMP is a modulator of the process are often described as irreversible. For instance, olfactory signal-transduction cascade. The NPY is a populations of once-domestic species that have lived for widespread polypeptide in the central nervous system and many generations outside human influence (a process the autonomic nervous system. It participates to the regu- known as feralization) do not regain brain mass. This is lation of hunger, and to the modulation of the vasocon- true of feral (Groves 1989), dingoes (Schultz 1969), strictor response triggered by noradrenergic neurons and a variety of other mammals (Birks and Kitchener (Mousley et al. 2006; Di Bona 2002). The NPY is a key 1999). In contrast, a study of feral pigs in the Galapagos molecule in the development and regeneration of the that lived in the wild for 150 years, found a significant olfactory epithelium (Hansel et al. 2001). It was shown that size increase in brain structures related to olfaction, which NPY is expressed at the level of microvillous cells with still suggests that pigs may retain a large fraction of their axonal process of the olfactory mucosa (Montani et al. olfactory system functionality (Plogmann and Kruska 2006). 1990). In fact, the positive relationship between the pro- We compared the expression patterns of two olfactory portional size of brain structures related to olfaction and sensory neurons (OMP and NPY) in Sardinian FLP and olfactory capability was proven for a variety of mammals Southern Italian wild boar. In addition, we compared them (Barton and Harvey 2000), including carnivores (Gittle- to domestic pigs. We first verified that domestic pigs show man 1991), primates (Barton et al. 1995; Barton 1996), proportionally smaller brains than wild boar, as extensively ‘‘insectivores’’ (Barton et al. 1995), and chiropters (Barton reported in literature. Then, we tested if Sardinian FLPs 123 Evol Biol (2014) 41:229–239 231

Fig. 1 Morphology of Olfactory Mucosa and diversity of samples. a Sagittal section of a pig head; b transversal section of Olfactory Mucosa stained with Mayer’s Acid Alum Hematoxylin; the diversity of three forms is exemplified in pictures of c wild boar; d domestic pig; e FLP have regained brain mass and complete olfactory function slaughterhouses in peninsular Italy. All adult domestic pigs through the ‘‘feralization’’ process they underwent. If FLP were [1 years old and weighing between 160 and 200 kg. shows brain size and OMP and NPY functionality com- FLP and wild boar were both culled in the field. FLPs came parable to wild boar, our data would indicate that the loss from Sardinia, in the occidental part of the Ogliastra region of functioning in the olfactory system which is common in (Lanusei, Punta Marmora). All the culled individuals were domestic pigs was probably reversed by free living life adult (mean height at the withers = 50 cm, ran- style. ge = 44–62 cm), and weighing between 50 and 70 kg (mean = 58 kg). These are in fact FLPs, deliberately reared in open-air and infrequently provided with supple- Materials and Methods mental food by local people, yet used to search for food on their own. We urge the reader to consider that we were not Wild boars, domestic pigs, and FLPs were tested for interested here in sizing up the extent of olfactory system morphological and bio-molecular differences in the olfac- recovery (if any) in Sardinian FLP. Rather, we tested tory epithelium and brain size. To perform the experiments, whether the Sardinian FLP regained functional olfactory we sacrificed 10 individuals for each category (Fig. 1c, d, system as free-living animals, after they passed through e). It is obviously not possible to test the domestic ances- domestication. tors of Sardinian FLP, since they are no longer existent. As Wild boars came from wild populations living in the such, we compared Sardinian FLP, and wild boar to Cilento and Vallo di Diano National Park (Southern Italy). commercial pig individuals we sampled from 3 different They were all adults, [1 years old as judged from coat

123 232 Evol Biol (2014) 41:229–239 pattern and medium height at the withers (mean height at For each individual a tissue sample was isolated in order to the withers = 65 cm) and then confirmed by inspection of perform DNA extraction. We relied on known phenotypic the dentition after culling. The culled animals weighted changes with ontogeny in both wild boar and FLP to select between 65 and 80 kg (mean = 71 kg). animals that were approximately 1 year old. We analysed the highly polymorphic melanocortin receptor 1 (MC1R) coat colour gene because it might be a Histological and Immunohistochemical Analyses useful marker for detecting natural hybridisation between of the Olfactory System FLPs and wild boars (Frantz et al. 2012, 2013; Krause- Kyora et al. 2013). Various studies support the use of this We fixed strands of the olfactory mucosa for each speci- gene as a tool for distinguishing breeds in a range of animal men in Bouine’s fluid and stained as follows. After fixa- species. In particular, MC1R allelic variants are closely tion, horizontal sections (7 lm thick) were cut, mounted on associated to particular breeds and colour morphs in S. slides and stained using the classical histochemical meth- scrofa (Johansson Moller et al. 1996; Kijas et al. 1998, ods hematoxylin-eosin (HE). The One-Way ANOVA with 2001; Marklund et al. 1996; Giuffra et al. 2002; Pielberg post hoc was used to compare in the means in neuronal et al. 2002; Fang et al. 2009; Koutsogiannouli et al. 2010). counts among samples. For each specimen, we amplified fragments of MC1R with Immunohistochemical analyses were developed on two different pairs of primers to analyse all the polymorphisms markers of neurons’ functionality: NPY and OMP. The in the coding region [MERL1, EPIG1, EPIG3R (Kijas et al. sections of olfactory mucosa were incubated in serum at a 1998); MF1 MR1 (Li et al. 2010)]. We determined the 1:100 dilution in PBS (Normal Goat Serum and Normal entire MC1R coding sequence (963 bp). The amplifications Rabbit Serum for NPY and OMP, respectively) for 20 min, were carried out on 50 ng of genomic DNA in a 25 llfinal washed and then incubated in primary antibody (Rabbit volume containing 2.5 ll PCR buffer (10x), 200 mM of anti-NPY, 1:3000 in PBS, 1:10000 in PBS, Goat anti-OMP, each dNTP, 20 pmol of each primer and 1 U Phusion High- 1:10000 in PBS) for 24 h. After washings the sections were Fidelity DNA Polymerase (Thermo Scientific). PCR reac- incubated in secondary antibody 1:200 dilution in PBS tion times were: 4 min of initial strand denaturation at (Goat anti-Rabbit biotinylated and Rabbit anti Goat bio- 94 °C, followed by 40 cycles each comprising 30 s of tinylated, respectively) for 45 min. After second washings strand denaturation at 94 °C, 40 s of annealing at 58 °C the sections were incubated in streptavidine horseradish and 40 s of extension at 72 °C and a final extension of peroxidase (HRP) conjugated, 1:200 dilution in PBS. After 5 min. PCR products were purified by Exo/SAP digestion the HRP-diaminobenzidine reaction, sections were viewed were directly sequenced using the forward primer the and photographed using a Laica optical microscope. BigDye Terminator kit version 3.1 (Applied Biosystems). Fragments were finally purified in columns loaded with Gene Expression DyeEx 2.0 Spin Kit (Qiagen); and run in an ABI PRISM 3100 Avant automatic sequencer (Applied Biosystems). To determine OMP and NPY mRNA expression levels, Ambiguous positions were verified by re-sequencing the total cellular RNA was isolated from tissues using TRI target region with the internal reverse primer. The Reagent (SIGMA) according to manufacturer’s recom- sequences of MC1R were aligned and edited by Geneious mendations. The quantity and quality of total RNA was version 5.4 (Drummond et al. 2011) analysing nucleotide determined by spectrometry and agarose gel electrophore- composition and variable sites polymorphisms, compared sis, respectively. The RNA was purified using RQ1 RNase- with GenBank Sequences and assigned to different breeds. Free DNase (Promega). We performed all of the analyses on all specimens (n = 30). All the activities were performed in full respect of the Italian law. Table 1 Sequence of primers and size of amplicones used in the semi quantitative and quantitative PCR Primer Sequence Amplicone size Morphological, Histological and Immunohistochemical NPY 50CTCGGCGTTGAGACATTACA 157 bp Analyses 30CACTTCCCATCACCACACAG OMP 50ACCTCACCAACCTCATGACC 170 bp Brain Size 30CCCGAAGGAGATGAGGAAAT ACTB 50AGAGCGCAAGTACTCCGTGT 210 bp To assess the relative size of the brain, we weighed the 0 brain mass and computed the brain to body mass ratio. The 3 AAAGCCATGCCAATCTCATC olfactory mucosa was separated and fixed in Bouine’s fluid. NPY neuropeptide Y, OMP olfactory marker protein, ACTB beta actin 123 Evol Biol (2014) 41:229–239 233

Table 2 Mutations in MC1R gene in the three samples analyzed. On the top: genotype mutation pattern described in Fang et al. 2009

On the bottom: genotype mutation pattern in our samples. Domestic pigs can be assigned to Large white pig breed (0501 genotype). Wild boar is a typical wild type sequences of MC1R (0101). FLP were consistent with the 0401 genotype

cDNA was synthesised using a Superscript III First- Quantitative real time PCR analysis was carried out using Strand Synthesis System with Random Hexamers (Invit- the 2(-Delta Delta C(T)) method (Livak and Schmittgen rogen) and as control of the cleaning from DNA, RT(?) 2001). In all qPCR experiments the data were normalized and RT(-) in order to amplify the collinear 5S gene were to the expression of the b-actin housekeeping gene. performed. Gene expression levels were measured using Negative controls included omission of template dissoci- semi-quantitative PCR performed with Dream Taq (Fer- ation curves, gel analysis and sequencing of certain PCR mentas). The actin gene was analysed simultaneously as a products confirmed gene specific product amplification. control, and used for normalisation of data. The PCR Three different RNA preparations were tested for each conditions were 94 °C for 2 min, followed by 28 cycles at sample, and each reaction was run in triplicate. Data are 94 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s and 72 °C representative of three independent experiment. for 1 min in the end. The primers used for each gene are described in Table 1. To examine whether differences in expression levels of Results NPY and OMP can discriminate activity of cells in olfactory mucosa of wild boar, pig and FLP we conducted No interbreeding in domestic pigs and wild boars was gene expression assays through quantitative-PCR (q- detected in our samples by using MC1R assay, in the entire PCR). Quantitative analysis was performed by using the MC1R coding sequence (963 bp). The nuclear markers iQTM 5 Multicolor Real-Time PCR Detection System show that wild boar have a typical wild type sequences of (Bio-Rad). The PCR reactions were performed in a final MC1R (Accession Number KF780580; 0101 genotype, volume of 15 microliters using 1 microliter of cDNA, according to Fang et al. 2009 allele nomenclature). The 5 pmol of each primer and 7.5 microliter of iQTM SYBR sequences of FLP revealed nucleotide substitutions at two Green Supermix 2X (Bio-Rad). PCR cycling profile diagnostic position (one of them heterozygote) consistent consisted of a cycle at 95 °C for 3 min and 40 two-step with recessive red phenotype (Accession number cycles at 95 °C for 10 s and at 60 °C for 30 s. KF780579; 0401–0401* genotype). The domestic pigs can 123 234 Evol Biol (2014) 41:229–239

and FLP’ brains were not significantly smaller than wild boar brains (p = 0.225). The olfactory mucosa in S. scrofa, as in all the other mammals, is greatly folded on turbinate and ethmoid bones (Fig. 1a). After several trials to get sagittal sections of the entire mucosa, we standardized the procedure by taking just a 5 mm2 piece of olfactory mucosa from the second lamella (Fig. 1b). In homolog sections of domestic pigs mucosa, we found drastically smaller number of neurons as compared to wild boars and FLPs (Figs. 3a, b, c, 4). The density of cells changed statistically across wild boar, FLP and domestic pig (ANOVA: F = 36.23 df = 2, 27 p 0.001). Post-hoc tests indicated that both wild boars and FLPs have signif- icantly higher cell density than pigs (wild boar/domestic pig, p \ 0.001, FLP/domestic pig, p \ 0.001), and FLP had no fewer neurons per unit area than wild boars (p = 0.459). Olfactory marker protein immunoreactivity (OMP-ir) is diffuse within the olfactory epithelia (Fig. 3d, e, f) showing a qualitatively higher reactivity in the wild boar than in wild and domestic pig. Using an antibody against NPY, we found different immunestaining among the three samples (Fig. 3 g, h, i). Wild boar showed a high NPY immunoreactivity within several olfactory receptor neurones; to the extent that even some basal cells were labelled. In the FLP olfactory sen- sory neurons NPY-ir and in the domestic pig the staining was much weaker (Fig. 3g, h, i). A rapid screenings using semi-quantitative PCR, target to assay mRNA, showed the same differences seen with immuno-technique assaying within the three samples. Primers for actin were used to normalise the reaction (Fig. 5a). Moreover a quantitative total RNA was screened for NPY and OMP mRNA transcripts by real-time RT-PCR. A far larger amount of mRNA was seen in wild boars (11.4 NPY and 6.0 OMP) than in FLPs (2.7 NPY and 3.1 OMP) as a fold of transcript in domestic pigs (Fig. 5b); (ANOVA: F = 161.3 df = 2 p 0.001). Fig. 2 Brain size. The relative brain size of wild boar (a), FLP (b) and domestic pig (c) is shown in three typical sections of head. Scale bar indicate 5 cm Discussion be assigned to Large white pig breed (Accession number KF780581; 0501 genotype) (Table 2). The timing of sensory systems adaptation to external On average, the relative brain size of FLP (accounting conditions ranges from milliseconds (receptor desensitiza- for body mass) was approximately 96 % of the wild boar tion) to years (plasticity of central representations). Pro- brain (Figs. 2, 4). In stark contrast, domestic animals longed sensory stimulation can strengthen, weaken, or even showed an extremely reduced brain size that is only 35 % eliminate the responsiveness of sensory neurons. The of the wild boar brain size. These differences are signifi- consequences of stimulation depend on the strength and cant (ANOVA: F = 447.6 df = 2, 27, p 0.001). Post- duration of the sensory stimulus. They also vary with hoc test indicated that both wild boars and FLP have sig- developmental stage and stimulus context (Dudai 1989; nificantly larger brains per unit mass than pigs (wild boar/ Ochoa et al. 1990; Hadcock and Malbon 1993; Raus- domestic pig, p \ 0.001, FLP/domestic pig, p \ 0.001), checker and Korte 1993). 123 Evol Biol (2014) 41:229–239 235

Fig. 3 Olfactory Mucosa sections. Olfactory Mucosa sections of wild boar (a, d, g), FLP (b, e, h), and domestic pig (c, f, i). Scale bar 50 lm. Hematoxylin–eosin (HE) shows the histological differences in a section of the olfactory mucosa (a, b, c) quantitative analysis are in Fig. 4. Olfactory marker protein (OMP) immunoreactivity shows a few and comparable stained cells in FLP (e) and domestic pig (f) than wild boar (d), in this latter signal is evident and strong. A comparable status was found using neuropeptide Y (NPY) immunoreactivity in wild boar (g), FLP (h) and domestic pig (i)

Fig. 4 Brain size and number of cells in Olfactory mucosa. Black bars indicate the brain size and gray bars indicate number of cells in the olfactory mucosa in wild boar, FLP and domestic pig (Values on the right axis and left axis respectively). Error bars represent the standard deviation. Asterisks indicate a statistically significant difference (p \ 0.001, see in the text)

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Cummings and Brunjes 1997;Cummingsetal.1997). This means that living in the wild may have directly increased neuron density in FLP olfactory mucosa via reduced neurons degeneration during growth. The full recovery in relative brain mass in FLP is yet another indication that life in the wild promoted an inversion in evolution towards the ‘‘wild’’ phenotype. The quality of this recovery, though, is signifi- cantly limited. Otherwise, if Sardinian FLP were similar to wild boar once they became free on the island (rather than having a fully domestic ancestry) it remains striking the difference in NPY and OMP in FLP as compared to wild boars. Whatever the case, FLPs are very much different from wild boars at the functional, but not at the morpho- logical (in terms of brain mass) level, which still suggest that the two evolve at different rates through domestication, in keeping with the notion of incomplete reversibility. Both OMP and NPY do not show the same pattern of recovery (in FLPs as compared to wild boars) than struc- tural components. The binding of these proteins to the cell receptors in the olfactory mucosa of both wild and domestic pigs is highly inefficient. Although OMP and NPY expression in FLPs are some 3 times as much as in Fig. 5 Gene expression analysis. a Agarose gel of Reverse Trascrip- domestic pigs (Fig. 1), they are still 2–4 times as small as tase PCR amplification of NPY, OMP and Actin mRNA from the in the wild boars. That is, the full recovery in olfactory Olfactory Mucosa among three forms of Sus scrofa. NPY expression neurons density, and the huge recovery in brain mass is not in the wild boar was about three times higher than observed in wild and domestic pig, in which value are comparable. OMP mRNA paralleled at the biomolecular level. This strongly suggests expression was appreciable only in wild boar. b OMP and NPY real- that i) the functionality of the sense of smell in Sardinian time RT-PCR amplification from wild boar, FLP, and domestic pig FLPs is very reduced as compared to wild boars, and ii) olfactory mucosa. Relative fold change in gene expression was although a structural recovery (in terms of density of calculated relative to gene expression 100 mg of olfactory mucosa. Gene expression are statistically significant. Error bars represent the neurons in the olfactory mucosa and total brain mass) is in standard deviation fact evident in the FLPs the sense of smell in the latter is most probably not even comparable to wild boars’. This is We found that the sense of smell in FLP in Sardinia, particularly intriguing since the brain regions linked to now living in the wild, adapt to the far richer range of smell in pigs are comparatively far less reduced than in sensory stimuli they experience, as compared to their fully other domestic species (Plogmann and Kruska 1990). We captive relatives. The extent of this sensory recovery, cannot rule out a priori that the reduction of the olfactory though, is limited. functions in the Sardinian FLP is associated to the envi- We examined (1) to what extent the olfactory mucosa ronmental conditions they experience at the ontogenetic structure differs among wild boars, domestic pigs, and level (i.e. it is not heritable). Yet, it is now fairly evident FLPs in terms of the density of olfactory neurons; and (2) from the literature that genes linked to the sense of smell the functionality of the mucosa in terms of expression are under the influence of selection, both natural and arti- levels of OMP and NPY (i.e. how much functional the ficial (Amaral et al. 2012; Groenen et al. 2012). This olfactory neurons are in the three S. scrofa strains). suggests that selection, rather than individual reaction In our samples the density of neurons in the olfactory norms during ontogeny are responsible for the loss of NPY mucosa is much higher in wild boar and FLP than in and OMP functioning in wild Sardinian pigs. domestic pig. This indicates that FLP reacquired high The case study presented here is mostly consistent with cellular density in the mucosa through feralization. This is Dollo’s law, reversibility in the evolutionary loss in the probably important to FLP individuals’ fitness in the wild, sense of smell quality in pigs is very limited, and very where a good sense of smell is crucial for survival (Man- probably linked to the fact that the ontogeny of olfactory dairona et al. 2003). mucosa in S. scrofa is retained unaltered in the domestic The density of neurons in the Main Olfactory Bulb breeds. depends on the intensity and variation in olfactory stimuli These findings remain valid regardless of the extent to during ontogeny (Frazier and Brunjes 1988; Brunjes 1994; which the first domestic pigs introduced to Sardinia (the 123 Evol Biol (2014) 41:229–239 237 ancestors of the modern Sardinian FLP population) pos- Barton, R. A. (1996). Neocortex size and behavioural ecology in sessed a similar extent and quality of brain reduction to the primates. Proceedings of the Royal Society B: Biological Sciences, 263, 173–177. modern domestic pigs used in our analyses. It is unlikely Barton, R. A., & Harvey, P. H. (2000). Mosaic evolution of brain that aboriginal Sardinian domestic pigs were much differ- structure in mammals. Nature, 405, 1055–1058. ent from modern domestic pigs. For instance, it is known Barton, R. A., Purvis, A., & Harvey, P. H. (1995). Evolutionary that brain mass reduction with domestication is a rapid radiation of visual and olfactory brain systems in primates, bats and insectivores. Philosophical Transactions: Biological Sci- process (Kruska 1996). This also means that the chance ences, 348, 381–392. that the FLP population in Sardinia was established from Birks, J. D. S., & Kitchener, A. C. (1999). The Distribution and status large-brained pigs, presumably very shortly after pigs were of the polecat Mustela putorius in Britain in the 1990s. National imported to the island, is small. Additionally, we know of Museums of Scotland: The Vincent Wildlife Trust. Bo¨ko¨nyi, S. (1974). History of domestic mammals in Central and no pig breed today that possesses large brains. Indeed, Eastern Europe (p. 597). Budapest: Akade´miai Kiado´. Albarella et al. (2006) suggested on biometric grounds Borowsky, R. (2008). Restoring sight in blind cavefish. Current (teeth and long bone measurements) that Sardinian FLPs Biology, 18, R23–R24. escaped from captivity very early, preserving most of the Brunjes, P. C. (1994). Unilateral naris closure and olfactory system development. Brain Research Reviews, 19, 146–160. wild boar morphological status. In our case, rather than a Collin, R., & Miglietta, M. P. (2008). Reversing opinions on Dollo’s partial recovery from a fully domestic status (as we Law. Trends in Ecology and Evolution, 23(11), 602–609. assumed here), wild life style in the FLP were not enough Cummings, D. M., & Brunjes, P. C. (1997). The effects of variable to recover a fully functional sense of smell. This is still periods of functional deprivation on the olfactory bulb develop- ment in rats. Experimental Neurology, 148, 360–366. consistent with Dollo’s law. It is worth noticing, though Cummings, D. M., Henning, H. E., & Brunjes, P. C. (1997). Olfactory that teeth and long bone measurements are not the same bulb recovery after early sensory deprivation. The Journal of thing as (neither they are a proxy for) brain size and Neuroscience, 17, 7433–7440. functioning. Di Bona, G. F. (2002). Neuropeptide Y. American Journal of Physiology—Regulatory, Integrative and Comparative Physiol- Our results provide a close look to the anatomy of ogy, 282(3), R635–R636. Dollo’s law. Sardinian FLPs are pressed by their life in the Dollo, L. (1893). Les Lois de l’e´volution. Bulletin de la Socie´te´ beige wild to have a full-functional sense of smell. 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