# Roman dogs from the Iberian Peninsula and the Maghreb - A glimpse into their morphology and genetics

Ana Elisabete Pires, Cleia Detry, Carlos Fernandez-Rodriguez, Silvia Valenzuela-Lamas, Ana Margarida Arruda, Jacopo de Grossi Mazzorin, Morgane Ollivier, Catherine Hanni, Fernanda Simoes, Catarina Ginja

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## To cite this version:

Ana Elisabete Pires, Cleia Detry, Carlos Fernandez-Rodriguez, Silvia Valenzuela-Lamas, Ana Margarida Arruda, et al.. Roman dogs from the Iberian Peninsula and the Maghreb - A glimpse into their morphology and genetics. Quaternary International, 2018, 471, pp.132-146. 10.1016/j.quaint.2017.11.044. hal-01806865

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Quaternary International xxx (2017) 1–15

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# Roman dogs from the Iberian Peninsula and the Maghreb – A glimpse into their morphology and genetics

Ana Elisabete Pires a, b, c, *, Cleia Detry c, Carlos Fernandez-Rodriguez d,
Silvia Valenzuela-Lamas e, Ana Margarida Arruda c, Jacopo De Grossi Mazzorin f,
Morgane Ollivier g, 1, Catherine Hänni h, Fernanda Simões i, Catarina Ginja b

## ARTICLE INFO

Article history:

Received 23 January 2017

Received in revised form

27 November 2017

Accepted 29 November 2017

Available online xxx

## ABSTRACT

In this study, we integrate osteometric and palaeogenetic data to investigate dog variability in the Roman Empire in Iberia and North Africa. Osteometry was used to distinguish the status—domestic or wild, of approximately 2000 years old *Canis* remains and to understand to what extent teeth and long bones varied in dogs in the Roman provinces of *Mauretania Tingitana*, *Lusitania* and *Tarragonensis*. High-throughput 454-DNA sequencing technology was used to obtain mitochondrial DNA (mtDNA) sequences from 15 bone and teeth samples. We identified five dog haplotypes from partial sequences of the hypervariable *D-loop* region. MtDNA haplotypes were grouped into two of the four major clades found in present-day dogs. We detected three clade A haplotypes in 12 samples from Portugal, Spain and Morocco, and a single clade D haplotype in 3 samples from Spain. So far, this is the oldest evidence for the presence of dog clade D in Iberia. It is dated to the late Roman occupation in the 4th-5th cent. AD (ca. 1,600 years ago).

Our results confirm the existence of distinct dog morphotypes in Roman times that also harboured distinct genetic lineages. According to our data, dogs from distinct mtDNA lineages (clades A and D) have been continuously bred in the Iberian Peninsula since at least 1600 years ago. Moreover, the sharing of matrilines between dogs from Spain and North Africa may indicate gene flow. Dogs could have been easily transported between these regions by humans along maritime and terrestrial trade routes. These results provide new insights into pre-Roman and Roman domestication practices, confirming selection practices were extensively applied to dogs during the first centuries of our era in the Iberian Peninsula. We show that the greater size variability of teeth length (and consequently cranium) and long bone breadths (and consequently phenotype) of Roman dogs in the Iberian Peninsula, is concomitant with the detection of diverse and rare maternal lineages. This would reflect an intensification of dog breeding and the use of non-local dogs for breeding.

© 2017 Elsevier Ltd and INQUA. All rights reserved.

## 1. Introduction

Dogs (*Canis lupus familiaris*), the oldest domesticate, show huge morphological variability today. But when did this domestic animal begin to vary so much in size and shape? A variety of dog statures, with small dogs having a withers height less than 30 cm and others

<footnote>a LARC - Laboratório de Arqueociências, Direcção Geral do Património Cultural, Lisboa, Portugal</footnote>

<footnote>b CIBIO-InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Vairão, Portugal</footnote>

<footnote>c UNIARQ - Centro de Arqueologia da Universidade de Lisboa, Faculdade de Letras de Lisboa. Universidade de Lisboa, Portugal</footnote>

<footnote>d Departamento de Historia, Facultad de Filosofia y Letras, Universidad de León, León, Spain</footnote>

<footnote>e CSIC - Consejo Superior de Investigaciones Científicas, Institució Milà i Fontanals, Barcelona, Spain</footnote>

<footnote>f Università del Salento, Lecce, Italy</footnote>

<footnote>g CNRS-ENS, French National Platform of Paleogenetics, PALGENE, Ecole Normale Supérieure de Lyon, Lyon, France</footnote>

<footnote>h LECA - Laboratoire d'Ecologie Alpine, Université Grenoble Alpes, Grenoble, France</footnote>

<footnote>i Instituto Nacional de Investigação Agrária e Veterinária, I.P., Lisboa, Portugal</footnote>

<footnote>* Corresponding author. Laboratório de Arqueociências (LARC) / DGPC and CIBIO / InBIO – EnvArch group, Rua da Bica do Marquês (à Ajuda), n° 2, 1300-087, Lisboa, Portugal.

E-mail address: ana.elisabete.pires@gmail.com (A.E. Pires).</footnote>

<footnote n="1">¹ Present address: UMR CNRS 6553 Ecobio, OSUR (Observatoire des Sciences de l'Univers de Rennes), University of Rennes 1, Rennes Cedex, France.</footnote>
over 60 cm, have been reported from the Late Glacial Period ca. 18,000–10,000 years ago (Horard-Herbin et al., 2014). Although not as variable as extant dogs, greater variability in both size and shape is also well known in dogs from Roman times ca. 2,000 years ago (Altuna and Mariezkurrena, 1992; Bennett et al., 2016; Colominas, 2015; De Grossi Mazzorin and Tagliacozzo, 2000; MacKinnon, 2010; Peters, 1998). This process probably originated in preceding times, i.e., in the late Iron Age ca. 2,500 years ago (Bennett et al., 2016), but the very small lap, or toy dogs, appear for the first time, and in large numbers, throughout the Roman Empire ca. 2,000 years ago (e.g. Boessneck and von den Driesch, 1980; Altuna and Mariezkurrena, 1992; Fernández-Rodriguez, 2003; MacKinnon, 2010). In the Western Mediterranean, the differentiation of dog populations into different phenotypes similar to breeds may have its roots in the Roman period (De Grossi Mazzorin and Tagliacozzo, 2000, 1998). In fact, the extent of morphological variation is most evident in Roman times and today—perhaps because artificial selection was and is extensively exercised over dogs by humans for aesthetic/emotional reasons (e.g. dogs just for companionship such as lap dogs) and not only for functional/working purposes (dogs used for hunting or herding). The definition of today's dog breeds follows criteria defined in the 19th century AD and it is not possible to recognize extant dog breeds in ancient periods merely through osteological morphology (Harcourt, 1974; Morales et al., 2015). Nevertheless, *Varro* in his *De re rustica* book (1st cent. AD) provided a dog classification based on their function.

Coat colour also varied since early times. The investigation of molecular markers associated with dark versus light coat colour, i.e., *Mc1r* (melanocortin 1 receptor) and *CBD103* (canine-β-defensin) genes, showed the presence of dogs with light and dark coat colours as early as the beginning of the Mesolithic, over 8000 years ago (Ollivier et al., 2013). Well known Roman iconography—mosaic floors and wall paintings—confirm a diversity of morphologies and coat colours in dogs (Fig. 1). The dog morphotypes represented in mosaics do not necessarily reflect local dogs, as the artisans tended to move around the Empire possibly producing representations of what they had seen in other places; however, in the Roman city of Conímbriga, in *Lusitania*, Portugal, there are indications that the artisans were local (Correia, 2013). Thus, it is probable that these dog morphotypes existed across the Roman Empire.

Zoarchaeological studies aid our understanding of size and morphological variation in Roman *Canis* (e.g., Altuna and Mariezkurrena, 1992; Bennett et al., 2016; Colominas, 2015; MacKinnon, 2010). The various anatomical parts are preserved differently in the archaeological record. For instance, the first molar tooth—M1—is frequently found intact in the archaeological record. However, the M1 length variability is not necessarily correlated with body size, but it reflects to some extent the cranium size and shortening of the snout. This latter characteristic is a marker of the domestication process, since dogs tend to have shorter snouts than wolves (Tchernov and Valla, 1997). Measurements of the long bones (femura) are a better proxy for the overall body size variability (Wayne, 2001) and therefore can indicate the presence of different morphotypes. However, in the archaeological record these bones are frequently found fragmented.

Genetic studies using ancient DNA (aDNA) can provide important insights into the understanding of population genetics, gene flow and human-driven selection for certain traits in animals of the past (Botigué et al., 2017; Ollivier et al., 2016, 2013; Pilot et al., 2014; Svensson et al., 2007), particularly when bones are morphologically indistinct. However, these kinds of studies are still scarce for Roman material, particularly in the Iberian Peninsula, a region where samples tend to be poorly preserved (but see Catagnano, 2016). Mitochondrial DNA (mtDNA) has been particularly useful for studying the genetic composition of archaeological specimens (e.g.,

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**Fig. 1.** Details of Roman mosaics from Conímbriga (Portugal) depicting dogs with different shape and colour: A) Two dogs on a leash with different coat colours, one brown and the other black (detail of mosaic nº 11 of Casa dos Repuxos, (Oleiro, 1992), Photo by Humberto Rendeiro@MMC/DGPC); B) A hunter carrying a hare and accompanied by a dog with a short snout (detail of mosaic nº 15 of Casa dos Repuxos, (Oleiro, 1992), Photo by Delfim Ferreira@MMC/DGPC). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Frantz et al., 2016; Leonard et al., 2002), especially in hot climates where DNA is poorly preserved. MtDNA sequence variability does not reflect directly variation in morphological traits, but regions such as the D-loop generally show a large degree of sequence variation between individuals, and it is most useful for addressing intraspecific evolutionary questions (e.g. Frantz et al., 2016; Leonard et al., 2002).

Worldwide mtDNA diversity of extant dogs indicate the existence of six clades or haplogroups: A, B, C, D, E and F (Savolainen et al., 2002). Clades A, B, C and D represent the majority of the maternal diversity found in dogs. Clade A is the most frequent lineage currently observed in dogs from Europe (including Iberia) and North Africa, whereas clade D is the less represented and geographically restricted to Portugal, Spain, Morocco, Turkey and Scandinavia in present times (Ardalan et al., 2011; Pires et al., 2006; Savolainen et al., 2002). Extant dogs from Iberia and North Africa have high genetic diversity, carrying haplotypes belonging to clades A, B, C and D (Pires et al., 2006). Clades E and F were only detected in dogs from Asia. Concerning clade D in ancient European dogs, it was first detected in the Near East and later in South-eastern and Central Europe (Pionnier-Capitan, 2010) during the Neolithic ca. 10,000 BC. This clade would most likely have been introduced in
Europe in the Neolithic along with other domesticated animals used for food, such as pig, sheep, goat and cattle (M. Ollivier, pers. com.).

In this study, we integrate osteometric, morphological and palaeogenetic data to characterize dog phenotypic and genetic variability of dogs of Roman times from Iberia and North Africa. Our hypothesis is that the greater morphological variation in dogs attributed to the Romans coincides with the presence of various dog mtDNA lineages. This would be in line with the Romans' interest in improving their domesticated animals—known to be more efficient and diverse, even extravagant.

## 2. Material and methods

### 2.1. Zooarchaeological study

The sites considered cover a time span from the Mesolithic (ca. 7,500 years ago) to present day dogs and wolves in order to assess the diversity of dog sizes in each period including Roman, and how it compares with extant populations. We included measurements of the lower first molar tooth (M₁) length and the humerus distal breadth of dog specimens from 56 archaeological sites including 13 sites dated to the Roman period (Tables 1 and 2). We selected the M₁ and distal humerus because these parts are frequently found in archaeological sites. M₁ crown length and humerus distal breadth were measured following von den Driesch (1976).

Lengths of archaeological specimens of M₁ were compared with measurements taken from Portuguese reference collections of skeletons of extant wolves (n = 45, 19 males, 18 females and 8 of unknown sex) and dogs (n = 29, unknown sex). These portuguese collections are held at Laboratório de Arqueociências, Direcção Geral do Património Cultural, Museu Nacional de História Natural e da Ciência and the Laboratório de Anatomia, Universidade Lusófona de Humanidades e Tecnologias, in Lisbon. In dogs, sexual dimorphism is present but it is not very strong (Cruz, 2007), and therefore this parameter does not affect size greatly. Adding to that, the variation of phenotypes and breeds also overlap any size variation derived from sexual dimorphism. Consequently, we did not consider sexual dimorphism as a variable in the analysis.

We measured the tooth and humerus from reference collections in Portugal and Spain and archaeological collections from Portugal. Measurements taken by other authors were used for comparisons (see Tables 1 and 2). The Coefficient of Variation (CV), being CV = (standard deviation/average) \* 100, for the M₁ length and humerus breadth was calculated for each period.

We used the non-parametric test of Siegel-Tukey (Siegel and Tukey, 1960) in order to test whether differences between the populations existed regarding the diversity of the measurements. This test measures the dispersion of values in two populations, that is, whether a population is more homogeneous than another.

## 3. Genetic study

### 3.1. Archaeological sites

The dog remains of this study derive from three archaeological sites located in the western range of the Roman Empire, namely (from south to north): Thamusida in northern Morocco (Mauretania Tingitana) in 1999; Monte Moliao, in Lagos in southern Portugal (Lusitania) in 2006/2008; and Lugo in Galicia in northern Spain (Tarraconensis) in 2003. The exact location of these sites and the sampled materials are shown in Fig. 2. A description of the archaeological sites from where these samples were excavated is presented below. It should be noted that the archaeological remains of dog from Morocco and Spain are contemporaneous (3rd-

4th cent. AD - ca. 1,600 years ago) whereas those from Portugal are older (1st cent. BC-1st cent. AD - ca. 2,000 years ago).

**Thamusida (Morocco)** The ruins of the city, already known from the end of the 19th century AD, were systematically investigated in the course of the 1930s and 1960s. Archaeologists discovered the most ancient layers of Mauro settlement and the main Roman monuments. In the year 40 AD, the province of Mauretania Tingitana was founded by Rome; the ancient Mauro settlement was destroyed during military operations aimed at controlling the region. During the second half of the 1st century AD, a military camp, close to a ford on the Sebou River, was established there. Around this camp a new urban centre was born. This new small city included different facilities such as houses, baths, temples, taverns and shops. Walls were constructed during the second half of the 2nd cent. AD. These were probably needed to resist attacks from the Berber tribes of the Gharb (west). It is also worth noting that at the end of the 3rd century AD the Roman army left the area, but the site continued to be inhabited. Later on, during the 7th-8th cent, Arabs arrived at this settlement, but they chose to live outside the ancient city walls.

**Monte Moliao (Lagos, Portugal)** was a small Roman town located on the south coast of Portugal, which was built over an Iron Age *oppidum*. The Roman occupation began in the 2nd century BC (Republican period), with intensive connections to other Mediterranean areas including the Italian Peninsula, North Africa and Bay of Cadiz, and lasted until the end of the 2nd century AD (Imperial period). Chronologies were obtained from the datable imported materials (ceramics) found on the site. Trading between Monte Moliao and North Africa existed, namely with the area of present-day Tunisia during the Republican and the Imperial Roman periods, as attested through imported food products transported in amphorae (Arruda and Sousa, 2013) as well as the presence of abundant imported ceramics from this area (Arruda, 2007). Dog remains were found in layers related to the site's occupation, especially the Imperial period (1st -2nd century A.D.; Detry and Arruda, 2013).

**Lugo (Lucus Augusti) (Spain)** was a Roman city that developed during the Early Imperial period (late 1st century BC) in the north-western region of the Iberian Peninsula. It eventually became Conventus Lucensis capital, one of the administrative units in which the *Gallaecia* province was divided (Rodríguez Colmenero, 2011). Excavations at the site *Domus del Mitreo*, revealed a diachronic occupation since the Roman period. Regarding the oldest records, they reflect a construction of the Early Imperial period, and a *domus* identified as a *mitreo* (for cult) was recorded (Alvar et al., 2006; Rodríguez and Cordeiro, 2014; Rodríguez Cao, 2006). This construction was renewed several times and it was damaged due to a Roman wall construction in the late 3rd cent. AD. It eventually disappeared at the end of 3rd cent. - 4th cent. AD during a refurbishment of the city. Dog remains excavated from deposition levels related to the renovation and destruction of the *domus*, can be dated to the 4th century, as indicated by other associated archaeological materials. The presence of camelids (possibly dromedary) among the faunal remains at *Domus del Mitreo* excavation site (C Fernandez-Rodriguez, pers. com.) may reflect connections with North Africa. There are also references to other parts of the Lugo city for the presence of North African products (Naveiro López, 1991) such as amphorae (Carreras Monfort, 2011; Naveiro López, 1991).

### 3.2. Sub-sampling for aDNA analysis

A total of 15 dog remains were sampled for ancient DNA (aDNA) analyses: one upper third pre-molar from Thamusida; two proximal femora and a mandible fragment from Monte Moliao; and two
<table><caption>**Table 1**
Measurements of the lower first molar tooth (M₁) from various archaeological sites in Portugal, Spain, Morocco and Tunisia. N – number of specimens measured; Min. – minimum value; Max. – maximum value; SD – Standard Deviation; CV – Coefficient of Variation (%).</caption><thead><tr><th>Archaeological Site</th><th>Location</th><th>Country</th><th>Roman province</th><th>Chronology</th><th>N</th><th>Min.</th><th>Max.</th><th>Average</th><th>SD</th><th>CV (%)</th><th>References</th></tr></thead><tbody><tr><td>20th Century Wolves</td><td></td><td>Portugal</td><td></td><td></td><td>45</td><td>23.8</td><td>30.7</td><td>27.3</td><td>1.5</td><td><strong>5.7</strong></td><td>(Detry and Cardoso, 2010)</td></tr><tr><td>20th Century Dogs</td><td></td><td>Portugal and Spain</td><td></td><td></td><td>29</td><td>14.6</td><td>25.3</td><td>19.4</td><td>3.0</td><td><strong>15.5</strong></td><td>C. Detry, personal communication</td></tr><tr><td>Silos de Carnide</td><td>Lisboa</td><td>Portugal</td><td></td><td>Modern Period</td><td>8</td><td>17.9</td><td>20.2</td><td>18.4</td><td>1.0</td><td>5.3</td><td>C. Detry and S.J.M. Davis, personal communication</td></tr><tr><td>Mosteiro de Santo André</td><td>Amares</td><td>Portugal</td><td></td><td>Modern Period</td><td>1</td><td>17.9</td><td>17.9</td><td>0</td><td>0.0</td><td>0.0</td><td>Davis, unpublished</td></tr><tr><td>Mosteiro do Bouro</td><td>Amares</td><td>Portugal</td><td></td><td>Modern Period</td><td>2</td><td>19.7</td><td>22.1</td><td>20.9</td><td>1.2</td><td>5.7</td><td>Davis, unpublished</td></tr><tr><td></td><td></td><td></td><td></td><td><strong>Modern Period</strong></td><td><strong>11</strong></td><td><strong>17.9</strong></td><td><strong>22.1</strong></td><td><strong>18.8</strong></td><td><strong>1.4</strong></td><td><strong>7.4</strong></td><td></td></tr><tr><td>Lixeira</td><td>Silves</td><td>Portugal</td><td></td><td>Medieval Islamic</td><td>3</td><td>22.6</td><td>23.1</td><td>22.8</td><td>0.2</td><td>1.0</td><td>(Davis et al., 2008)</td></tr><tr><td>Alcáçova de Santarém</td><td>Santarém</td><td>Portugal</td><td></td><td>Medieval Islamic</td><td>1</td><td>23.8</td><td>23.8</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Davis, 2006)</td></tr><tr><td></td><td></td><td></td><td></td><td><strong>Medieval Period</strong></td><td><strong>4</strong></td><td><strong>22.6</strong></td><td><strong>23.8</strong></td><td><strong>23.0</strong></td><td><strong>0.5</strong></td><td><strong>2.2</strong></td><td></td></tr><tr><td>Astorga</td><td>Astorga</td><td>Spain</td><td>Tarraconensis</td><td>Late Roman (3rd-4th cent. AD)</td><td>14</td><td>16.1</td><td>23.7</td><td>20.5</td><td>2.5</td><td>12.2</td><td>(Fernandez-Rodriguez, 2000)</td></tr><tr><td>Els Mallols</td><td>Barcelona</td><td>Spain</td><td>Tarraconensis</td><td>Late Roman (3rd-4th cent. AD)</td><td>3</td><td>19.5</td><td>21.0</td><td>20.2</td><td>0.6</td><td>3.1</td><td>(Catagnano, 2016)</td></tr><tr><td>Odrinhas</td><td>Sintra</td><td>Portugal</td><td>Lusitania</td><td>Late Roman (3rd-4th cent. AD)</td><td>14</td><td>15.6</td><td>22.0</td><td>19.0</td><td>2.0</td><td>10.5</td><td>(Davis and Gonçalves, 2017)</td></tr><tr><td>Monte Molião</td><td>Algarve</td><td>Portugal</td><td>Lusitania</td><td>Imperial Roman (1st-2nd cent. AD)</td><td>1</td><td>17.5</td><td>17.5</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Detry and Arruda, 2013)</td></tr><tr><td>Thamusida</td><td>Morroco</td><td>Mauretania Tingitania</td><td></td><td>Imperial Roman (1st-2nd cent. AD)</td><td>3</td><td>19.2</td><td>19.9</td><td>19.6</td><td>0.3</td><td>1.5</td><td>(De Grossi Mazzorin and De Venuto, 2010)</td></tr><tr><td>Calle Almendralejo</td><td>Mérida</td><td>Spain</td><td>Lusitania</td><td>Imperial Roman (1st-2nd cent. AD)</td><td>50</td><td>14.8</td><td>22.7</td><td>19.2</td><td>1.8</td><td>9.6</td><td>C. Detry and F.J. Heras, personal communication</td></tr><tr><td>Alcáçova de Santarém</td><td>Santarém</td><td>Portugal</td><td>Lusitania</td><td>Republican Roman (1st-2nd cent. BC)</td><td>1</td><td>20.6</td><td>20.6</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Davis, 2006)</td></tr><tr><td>Odemira</td><td>Odemira</td><td>Portugal</td><td>Lusitania</td><td>Republican Roman (1st-2nd cent. BC)</td><td>1</td><td>26.0</td><td>26.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Davis and Vilhena, 2017)</td></tr><tr><td></td><td></td><td></td><td></td><td><strong>Roman Period</strong></td><td><strong>87</strong></td><td><strong>14.8</strong></td><td><strong>26</strong></td><td><strong>19.5</strong></td><td><strong>2.1</strong></td><td><strong>10.8</strong></td><td></td></tr><tr><td>Althiburos</td><td></td><td>Tunisia</td><td></td><td>Iron Age</td><td>3</td><td>18.4</td><td>21.7</td><td>20.5</td><td>1.5</td><td>7.3</td><td>(Valenzuela-Lamas, 2016)</td></tr><tr><td>R. dos Correeiros</td><td>Lisbon</td><td>Portugal</td><td></td><td>Iron Age</td><td>1</td><td>19.0</td><td>19.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Detry et al., 2016)</td></tr><tr><td>Hereuet</td><td>Tarragona</td><td>Spain</td><td></td><td>Iron Age</td><td>3</td><td>22.4</td><td>23.3</td><td>22.9</td><td>0.4</td><td>1.7</td><td>S. Valenzuela-Lamas, personal communication</td></tr><tr><td>Estinclells</td><td>Urgell</td><td>Spain</td><td></td><td>Iron Age</td><td>1</td><td>26.7</td><td>26.7</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Valenzuela-Lamas, 2010)</td></tr><tr><td>Alorda Park</td><td>Barcelona</td><td>Spain</td><td></td><td>Iron Age</td><td>2</td><td>22.8</td><td>23.0</td><td>22.9</td><td>0.0</td><td>0.0</td><td>(Valenzuela-Lamas, 2008)</td></tr><tr><td>Cerro Macareno</td><td>Sevilla</td><td>Spain</td><td></td><td>Iron Age</td><td>4</td><td>20.0</td><td>23.0</td><td>21.4</td><td>1.4</td><td>6.5</td><td>(Amberger, 1985)</td></tr><tr><td>Cerro de la Tortuga</td><td>Malaga</td><td>Spain</td><td></td><td>Iron Age</td><td>1</td><td>22.0</td><td>22.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(von den Driesch, 1973)</td></tr><tr><td>Mas Castellar</td><td>Gerona</td><td>Spain</td><td></td><td>Iron Age</td><td>6</td><td>15.5</td><td>21.6</td><td>19.6</td><td>2.0</td><td>10.2</td><td>(Catagnano, 2016)</td></tr><tr><td>Los Toscanos</td><td>Malaga</td><td>Spain</td><td></td><td>Iron Age</td><td>1</td><td>19.0</td><td>19.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(von den Driesch, 1973)</td></tr><tr><td></td><td></td><td></td><td></td><td><strong>Iron Age</strong></td><td><strong>22</strong></td><td><strong>15.5</strong></td><td><strong>26.7</strong></td><td><strong>21.2</strong></td><td><strong>2.3</strong></td><td><strong>10.8</strong></td><td></td></tr><tr><td>Castelon Alto</td><td>Granada</td><td>Spain</td><td></td><td>Bronze Age</td><td>4</td><td>18.0</td><td>21.0</td><td>19.9</td><td>1.1</td><td>5.7</td><td>(Milz, 1986)</td></tr><tr><td>Cerro de la Encina</td><td>Granada</td><td>Spain</td><td></td><td>Bronze Age</td><td>4</td><td>20.2</td><td>21.3</td><td>20.7</td><td>0.5</td><td>2.4</td><td>(Friesch, 1987)</td></tr><tr><td>Terrena del Reloj</td><td>Granada</td><td>Spain</td><td></td><td>Bronze Age</td><td>3</td><td>18.5</td><td>20.5</td><td>19.7</td><td>0.8</td><td>4.3</td><td>(Milz, 1986)</td></tr><tr><td>Fuente Álamo</td><td>Almeria</td><td>Spain</td><td></td><td>Bronze Age</td><td>3</td><td>19.5</td><td>21.5</td><td>20.3</td><td>0.8</td><td>4.2</td><td>(von den Driesch et al., 1985)</td></tr><tr><td>Purullena</td><td>Granada</td><td>Spain</td><td></td><td>Bronze Age</td><td>8</td><td>17.2</td><td>23.0</td><td>20.5</td><td>1.8</td><td>8.5</td><td>(Lauk, 1976)</td></tr><tr><td>Monachil</td><td>Granada</td><td>Spain</td><td></td><td>Bronze Age</td><td>2</td><td>20.5</td><td>21.7</td><td>21.1</td><td>0.6</td><td>2.8</td><td>(Lauk, 1976)</td></tr><tr><td>Cabezo Redondo</td><td>Alicante</td><td>Spain</td><td></td><td>Bronze Age</td><td>5</td><td>16.5</td><td>20.8</td><td>18.7</td><td>1.5</td><td>7.8</td><td>(Boessneck et al., 1969)</td></tr><tr><td>Sacaojos Bei la Baneza</td><td>Léon</td><td>Spain</td><td></td><td>Bronze Age</td><td>3</td><td>22.5</td><td>23.5</td><td>23.0</td><td>0.4</td><td>1.8</td><td>(Boessneck and von den Driesch, 1980)</td></tr><tr><td>Los Palacios</td><td>Ciudad Real</td><td>Spain</td><td></td><td>Bronze Age</td><td>2</td><td>19.5</td><td>21.0</td><td>20.3</td><td>0.8</td><td>3.7</td><td>(Boessneck and von den Driesch, 1980)</td></tr><tr><td>Azuer</td><td>Ciudad Real</td><td>Spain</td><td></td><td>Bronze Age</td><td>5</td><td>17.5</td><td>21.5</td><td>20.4</td><td>1.5</td><td>7.2</td><td>(Boessneck and von den Driesch, 1980)</td></tr><tr><td>Lloma de Baxi</td><td></td><td>Spain</td><td></td><td>Bronze Age</td><td>2</td><td>19.7</td><td>23.0</td><td>21.4</td><td>1.7</td><td>7.7</td><td>(Sanchis and Sarrión, 2004)</td></tr><tr><td>Leceia</td><td>Oeiras</td><td>Portugal</td><td></td><td><strong>Bronze Age</strong></td><td><strong>41</strong></td><td><strong>16.5</strong></td><td><strong>23.5</strong></td><td><strong>20.4</strong></td><td><strong>1.6</strong></td><td><strong>7.8</strong></td><td></td></tr><tr><td>Zambujal</td><td>Torres Vedras</td><td>Portugal</td><td></td><td>Chalcolithic</td><td>2</td><td>19.0</td><td>20.0</td><td>19.5</td><td>0.4</td><td>1.9</td><td>(Pires et al., 2001)</td></tr><tr><td></td><td></td><td></td><td></td><td>Chalcolithic</td><td>5</td><td>17.7</td><td>23.0</td><td>20.6</td><td>0.0</td><td>0.0</td><td>(von den Driesch and Boessneck, 1976)</td></tr><tr><td>P. Torrào</td><td>Alentejo</td><td>Portugal</td><td></td><td>Chalcolithic</td><td>2</td><td>20.3</td><td>20.6</td><td>20.5</td><td>0.2</td><td>0.7</td><td>Davis, unpublished</td></tr><tr><td>Paraíso</td><td>Alentejo</td><td>Portugal</td><td></td><td>Chalcolithic</td><td>2</td><td>21.0</td><td>22.0</td><td>21.5</td><td>0.5</td><td>2.3</td><td>Davis, unpublished</td></tr><tr><td>Camino de las Yeseras</td><td>Madrid</td><td>Spain</td><td></td><td>Chalcolithic</td><td>12</td><td>18.1</td><td>22.2</td><td>20.2</td><td>1.4</td><td>6.7</td><td>(Daza, 2015)</td></tr><tr><td>Cerro de la Virgen</td><td>Granada</td><td>Spain</td><td></td><td>Chalcolithic</td><td>5</td><td>18.2</td><td>21.0</td><td>19.9</td><td>0.0</td><td>0.0</td><td>(von den Driesch, 1972)</td></tr><tr><td>Marizulo</td><td>Guipuzcoa</td><td>Spain</td><td></td><td>Chalcolithic</td><td>1</td><td>21.4</td><td>21.4</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Altuna, 1967)</td></tr><tr><td>Camino del Molino</td><td>Murcia</td><td>Spain</td><td></td><td>Chalcolithic</td><td>56</td><td>17.2</td><td>21.8</td><td>20.0</td><td>1.0</td><td>5.0</td><td>(Catagnano, 2016)</td></tr><tr><td></td><td></td><td></td><td></td><td><strong>Chalcolithic</strong></td><td><strong>83</strong></td><td><strong>17.2</strong></td><td><strong>23</strong></td><td><strong>20.1</strong></td><td><strong>1.1</strong></td><td><strong>5.5</strong></td><td></td></tr><tr><td>SJA Portam Latinam</td><td>Bilbao</td><td>Spain</td><td></td><td>Neolithic</td><td>1</td><td>18.0</td><td>18.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Altuna and Mariezkurrena, 2007)</td></tr><tr><td>Cova de L'Or</td><td>Alacant</td><td>Spain</td><td></td><td>Neolithic</td><td>1</td><td>20.0</td><td>20.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Sanchis and Sarrión, 2004)</td></tr><tr><td>Sierra del Mas Bonet</td><td>Gerona</td><td>Spain</td><td></td><td>Neolithic</td><td>1</td><td>21.0</td><td>21.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Catagnano, 2016)</td></tr><tr><td>Lameiras</td><td>Sintra</td><td>Portugal</td><td></td><td>Neolithic</td><td>1</td><td>20.7</td><td>20.7</td><td>0.0</td><td>0.0</td><td>0.0</td><td>S.J.M. Davis personal communication</td></tr><tr><td>Belas</td><td>Sintra</td><td>Portugal</td><td></td><td>Neolithic</td><td>1</td><td>20.0</td><td>20.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>Davis, unpublished</td></tr><tr><td></td><td></td><td></td><td></td><td><strong>Neolithic</strong></td><td><strong>5</strong></td><td><strong>18.0</strong></td><td><strong>21.0</strong></td><td><strong>19.9</strong></td><td><strong>1.0</strong></td><td><strong>5.0</strong></td><td></td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Archaeological Site</th><th>Location</th><th>Country</th><th>Roman province</th><th>Chronology</th><th>N</th><th>Min.</th><th>Max.</th><th>Average</th><th>SD</th><th>CV (%)</th><th>References</th></tr></thead><tbody><tr><td>Muge (C. Arruda &amp; C. Amoreira)</td><td>Salvaterra de Magos</td><td>Portugal</td><td></td><td>Mesolithic</td><td>2</td><td>20.5</td><td>21.6</td><td>21.1</td><td>0.6</td><td>2.6</td><td>(Detry and Cardoso, 2010)</td></tr><tr><td>Sado (Poças de São Bento)</td><td>Alcácer do Sal</td><td>Portugal</td><td></td><td>Mesolithic</td><td>1</td><td>21.7</td><td>21.7</td><td>0.0</td><td>0.0</td><td>0.0</td><td>C. Detry and S.J.M. Davis, personal communication</td></tr><tr><td></td><td></td><td></td><td></td><td><strong>Mesolithic</strong></td><td><strong>3</strong></td><td><strong>20.5</strong></td><td><strong>21.7</strong></td><td><strong>21.3</strong></td><td><strong>0.5</strong></td><td><strong>2.6</strong></td><td></td></tr></tbody></table>

tibiae, four humeri, two radii, one scapula, one pelvis and one femur from Lugo. These specimens were labelled and stored in individual zip-lock plastic bags and kept separately from modern samples.

We followed specific protocols for sub-sampling and aDNA extraction and took adequate precautions to avoid contamination: sub-sampling of archaeological remains was performed in the aDNA dedicated facilities at PALGENE, French National Platform of Palaeogenetics at the École Normale Supérieure de Lyon (France) and the Archaeological Research Laboratory of Stockholm University (Sweden). After ultra-violet sterilization of the outermost surfaces and removal of an approximately 1 mm layer from the surface, some powder of bone/tooth was collected from each sample using a sterile scalpel or a Dremel tool. Replicas were kept in a freezer at -20 °C for subsequent analyses.

### 3.3. Isolation of aDNA

Bone or tooth powder (100–200 mg) was digested overnight with 1 mL of buffer EDTA (ethylene diamine tetra acetic acid, pH 8) 0.5 M and Urea 1 M with 10 µL of proteinase K (20 mg/mL in water) at 38 °C with constant agitation. For each sample, the lysate volume was concentrated down to 100 µl with Amicon columns (Millipore-Amicon Ultra-4 30 k Da) through centrifugation at 4,000 g for 10–15 min. The sample volume was then mixed with 5× PB buffer from the commercial kit QIAquick PCR Purification Kit from Qiagen. Ancient DNA was recovered, following the manufacturer's protocol, in a final volume of 100 µL. Two negative extraction controls were included in every batch of six samples. Duplicates of aDNA extracts were obtained independently from each specimen.

### 3.4. Mitochondrial DNA amplification

We analysed a D-loop fragment of 181 base pairs (bp). In extant dogs, this region is amplified by Polymerase chain reaction (PCR) with DL1-DL2 primers (Leonard et al., 2002), and recovers the majority of the mtDNA haplotypic variability included in the most frequent dog haplogroups (i.e., Hgs A, B, C and D). These primers were also useful in ancient samples of dogs and wolves. We used primer pairs DL1/DL3 and DL7/DL2 (Table 3) in two separate PCR reactions to amplify the mtDNA region between positions 15,495 and 15,676 in the dog reference sequence (EU789784). After amplification, these two fragments of 187 and 108 bp, respectively, superimpose for an extension of 94 nucleotides.

PCR mixtures contained 2 µL of genomic DNA, 1× Taq polymerase buffer (Applied Biosystem), 2.5 mM of MgCl₂ (Applied Biosystem), 0.25 mM of each deoxynucleoside triphosphate (dNTP) (Sigma), 0.02 mg Bovine Serum Albumin (Roche), 0.5 µM of each PCR primer, 2.5 U of Hot start Taq DNA polymerase (Applied Biosystem) and PCR water (Qiagen) in a total of a 25 µL PCR reaction. In all amplification reactions, the primer sequences included multiplex identifiers for libraries (MIDs), i.e., 10 nucleotide sequence tags that allow for multiplexing in emulsion PCR during 454-sequencing (Roche). To monitor for possible aerosol contaminations, a specific control was included in every PCR assay, which were limited to

eight aDNA extracts. The aerosol control was a single tube that was kept opened during the PCR set up. We also monitored possible contaminations from reagents by setting up a negative control with water as a replacement to DNA extract (PCR-mix control). Negative controls were systematically used, and all extraction blanks were also subject to PCR amplification.

Amplification reactions were performed in an Applied Biosystems thermal cycler (Veriti), with an initial denaturation step of 94 °C for 10 min; followed by 55 cycles of denaturation at 94 °C (30 s), annealing at 52 °C (30 s for DL1/DL3 primer pair) or 48 °C for (30 s for DL7/DL2 primer pair), and extension at 72 °C (45 s); followed by a final extension step at 72 °C for 10 min.

Amplifications were verified by the electrophoresis of 5 µL of each PCR product in 2% ethidium bromide agarose gels in Tris/Borate/EDTA (TBE) (0.5×) buffer. Electrophoresis ran at 10 V/cm for 30 min. Samples that provided positive signal at the expected size were sequenced. Negative controls remained blank through agarose gel inspection.

For DNA sequencing, 20 µL of the remaining PCR product were electrophoresed in a second 2% agarose gel and the amplified DNA excised from the gel with a sterile scalpel under UV light. The PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen) and eluted in 30 µL elution solution. If primer dimer was present, we used the MinElute PCR Purification Kit (Qiagen) following the manufacturer's protocol.

### 3.5. Mitochondrial DNA sequencing

We used the Quant-iT™ dsDNA High-Sensitivity Assay Kit (Invitrogen) to quantify the purified positive PCR products. Equimolar mixtures of pooled amplification products were sequenced using the 454-GS Junior technology (Roche) in accordance to the manufacturer's protocols. Sequence reads were identified through primers and MIDs and aligned against reference NCBI sequences. The GALAXY platform (http://galaxy.psu.edu/), a web-based genome analysis tool (Afgan et al., 2016) was used to obtain the sequence files for each pooled PCR product. Dog sequences were compared with the DNA sequence information stored in the GenBank database. This was done using the “basic local alignment search tool” (BLAST) program (http://blast.ncbi.nlm.nih.gov) (Altschul et al., 1990) and the blastn algorithm.

For multiple alignments, the Seaview software (Galtier et al., 1996) was used and 60% consensus sequences were generated for each individual from independent amplifications (see sequencing coverage for each dog specimen in Table 4). If any conflict persisted preventing haplogroup assignment the specimen was sequenced until resolution. Partial mtDNA sequences of ancient Canis remains were deposited in GenBank (accession numbers KY014672–KY014674; KY014684–KY014894 and KY464999) (Table 4).

### 3.6. Phylogenetic analyses

Relationships between haplotypes were investigated using NETWORK v5.0.0.0 (Fluxus Technology Ltd, 2004–2016) software
<caption>**Table 2**</caption>

Measurements of the humerus distal breadth from various archaeological sites in Portugal and Spain. N – number of specimens measured; Min. – minimum value; Max. – maximum value; SD – Standard Deviation; CV – Coefficient of Variation (%).

<table><thead><tr><th>Archaeological Site</th><th>Location</th><th>Country</th><th>Roman province</th><th>Chronology</th><th>N</th><th>Min.</th><th>Max.</th><th>Average</th><th>SD</th><th>CV (%)</th><th>References</th></tr></thead><tbody><tr><td>20th Century<br>Wolves</td><td>Portugal</td><td></td><td></td><td></td><td>30</td><td>32.4</td><td>48.9</td><td>41.8</td><td><strong>4.2 10.0</strong></td><td>(Detry and Cardoso, 2010)</td><td></td></tr><tr><td>20th Century<br>Dogs</td><td>Portugal&Spain</td><td></td><td></td><td></td><td>25</td><td>19.3</td><td>47.5</td><td>29.3</td><td><strong>7.7 26.3</strong></td><td>C. Detry, personal communication</td><td></td></tr><tr><td rowspan="2">Silos de Carnide</td><td rowspan="2">Lisboa</td><td rowspan="2">Portugal</td><td rowspan="2"></td><td>Modern period</td><td>8</td><td>20.9</td><td>28.6</td><td>24.7</td><td>3</td><td>12.1</td><td>C. Detry and S.J.M. Davis, personal communication</td></tr><tr><td>Modern period</td><td>1</td><td>22.3</td><td>22.3</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Davis, 2006)</td></tr><tr><td rowspan="2">Alcáçova de<br>Santarém</td><td rowspan="2">Santarém</td><td rowspan="2">Portugal</td><td rowspan="2"></td><td>Modern period</td><td><strong>9</strong></td><td><strong>20.9</strong></td><td><strong>28.6</strong></td><td><strong>24.4</strong></td><td><strong>3</strong></td><td><strong>12.3</strong></td><td></td></tr><tr><td>Christian period</td><td>1</td><td>36.6</td><td>36.6</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Davis, 2006)</td></tr><tr><td>Alcáçova de<br>Santarém</td><td>Santarém</td><td>Portugal</td><td></td><td>Islamic period</td><td>1</td><td>27.2</td><td>27.2</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Davis, 2006)</td></tr><tr><td>Alcáçova de<br>Santarém</td><td>Santarém</td><td>Portugal</td><td></td><td>Medieval period</td><td><strong>2</strong></td><td><strong>27.2</strong></td><td><strong>36.6</strong></td><td><strong>31.7</strong></td><td><strong>4.5 14.2</strong></td><td></td><td></td></tr><tr><td>Els Mallols</td><td>Barcelona</td><td>Spain</td><td>Tarragonensis</td><td>Late Roman (3rd-4th cent. AD)</td><td>3</td><td>26.1</td><td>38.7</td><td>33.1</td><td>5.3</td><td>16.0</td><td>(Catagnano, 2016)</td></tr><tr><td>Odrinhas</td><td>Sintra</td><td>Portugal</td><td>Lusitania</td><td>Late Roman (3rd-4th cent. AD)</td><td>10</td><td>20.1</td><td>31.6</td><td>26.4</td><td>3.7</td><td>14.0</td><td>(Davis and Gonçalves, 2017)</td></tr><tr><td>Astorga</td><td>Léon</td><td>Spain</td><td>Tarragonensis</td><td>Late Roman (3rd-4th cent. AD)</td><td>19</td><td>23.7</td><td>39.5</td><td>29.6</td><td>4.8</td><td>16.2</td><td>(Fernandez-Rodriguez, 2000)</td></tr><tr><td>Vila Madrid</td><td>Madrid</td><td>Spain</td><td>Lusitania</td><td>Imperial Roman (1st-2nd cent. AD)</td><td>17</td><td>21.3</td><td>38.4</td><td>31.3</td><td>4.9</td><td>15.7</td><td>(Colominas, 2009)</td></tr><tr><td>Baetulo</td><td>Barcelona</td><td>Spain</td><td>Lusitania</td><td>Imperial Roman (1st-2nd cent. AD)</td><td>1</td><td>26.2</td><td>26.2</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Colominas, 2009)</td></tr><tr><td>Munigua</td><td>Sevilla</td><td>Spain</td><td>Baetica</td><td>Imperial Roman (1st-2nd cent. AD)</td><td>1</td><td>20.0</td><td>20.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Boessneck and von den Driesch, 1980)</td></tr><tr><td>Calle<br>Almendralejo</td><td>Mérida</td><td>Spain</td><td>Lusitania</td><td>Imperial Roman (1st-2nd cent. AD)</td><td>24</td><td>20.1</td><td>31.6</td><td>26.4</td><td>3.7</td><td>14.0</td><td>C. Detry and F.J. Heras, personal communication</td></tr><tr><td>Lugo</td><td>Galiza</td><td>Spain</td><td>Tarragonensis</td><td>Imperial Roman (1st-2nd cent. AD)</td><td>2</td><td>25.6</td><td>32.4</td><td>29.0</td><td>3.4</td><td>11.7</td><td>(Fernandez-Rodriguez, 2000)</td></tr><tr><td>Mesas de<br>Castelinho</td><td>Almodóvar</td><td>Portugal</td><td>Lusitania</td><td>Republican Roman (1st-2nd cent. BC)</td><td>1</td><td>22.7</td><td>22.7</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Valenzuela-Lamas and Fabião, 2012)</td></tr><tr><td>Cerro de la<br>Tortuga</td><td>Malaga</td><td>Spain</td><td></td><td>Roman period</td><td><strong>79</strong></td><td><strong>20.0</strong></td><td><strong>40.9</strong></td><td><strong>29.9</strong></td><td><strong>5.3 17.7</strong></td><td></td><td></td></tr><tr><td>Can Xercanvins</td><td>Barcelona</td><td>Spain</td><td></td><td>Iron Age</td><td>1</td><td>26.2</td><td>26.2</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(von den Driesch, 1973)</td></tr><tr><td>Mas Castellar</td><td>Gerona</td><td>Spain</td><td></td><td>Iron Age</td><td>1</td><td>34.1</td><td>34.1</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Catagnano, 2016)</td></tr><tr><td>Rua dos<br>Correeiros</td><td>Lisbon</td><td>Portugal</td><td></td><td>Iron Age</td><td>5</td><td>23.6</td><td>33.3</td><td>28.5</td><td>3.3</td><td>11.6</td><td>(Catagnano, 2016)</td></tr><tr><td></td><td></td><td></td><td></td><td>Iron Age</td><td>1</td><td>25.4</td><td>25.4</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Detry et al., 2016)</td></tr><tr><td>Cabezo Redondo</td><td>Alicante</td><td>Spain</td><td></td><td>Iron Age</td><td><strong>8</strong></td><td><strong>23.6</strong></td><td><strong>34.1</strong></td><td><strong>28.5</strong></td><td><strong>3.5 12.3</strong></td><td></td><td></td></tr><tr><td>Monachil</td><td>Granada</td><td>Spain</td><td></td><td>Bronze Age</td><td>7</td><td>24.3</td><td>30.6</td><td>27.2</td><td>1.9</td><td>7.0</td><td>(Boessneck et al., 1969)</td></tr><tr><td>Purullena</td><td>Granada</td><td>Spain</td><td></td><td>Bronze Age (Argaric)</td><td>4</td><td>26.5</td><td>31.7</td><td>28.6</td><td>2.0</td><td>7.0</td><td>(Lauk, 1976)</td></tr><tr><td>Fuente Álamo</td><td>Almeria</td><td>Spain</td><td></td><td>Bronze Age</td><td>10</td><td>22.0</td><td>31.0</td><td>26.6</td><td>2.9</td><td>10.9</td><td>(Lauk, 1976)</td></tr><tr><td>Terrena del Reloj</td><td>Granada</td><td>Spain</td><td></td><td>Bronze Age</td><td>7</td><td>26.0</td><td>31.0</td><td>27.9</td><td>1.7</td><td>6.1</td><td>(von den Driesch et al., 1985)</td></tr><tr><td>Muntanya<br>Assolada</td><td>València</td><td>Spain</td><td></td><td>Bronze Age (Argaric)</td><td>2</td><td>23.5</td><td>29.0</td><td>26.3</td><td>2.8</td><td>10.6</td><td>(Milz, 1986)</td></tr><tr><td>Azuer</td><td>Ciudad Real</td><td>Spain</td><td></td><td>Bronze Age</td><td>1</td><td>25.8</td><td>25.8</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(von den Driesch, 1973)</td></tr><tr><td>Can Roqueta</td><td>Sabadell</td><td>Spain</td><td></td><td>Bronze Age</td><td>1</td><td>33.5</td><td>33.5</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(Boessneck and von den Driesch, 1980)</td></tr><tr><td>Cerro de la<br>Encina</td><td>Granada</td><td>Spain</td><td></td><td>Bronze Age</td><td>9</td><td>25.0</td><td>33.0</td><td>28.3</td><td>0.0</td><td>0.0</td><td>(Albizuri, 2011)</td></tr><tr><td></td><td></td><td></td><td></td><td>Bronze Age</td><td>5</td><td>25.9</td><td>33.2</td><td>29.0</td><td>2.5</td><td>8.6</td><td>(Friesch, 1987)</td></tr><tr><td>Zambujal</td><td>Torres Novas</td><td>Portugal</td><td></td><td>Bronze Age</td><td><strong>46</strong></td><td><strong>22.0</strong></td><td><strong>33.5</strong></td><td><strong>27.8</strong></td><td><strong>2.6 9.4</strong></td><td></td><td></td></tr><tr><td>Leceia</td><td>Oeiras</td><td>Portugal</td><td></td><td>Chalcolithic</td><td>1</td><td>26.0</td><td>26.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>(von den Driesch and Boessneck, 1976)</td></tr><tr><td>Cerro de la<br>Virgen</td><td>Granada</td><td>Spain</td><td></td><td>Chalcolithic</td><td>3</td><td>26.0</td><td>29.0</td><td>27.5</td><td>1.2</td><td>4.4</td><td>(Pires et al., 2001)</td></tr><tr><td>Les Jovades</td><td>Alacant</td><td>Spain</td><td></td><td>Chalcolithic</td><td>6</td><td>24.8</td><td>30.2</td><td>27.6</td><td>1.6</td><td>5.8</td><td>(von den Driesch, 1972)</td></tr><tr><td>Marizulo</td><td>Guipuzcoa</td><td>Spain</td><td></td><td>Chalcolithic</td><td>1</td><td>25.8</td><td>25.8</td><td>25.8</td><td>0.0</td><td>0.0</td><td>(Sanchis and Sarrión, 2004)</td></tr><tr><td>Camino del<br>Molino</td><td>Murcia</td><td>Spain</td><td></td><td>Chalcolithic</td><td>1</td><td>33.8</td><td>33.8</td><td>33.8</td><td>0.0</td><td>0.0</td><td>(Altuna, 1967)</td></tr><tr><td>Camino de las<br>Yeseras</td><td>Madrid</td><td>Spain</td><td></td><td>Chalcolithic</td><td>40</td><td>24.4</td><td>38.9</td><td>28.2</td><td>3.0</td><td>10.6</td><td>(Catagnano, 2016)</td></tr><tr><td></td><td></td><td></td><td></td><td>Chalcolithic</td><td>3</td><td>28.5</td><td>31.5</td><td>30.0</td><td>1.2</td><td>4.0</td><td>(Daza, 2015)</td></tr><tr><td>Muge</td><td>Salvaterra de<br>Magos</td><td>Portugal</td><td></td><td>Chalcolithic</td><td><strong>55</strong></td><td><strong>24.4</strong></td><td><strong>38.9</strong></td><td><strong>28.2</strong></td><td><strong>2.8 9.9</strong></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>Mesolithic</td><td><strong>1</strong></td><td><strong>23.5</strong></td><td><strong>23.5</strong></td><td><strong>23.5</strong></td><td><strong>0.0 0.0</strong></td><td></td><td></td></tr></tbody></table>

to construct a Median-Joining (MJ) network (Bandelt et al., 1999). Nucleotide (nt) substitutions weighted 10 for transitions and 30 for transversions so that the component with the lowest expected mutation rate was assigned the highest weight (Bandelt et al.,

2000). The identification of dog clades was undertaken through comparison with reference sequence data of extant Iberian dogs and wolves trimmed to the 181 base pairs fragment.

Identification of dog clades was done by comparison with
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** **A)** Location of the archaeological sites from where the 15 dog remains used in the genetic analysis were recovered. The sampling was carried out in the western-most range of the Roman Empire, namely (from south to north): **1** - Thamusida, Morocco (*Mauretania Tingitana*); **2** - Monte Molião, Portugal (*Lusitania*); **3** - Lugo, Spain (*Tarragonensis*). Shaded in black is the maximum extent of the Roman empire. **B)** Sampled materials from Lugo and Monte Molião. Note: The sample from Thamusida was fragmented after sub-sampling for DNA analysis and no picture was taken. Photos by Carlos Fernandez-Rodriguez (material from Lugo) and José Paulo Ruas (material from Monte Molião).</figcaption></figure>

<table><caption>**Table 3**
Primer sequences for mtDNA-control region amplification in *Canis*.</caption><thead><tr><td>Primer Name</td><td>Primer Sequence 5' – 3'</td></tr></thead><tbody><tr><td>DL1F forward</td><td>GTG CTA TGT CAG TAT CTC CAG G</td></tr><tr><td>DL3R reverse</td><td>CCC TTA TTG GAC TAA GTG ATA TGC AT</td></tr><tr><td>DL7F forward</td><td>TAT TAT ATC CTT ACA TAG GAC</td></tr><tr><td>DL2R reverse</td><td>GCA AGG GTT GAT GGT TTC TCG</td></tr></tbody></table>

reference sequence data on extant dogs from Iberia and North Africa, and comprising breed and village dogs from 1) Pires et al. (2006), 49 haplotypes, 887 base pair long with GenBank accession numbers AY706476–AY706524 (Pires et al., 2006); and 2) Pang et al. (2009), haplotypes A4, A5, A10, A11, A19, A20, A22, A33, A34, A127, B1, D6, D7 (Pang et al., 2009).

An alignment of all these sequences/haplotypes is available from Partial_MtDNA-SequeneAlignment_DOG_Roman_QInt (fasta).

Short mtDNA fragments may not exactly reflect the phylogeny as determined by complete mitogenomes. We thus constructed a Bayesian phylogenetic tree to confirm haplogroup assignment of

the partial mtDNA sequences obtained in this study by comparison with well-defined dog matrilines from GenBank. The software MrBayes (Huelsenbeck et al., 2001; Ronquist and Huelsenbeck, 2003) was used with the evolutionary model GTR + I + gamma. The shape parameter of the gamma distribution was 0.7424 and the proportion of invariable sites (I) was 0.6851. We used 10,000,000 generations, sampling every 1,000th generation, and default settings for the remaining options. Convergence of the Monte Carlo Markov Chain and burn-in were determined through the analysis of the generations versus the log probability plot using the trace analysis tool TRACER v1.6. (Rambaut et al., 2014). The initial burn-in step discarded 20% of the samples. This allowed for haplogroup assignment of all our Roman dog partial mtDNA sequences with high confidence.

## 4. Results

### 4.1. Zooarchaeological analyses

Tables 1 and 2 and the associated histograms (Figs. 3 and 4)
<caption>**Table 4**</caption>

Details concerning the 15 Roman dog remains examined at the molecular level in this study. PT stands for Portugal, ES for Spain and MA for Morocco. The molecular analyses were performed in two independent laboratories: AFL in Stockholm, Sweden and ENS in Lyon, France.

<table><thead><tr><th>Sample</th><th>Archaeological site, Country</th><th>Chronology</th><th>Skeletal element recovered</th><th>aDNA Laboratory</th><th>mtDNA sequence (base pairs)</th><th>454 sequencing coverage</th><th>GenBank Accession number</th><th>mtDNA Haplogroup (Hg)</th></tr></thead><tbody><tr><td>SEP013</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>tibia</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>559</td><td>KY014684</td><td>A</td></tr><tr><td>SEP014</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>humerus</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>155</td><td>KY014685</td><td>A</td></tr><tr><td>SEP015</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>radius</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>42</td><td>KY014686</td><td>A</td></tr><tr><td>SEP016</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>scapula</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>83</td><td>KY014687</td><td>A</td></tr><tr><td>SEP017</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>pelvis</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>48</td><td>KY014688</td><td>A</td></tr><tr><td>SEP018</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>humerus</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>29</td><td>KY014689</td><td>A</td></tr><tr><td>SEP019</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>humerus</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>50</td><td>KY014690</td><td>A</td></tr><tr><td>SEP020</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>radius</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>38</td><td>KY014691</td><td>D</td></tr><tr><td>SEP021</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>tibia</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>135</td><td>KY014692</td><td>A</td></tr><tr><td>SEP022</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>femur</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>126</td><td>KY014693</td><td>D</td></tr><tr><td>SEP023</td><td>Lugo, ES</td><td>3rd - 4th cent. AD</td><td>humerus</td><td>AFL, Stockholm University, Sweden</td><td>139</td><td>23</td><td>KY014694</td><td>D</td></tr><tr><td>LYEP57</td><td>Thamusida, MA</td><td>1st cent. AD</td><td>tooth - lower third pre-molar</td><td>ENS-Lyon, France</td><td>109</td><td>225</td><td>KY464999</td><td>A</td></tr><tr><td>LYEP59</td><td>Monte Molião, PT</td><td>1st cent. BC</td><td>proximal femur</td><td>ENS-Lyon, France</td><td>181</td><td>746</td><td>KY014672</td><td>A</td></tr><tr><td>LYEP60</td><td>Monte Molião, PT</td><td>1st cent. AD</td><td>mandible fragment</td><td>ENS-Lyon, France</td><td>166</td><td>110</td><td>KY014673</td><td>A</td></tr><tr><td>LYEP61</td><td>Monte Molião, PT</td><td>1st cent. BC</td><td>proximal femur</td><td>ENS-Lyon</td><td>181</td><td>479</td><td>KY014674</td><td>A</td></tr></tbody></table>

summarize information for each archaeological site and its period. Data from the Roman and Late Roman periods—eight archaeological sites for the M₁ length measurements and nine for the humerus distal breadth—confirm the wide variability in size for dogs at that time.

Among the 15 *Canis* specimens from the Roman period selected for aDNA analyses, only three were possible to measure and their measurements fall within the range of M₁ length (one sample, Monte Molião) and humerus distal breadth (two samples, Lugo) for dogs rather than for wolves. Besides this metrical criterion, all archaeological information from where these canid elements were found corresponds to domestic contexts, thus reinforcing their identification as the domestic subspecies.

The results suggest that Chalcolithic and Bronze Age dogs are significantly more uniform than Roman and present-day ones. The low number of measurements for the Iron Age and Modern times prevents us from knowing whether these dog populations were either diverse or homogeneous.

As for cranial variability, the CV calculated for the Roman period (eight distinct archaeological sites) indicates that M₁ length variability in Roman time is larger (CV = 10.8%) compared to other periods (1–7.4%), the same as in Iron Age (CV = 10.8%), but lower when compared to extant dogs (CV = 15.5%) (Table 1). The humerus distal breadth measurements show similar results: the CV calculated from the combined values of nine different archaeological sites show its highest value in the Roman period (CV = 17.7%) followed by extant dogs (CV = 26.3%). For this measurement, the CV value for the Iron Age is much lower (CV = 12.3%).

The stacked histograms (Figs. 3 and 4) also show that the amplitude of values seems higher in the Roman period with smaller dogs observed more frequently.

The results of the Siegel-Tukey test (for assemblages with more than 5 samples) suggest that molar size has greater variability than humerus breadth in different chronological periods (Table 5): 9

comparisons found no significant differences in the diversity of values of humerus breadth, and only one (Chalcolithic vs. Roman) had highly significant differences (p < 0.01), compared to 5 comparisons with high significant differences (p < 0.01) and 7 comparisons with no significant differences on the first molar. Interestingly, the length of M₁ is more homogeneous in Chalcolithic dogs than in any other periods.

## 4.2. Genetic analyses

We were able to recover DNA from a total of 15 *Canis* remains, as follows: Thamusida, Morocco (*Mauretania Tingitana*), n = 1; Monte Molião, Portugal (*Lusitania*), n = 3, and Lugo, Spain (*Tarraconensis*), n = 11. None of the controls showed any signs of contamination. Table 4 summarizes the samples analysed for ancient DNA.

Mitochondrial DNA data comprised sequences of 109–181 nucleotides and a total of 17 variable nucleotide sites. We observed five dog mitochondrial haplotypes and detected a single new variant, in Portugal, after comparison with data for extant dogs. These ancient haplotypes could be grouped into two of the major mtDNA lineages observed in extant dogs, i.e., clades A and D (Fig. 5).

More precisely, in Roman times, we detected dog remains carrying haplotypes that belong to mtDNA clades A (n = 12: Portugal 3, Spain 8 and Morocco 1) and D (n = 3, Spain) (Fig. 6). Interestingly, the older samples from Portugal, which are not contemporaneous with neither African nor Spanish samples, show two different haplotypes within clade A. One sequence (from Monte Molião Portugal, LYEP60) is considered a singleton and is not represented in extant dogs. So far, neither clade C nor B haplotypes were detected in the Roman dogs studied (Fig. 6). In Lugo, northwest Spain, we detect the highest number of haplotypes. These haplotypes differed by up to 7 nucleotides. Evidence for the presence of clade D in the Iberian Peninsula first appears in archaeological dog remains from Roman Spain.
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Stacked histograms with measurements for the length of the lower first molar (mm) of dogs and wolves from the Iberian Peninsula. A diachronic view from several archaeological sites in Iberia and North Africa are compared with measurements from reference skeletal collections (20th century). For more details about these sites and samples see Table 1. [Chalcolithic – 5,000–4,000 years cal BP; Neolithic – 7,500–5,000 years cal BP; Mesolithic – 8,000–7,500 years cal BP].</figcaption></figure>

**5. Discussion**

Teeth and bones generally preserve well in archaeological sites and can provide information about ancient animals using osteometrical and genetic analyses. The first lower molar generally provides a distinction between dogs and wolves (Tchernov and Valla, 1997; Detry and Cardoso, 2010), and it is a useful element also to assess the degree of variability in size and shape of canid populations.

The lengths of the lower molars (M₁) and the breadths of long bones of the *Canis* remains analysed in this study clearly show the wide variability of dog morphotypes in the Roman period, which include small sized “dwarf dogs”. The variability attested in Roman times is not observed in earlier periods, perhaps with the exception of the Iron Age. Only present-day dogs have a larger variability of sizes and shapes than the Roman ones.

In many organisms, the coefficient of variation (CV) for different traits in natural populations lies between 4 and 10% (Simpson et al., 1960). For the Roman period as well as for Iron Age and extant dogs, the CV is greater than 10%, as shown in the CV of M₁ length and distal humerus breadth (Tables 1 and 2). This is compatible with a high level of phenotypic variability, as a result of a strong artificial selection for these phenotypes indicating that human-directed selection pressure and consequent morphological changes were well established in Roman dogs. Such variability was expected since it has been observed in different areas of the Roman empire, namely in England (Bennett and Robert, 2016), North Africa (MacKinnon, 2010) and Iberia (Boessneck and von den Driesch, 1980; Altuna and Mariezkurrena, 1992; Colominas, 2015). A reduction of the CV after the Roman period also appears to be evident. This can be related to the absence of extreme dogs that only appear again in the 20th century due to artificial selection mainly for aesthetical purposes.

Regarding the genetic analysis, the site of Lugo provided most of the samples analysed (n = 11). Thus, in this site, higher mtDNA diversity was observed. Other than the number of samples, the fact that they date from Late Roman times may also have had an influence. To our knowledge, this is the first time that the presence of clade D haplotypes is attested among historic dogs from Iberia. This maternal lineage possibly originates in the Near-East, and has been detected in South-Eastern and Central Europe as early as the Neolithic period (Pionnier-Capitan, 2010). In Iberia, the detection of the first dog D-haplotypes occurs much later. The dog remains from Lugo are dated to Late Roman times—3rd-4th century AD (ca. 1,600–1,700 years ago)—but this haplotype could have come to the Iberian Peninsula any time after the Neolithic, given the continuous human contacts along the Mediterranean since then. This includes early contacts (Davis and Simões, 2016; Isern et al., 2017) as well as the more intense Mediterranean trade in the Iron Age, significantly Phoenicians. This is supported by the presence of numerous colonies and imports along the Southern coast of the Iberian Peninsula and North African (Aranegui, 2016; Aubet, 1987; Ponsich, 1968). First contacts between Iberia and North Africa date of much earlier, at least since the Early Iberian Neolithic (Martínez-Sánchez et al., 2017), probably with a diffusion of farming across the Straits of Gibraltar following a North-to-South direction (from Iberia to North Africa) (Zilhao, 2014). A more comprehensive sampling may reveal an earlier presence of clade D (and eventually other clades) in the Iberian Peninsula and North Africa.

The presence of dogs carrying more distinct mtDNA lineages in Lugo may also be explained by the fact that it was an administratively and politically very important Roman city (Rodríguez Colmenero, 2011). Considering the sites studied here, Lugo was probably the one with more contacts with other parts of the Roman
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Stacked histograms with measurements for the humerus distal breadth (mm) of dogs and wolves from the Iberian Peninsula. A diachronic view from several archaeological sites in Iberia and North Africa are compared with measurements from reference skeletal collections (20th century). For more details about these sites and samples see Table 2. [Chalcolithic – 5,000–4,000 years cal BP; Neolithic – 7,500–5,000 years cal BP; Mesolithic – 8,000–7,500 years cal BP].</figcaption></figure>
<caption>**Table 5**</caption>

Results of the Siegel-Tukey test (p-values) on the humerus distal breadth (HU Bd) and length of the first lower molar (M1 L) and per assemblage/period. Above diagonal, n.s. stands for non-significant (*p*>0.05), * = *p*<0.05, ** = *p*<0.01, *** = *p*<0.001.

<table><thead><tr><th>HU Bd</th><th>Chalcolithic</th><th>Bronze</th><th>Iron</th><th>Roman</th><th>Modern</th><th>Present-day</th><th>Number of measurements</th></tr></thead><tbody><tr><td>Chalcolithic</td><td></td><td>n.s.</td><td>n.s.</td><td>**</td><td>n.s.</td><td>*</td><td>55</td></tr><tr><td>Bronze</td><td>0.9375</td><td></td><td>n.s.</td><td>*</td><td>n.s.</td><td>*</td><td>46</td></tr><tr><td>Iron</td><td>0.2876</td><td>0.3804</td><td></td><td>n.s.</td><td>n.s.</td><td>*</td><td>8</td></tr><tr><td>Roman</td><td>0.005013</td><td>0.02842</td><td>0.3702</td><td></td><td>n.s.</td><td>*</td><td>79</td></tr><tr><td>Modern</td><td>0.1067</td><td>0.1327</td><td>0.5966</td><td>0.7203</td><td></td><td>n.s.</td><td>9</td></tr><tr><td>Present-day</td><td>0.01566</td><td>0.04019</td><td>0.03377</td><td>0.029</td><td>0.2261</td><td></td><td>25</td></tr><tr><th>M1 L</th><th>Chalcolithic</th><th>Bronze</th><th>Iron</th><th>Roman</th><th>Modern</th><th>Present-day</th><th>Number of measurements</th></tr><tr><td>Chalcolithic</td><td></td><td>n.s.</td><td>**</td><td>**</td><td>**</td><td>**</td><td>83</td></tr><tr><td>Bronze</td><td>0.2019</td><td></td><td>*</td><td>n.s.</td><td>n.s.</td><td>**</td><td>41</td></tr><tr><td>Iron</td><td>0.001734</td><td>0.03958</td><td></td><td>n.s.</td><td>n.s.</td><td>n.s.</td><td>22</td></tr><tr><td>Roman</td><td>0.001105</td><td>0.3372</td><td>0.1661</td><td></td><td>n.s.</td><td>*</td><td>87</td></tr><tr><td>Modern</td><td>0.00929</td><td>0.221</td><td>0.4914</td><td>0.7114</td><td></td><td>*</td><td>11</td></tr><tr><td>Present-day</td><td>0.009162</td><td>0.003261</td><td>0.09402</td><td>0.02602</td><td>0.03784</td><td></td><td>29</td></tr></tbody></table>

Empire. Additionally, the detection of the less frequent clade D may echo the consolidation of distinct dog types, as we describe herein for this period. Our results show the presence and breeding of dogs of distinct mtDNA haplogroups, A and D, in Iberia continuously for over 1,600 years.

Samples from Monte Moliao, dated to the 1st cent. BC - 1st cent. AD (ca. 2,000 years ago), also reveal interesting aspects of the genetic composition of dog populations in the past. The two mtDNA haplotypes genotyped are not shared with the other dog samples analysed, and one of them consists of a new variant not found in extant dogs. This suggests some level of mtDNA diversity in dogs at this site and that some dog-lineages may have been lost through time. However, we cannot completely discard the possibility that the nucleotide differences found between these single haplotypes and the others results from some level of DNA degradation. In particular, the Monte Moliao LYEP60 singleton (a mtDNA sequence found only in one individual), is defined by a nucleotide position that exhibits a thymine (T) while all others exhibit a cytosine (C), which could be a result of damage. In this case, thymine was the prevalent nucleotide in 79 out of the 110 reads for this sample.

The mtDNA sequence of the African dog specimen belongs to clade A, which is nowadays the most common lineage found in dogs worldwide, including North Africa (Boyko et al., 2009) and Iberia (Pires et al., 2006). Here we contribute only one sample from Thamusida: interestingly, it shared a clade A haplotype with three other samples from Lugo. This result may indicate that either this was a common lineage in each of these regions or the existence of gene flow between dogs from these regions. Dogs could have been easily transported across the Mediterranean by humans following maritime trading routes. There is considerable evidence for maritime trade between North Africa and North Western Iberia in Roman times (e.g. Naveiro López, 1991), that became even more consolidated in the Late Roman period (e.g. Fernández Fernández, 2014). However, the terrestrial routes could also have been relevant, namely through via de la Plata (linking Augusta Emerita – Mérida with Asturica Augusta – Astorga) and via XIX (linking Bracara Augusta – Braga with Asturica Augusta through Lucus Augusti (Lugo)) (De Soto, 2013).

Despite its geographic proximity, the Thamusida sample does not reflect evidence for gene flow between dogs from this region and Monte Moliao (no haplotype sharing). This is consistent with other archaeological materials (e.g. pottery), that suggest that, during the Republican and the Imperial Roman periods, imports at Monte Moliao from North Africa mainly originated in the

province of Africa proconsularis (present-day Tunisia) (Arruda, 2007; Arruda and Sousa, 2013). Nevertheless, this may also be related to the limited sampling in both sites and possible genetic drift.

Fig. 1, as well as data from Bennett and Robert (2016), show that Roman iconography reflects a large amount of morphological variability in dogs. The Roman mosaics from Conímbriga, a very important city in Roman Lusitania (Portugal), show several dog coat colours and even different shapes of the cranium (Fig. 1). These are probably accurate representations of Roman dogs, although we cannot be certain about their geographical origin. The artists that produced these mosaics could be itinerant specialists inspired by local or foreign models. In Conímbriga there are evidences that the artisans are local and the mosaics with the dogs are described as being depictions of the servants of the owner of the house (Correia, 2013). Additionally, various shapes of dogs are represented in ceramic decorations of the sigillata type found in the Iberian Peninsula (Bennett and Robert, 2016; Bustamante and Detry, 2017). However, taken together, iconographic, zooarchaeological and ancient DNA data, all coincide in reflecting the existence of different dog sizes, shapes and origins in Roman times.

## 6. Conclusion

This study confirms the utility of combining morphological, palaeogenetic and archaeological data to describe past phenotypic and genetic variability of dogs. We detected the presence of various mtDNA lineages during Roman times in Iberia and North Africa, which is in agreement with the documented high morphological diversity of dogs in the iconographic evidence—pottery and mosaics—from these areas. The zooarchaeological study also revealed that the diversity of morphologies was greater in Roman times compared to any other period, with the exception of present-day dogs. This suggests that artificial selection for morphologic traits was well established in Roman times and included lap dogs.

While mtDNA analysis is informative to investigate genetic affinities and demographic changes in domestic animal populations, autosomal DNA (i.e., markers linked to phenotypic traits) is needed to recover signatures of post-domestication changes in inheritable traits due to human selection. Subsequent genetic analysis of the dog specimens described in this study using such markers will allow us to describe some phenotypic traits of Roman dogs for a more comprehensive interpretation of their variability.
<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Bayesian phylogenetic tree used for haplogroup assignment of the 109–181 bp mitochondrial DNA sequences obtained in this study by comparison with well-defined dog matrilines from GenBank. Sequences from ancient Iberian *Canis* remains are depicted in bold. Extant dog data from GenBank include the following: 54 sequences trimmed from mitogenome data (Duleba et al., 2015), haplogroup/sub-haplogroup nomenclature precedes accession number information; 49 sequences from modern Iberian native breeds and Iberian village dogs (Pires et al., 2006), with haplogroup letters, accession numbers and haplotype numbers shown; 4 Portuguese, 10 Spanish and 14 North African dogs (Pang et al., 2009), with haplogroup letters and haplotype numbers also indicated. Major haplogroups are coloured as follows: A-orange; B-yellow; C-blue; and D-green. Bootstrap values are indicated for each node. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Fig. 6.** Median-joining network of mtDNA sequences. Clades A, B, C and D represent the major haplogroups found in extant dogs as defined by mitogenomes in Duleba et al. (2015). Five distinct ancient haplotypes were recovered from Roman dog samples (black) and which clustered with haplotypes A and D of extant dogs (gray). ES refers to haplotypes found in Roman Spain, MA in Morocco and PT in Portugal. The single white dot represents a median vector generated by the software to indicate an expected but unsampled haplotype.</figcaption></figure>

## Conflicts of interest

The authors declare no conflicts of interest.

## Acknowledgments

The authors wish to thank: Virgílio Hipólito Correia of the Conímbriga Museum for photographs and information regarding the Roman mosaics; David Gonçalves for help with the map of the Roman Empire and depiction of the archaeological sites; Javier Heras for zooarchaeological data from Mérida – Calle Almen-dralejo; to Simon Davis for a critical review of the manuscript and also for revising the English language, and to two anonymous re-viewers whose comments contributed greatly to the quality of this manuscript. This study was funded by the FCT (Fundação para a Ciência e a Tecnologia) within the research PTDC/HIS-ARQ/100225/ 2008; and AE Pires grant SFRH/BPD/112653/2015; C Detry grant SFRH/BPD/108326/2015 and C Ginja contract grant IF/00866/2014.

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