Microscopic and ancient DNA profiling of Polynesian dog (kurī) coprolites from northern New Zealand

Journal of Archaeological Science: Reports 6 (2016) 496–505

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Microscopic and ancient DNA profiling of Polynesian dog (kurī) coprolites from northern New Zealand Jamie R. Wood a,⁎, Andrea Crown b, Theresa L. Cole a,d, Janet M. Wilmshurst a,c a

Long-term Ecology Lab, Landcare Research, PO Box 69040, Lincoln 7640, New Zealand Geometria, PO Box 68653, Newton, 1045 Auckland, New Zealand School of Environment, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand d Department of Zoology, University of Otago, PO Box 56, Dunedin 9054, New Zealand b c

a r t i c l e

i n f o

Article history: Received 25 November 2015 Received in revised form 17 February 2016 Accepted 17 March 2016 Available online xxxx Keywords: Ancient DNA Commensal species Coprolites Cultivars Diet Māori Pollen

a b s t r a c t Dogs (known to Māori as kūri) were first brought to New Zealand by Polynesian settlers ~750 years ago, and served both important functional and cultural roles. Purebred kūri became extinct soon after European settlement, by cross-breeding with dogs of European origin. Thus, knowledge about kūri relies heavily upon interpretation of their archaeological remains. The diet of kūri has previously been inferred from coprolite analyses, yet some uncertainty remains as to whether these coprolites were from dog or human origins. Here, we provide the first study of coprolites from New Zealand archaeological contexts to combine both microscopic and ancient DNA analyses. We confirm the depositor of the coprolites was kūri, and reveal a diet comprised dominantly of marine (fish, shellfish) components and plant matter. Plants identified from the coprolites included both native (e.g. Macropiper, Carex, Juncus, Potamogeton) and cultivated commensal (e.g. Lagenaria siceraria, Broussonetia papyrifera) taxa. The results suggest that kūri diets overlapped with typical early Māori diets in the same region. Our study has demonstrated the potential for combined microscopic and ancient DNA analysis of coprolites to provide deeper insights into the biology, behaviour and husbandary of kūri in prehistoric New Zealand. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Dogs (Canis lupus familiaris) were one of several commensal species transported to islands throughout East Polynesia by the first human settlers approximately 1000–750 years ago (Anderson and Clark, 2001; Wilmshurst et al., 2011; Greig et al., 2015). In New Zealand, dogs (known to Māori as kurī) were an important resource for the early settlers. Kurī bones are common in pre-European archaeological sites across New Zealand, including the subantarctic Auckland Islands (Anderson, 2005), and the animals held both important functional (e.g. meat for consumption, skins for cloaks) and cultural roles (Clark, 1997). During the mid-late 19th Century there was widespread interbreeding between kurī and dogs of European origin, and roaming packs of feral dogs of mixed bloodlines became frequently encountered throughout New Zealand (White, 1889). With the subsequent extermination of these packs the last bloodlines of kurī likely also became extinct (Anderson and Clark, 2001). The absence of kurī bones from natural fossil deposits (e.g. caves and tomos) suggests that purebred (i.e. pre-European) kurī were never feral, but remained in close association with people (Anderson, 1981;

⁎ Corresponding author. E-mail address: [email protected] (J.R. Wood).

http://dx.doi.org/10.1016/j.jasrep.2016.03.020 2352-409X/© 2016 Elsevier Ltd. All rights reserved.

Worthy and Holdaway, 2002). However, there are few documented accounts of kurī biology, behaviour and husbandary, and so such details must largely be derived from their archaeological remains. These remains are mainly in the form of bones, but a number of archaeological sites throughout New Zealand have also yielded coprolites inferred to have been deposited by kurī (Clark, 1995, 1997; Horrocks et al., 2003). Both macroscopic and microscopic examinations of these coprolites have provided some details of kurī diets, foraging and parasitology (Clark, 1995, 1997; Horrocks et al., 2003; Irwin et al., 2004), although in general there has been relatively little research performed on this resource. Some of the coprolites recovered from New Zealand archaeological sites are clearly attributable to carnivores, on the basis that they contain large fragments of chewed bone. While kurī were the only large terrestrial mammalian carnivore in New Zealand prior to European arrival, in coastal sites the New Zealand sea lion (Phocarctos hookeri) may also represent a potential depositor. Yet for many coprolites without obvious bone fragments, uncertainty remains as to whether they were deposited by dogs or humans, because of a large degree of potential overlap in their diets (e.g. Horrocks et al., 2002, 2003). Assuming the identity of a depositing species from the content of a coprolite, however, can create circular interpretations of diet, and can be problematic when species, such as dog, may have had meat- or plant-dominated diets at different times or places (Clark, 1997). Ancient DNA analysis has

J.R. Wood et al. / Journal of Archaeological Science: Reports 6 (2016) 496–505

become a widely used tool for studying coprolites, and in addition to providing insights into diets can provide a means to identify the depositing species (e.g. Bon et al., 2012; Wood et al., 2012, 2013). The additive benefits of combining ancient DNA with traditional palaeoecological techniques, such as pollen analysis, are now widely appreciated (Wood et al. 2012; Pedersen et al., 2013; Wilmshurst et al., 2014; Parducci et al., 2015; Birks and Birks, 2016). Here, we provide the first combination of microscopic and ancient DNA analyses on coprolites collected from an archaeological context in New Zealand. The study confirms the identity of these coprolites as being from kurī, and provides further insights into the diets and husbandry practices of dogs in prehistoric New Zealand. 2. Materials and methods 2.1. Study site and coprolites The coprolites examined in this study are from the Masonic Tavern archaeological site, located on the shoreline of Torpedo Bay in north Auckland, North Island, New Zealand (Fig. 1). The Masonic Tavern archaeological site includes several phases of human occupation (from prehistoric and early historic [mid 19th century] through to the present day). Archaeological excavation of the carpark area adjacent to the Masonic Tavern (encompassing sites: R11/2517 [Masonic Tavern and Boarding House], R11/2518 [cottage moved in 1866], R11/2519 [first cottage on the site], and R11/2404 [pre-European Māori burial]), were performed from May–Aug 2013 (Stage 1), and Nov–Dec 2013 (Stage 2). The remains of two 19th century dwellings, remnants of old associated outbuildings, rubbish pits, historic services (drains, pipes etc.), and evidence of an earlier cottage were discovered under the tavern carpark during the course of Stage 1 archaeological excavations. Pre-European Māori occupation layers, stratigraphically below the historic layers, were also excavated, and centred around two specific areas: the foredune at the front of the site containing several human burials and a slightly depressed, artefact rich swale behind the foredune - immediately west of the tavern building itself. The cultural “cap” of charcoal and artefact dense layers behind the foredune comprised two distinct activity areas: 1) a concentration of imported stone and obsidian flakes with stone tools and fishhooks, and 2) a semicircular area with fire features and rake-out surrounding a large, central, stone-lined hearth. Obsidian, greywacke, chert, worked bone, shell, faunal material and human bone were found scattered across this part of the site. Stage 2 excavations beneath the boarding house area revealed numerous pre-European fire features, lithic working areas, nondescript pit features and large quantities of faunal material. Artefactual and faunal analyses from the site are

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still ongoing, and a full description of the site and materials contained within it is still in progress (Crown, 2013, 2014). Briefly, the faunal assemblage from the Stage 1 excavations contains a large proportion of dog (at least 2529 bones of 289 individuals, representing 16.05% of the faunal assemblage). Fish dominate, representing 56.39% of the assemblage. Australasian snapper (Pagrus auratus) was the dominant fish species, with bones from at least 320 individuals identified (however this is likely to be significant under-estimate of its true abundance, as we assume that much of the still unidentified fish bones from Stage 1 are also snapper). A significant number of coprolites were excavated from the tavern carpark and boarding house area. These were distributed across the sandy foredune, the swale area, and the area beneath the relocated boarding house (Fig. 2). The largest concentration of coprolite samples was collected from the swale area in the centre of the site – a zone dominated by numerous fire features, lithic debris and an abundance of faunal material (including fish, dog, rat, and bird bones), which was scattered across the occupation floor. Five of these coprolites were selected for analysis (Table 1; Fig. 3). 2.2. Subsampling To reduce the chance of detecting post-depositional contamination of the coprolites in our analyses we subsampled the specimens according to a strict protocol. The subsampling procedure was performed in a clean still-air perspex box in a laboratory where DNA extractions and amplifications are not performed, following the protocol of Wood & Wilmshurst, (2016). The interior of the box was cleaned between samples using bleach, ethanol and UV irradiation. External surfaces of the coprolites (to a depth of ~3 mm) were removed by scraping with a sterile scalpel blade, and freshly exposed surfaces were UV irradiated for 20 min. The coprolites were then bisected, and subsamples from the interior-most portions were taken for microscopic and ancient DNA analyses. Of the remaining coprolite, half was used for analysis of macroremains and the other half was retained as a voucher sample for future study (held at the Long-term Ecology Lab, Landcare Research, Lincoln, New Zealand, site code X14/4). 2.3. Microscopic analyses Subsamples from each coprolite were prepared for microscopic analysis by heating in potassium hydroxide for 10 min, followed by treatment with hydrochloric acid, heavy-liquid flotation (lithium polytungstate at a specific gravity of 2.2). The float was split into two fractions. The first fraction was directly mounted onto a microscope slides for analysis of parasite eggs. The second was acetolysed, stained

Fig. 1. Location of Masonic Tavern site, Auckland, North Island, New Zealand.

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Fig. 2. Distribution of coprolite samples (numbered black filled circles) across the Stage 1 and Stage 2 excavation areas, Masonic Tavern site, Auckland, New Zealand. Stage 2 area is shown in the red box at the top right of the map.

with fuchsin, and mounted on a microscope slide for analysis of the coprolite pollen content. Slides were scanned systematically under a binocular microscope at 400× magnification. The subsamples for analysis of macroremains were rehydrated in distilled water for three weeks, and placed into a petri dish where they were gently disaggregated using a fine metal probe. The disaggregated material was examined under a dissecting microscope at 10–60× magnification before being air dried and stored in small plastic vials.

2.4. Ancient DNA analyses DNA extractions and PCR setups were performed in a physically isolated, purpose-built ancient DNA facility at Landcare Research, Lincoln, New Zealand. Subsamples of each coprolite (0.033–0.044 g) were placed in 1.5 ml tubes and digested by rotating at 55 °C for 24 h in a buffer containing 945 μl of 0.5 M EDTA (pH 8), 20 μl of 10% SDS, and

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Table 1 Descriptions and contexts of prehistoric dog (kurī) coprolites analysed from the Masonic Tavern site, Auckland, New Zealand. Coprolite DNA lab code

Length (mm)

Width (mm)

Physical description

Archaeological context

AD29

34.2

18.5

Noticeably damper than other samples, dark brown exterior but broken surfaces yellow-brown. Large charcoal fragment visible. Fig. 3c

AD30

48.9

16.7

AD31

43.7

20.1

Complete, cylindrical, rounded ends. Yellow-brown with organic sand covering external surface. Fig. 3a Broken cylindrical, pointed end. Bone visible on broken surfaces. Fig. 3f

AD33

26.9

21.9

AD34

39.5

26.9

Excavated 22 Jul 2013 (sample 2574); unit/feature 168; Layer III; from level III of feature 168, 23–28 cm below surface Excavated 22 Jul 2013 (sample 2807); unit/feature 169; from over the torso of burial #3 Excavated 5 Aug 2013 (sample 2794); unit/feature 169; from burial #3 Excavated 12 Jul 2013 (sample 2806); unit/feature F94; from grave R11 B2 Excavated 18 Jul 2013 (sample 2520); unit/feature 163; layer trench 9–11 m

Oval, end broken to show white objects (bone fragments?) in cross section. Yellow-brown. Fig. 3b Cylindrical, broken. Rounded at complete end. Light brown-grey. Large charcoal fragment visible. Figs. 3d and 3e

35 μl of 20 mg/mL Proteinase-K. The tubes were centrifuged for 5 min at 10,000 rpm to pellet any undigested material, and DNA was extracted from the supernatant using the Dneasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). PCR reactions contained 10 μl of 5 PRIME MasterMix (5 PRIME, Inc., Gaithersburg, MD, USA), 0.5 μl of each primer mix (10 mM starting concentration), 2 ng of DNA template and 12 μl of H2O. Eukaryote 18S rDNA and plant chloroplast trnL were amplified using fusion primers incorporating the locus-specific primers Euk1391f and EukBr (Caporaso et al., 2012) and trnLc and trnLh (Taberlet et al., 2007). To ensure sequence complexity in the amplified libraries, four versions of each fusion primer were pooled (containing 0, 1, 2 and 3 degenerate (N) nucleotides prior to the linker sequences). Cycling conditions were as follows: 94 °C for 3 min, followed by 42–50 cycles of 94 °C for 30 s, 57 °C for 60 s and 72 °C for 90 s, with a final extension step of 72 °C for 10 min. A blank extraction control was included to detect sequences originating from potential reagent contamination. PCR products were purified using the right-side size selection (0.7 ×) protocol of the SPRI-select magnetic bead system (Beckman Coulter, CA, USA). Length profiles of purified pooled amplicons for each coprolite were assessed using a bioanalyser prior to a subsequent PCR to attach indexed Illumina adapters (Nextera Index kit). DNA sequencing was performed on an Illumina MiSeq (by NZ Genomics Ltd.), using the MiSeq reagent kit v.2 (2 × 250 bp read length).

Sequence files were demultiplexed using MiSeq software, and as a quality control step only sequences with no mismatches in the barcode regions were retained. Corresponding paired end reads were assembled using PANDAseq (Masella et al., 2012). Primer trimming and removal of anomalously short (b 75 bp) and long (N200 bp) assembled reads were performed using Geneious v.7.1.7 (Biomatters, Auckland, NZ). The top 50 alignments and descriptions with a maximum e value of 0.0001 were recorded for the remaining sequences using Blast v.2.2.29 against the NCBI nucleotide database. Taxonomies were assigned to sequences and visualised using MEGAN v.5.7.0 (Huson et al., 2011). To reduce the chance of misassignment of identities to the sequences, we used relatively conservative minimum bit score cutoff values of 220 for 18S and 200 for trnL, and only considered taxa within 1% of the nearest match that met these bit score criteria.

2.5. Radiocarbon dating Samples of charcoal from twigs or short-lived tree species were selected from five fire features in the Stage 1 excavation area, where the majority of the coprolites were encountered. These were submitted to the University of Waikato Radiocarbon Dating Laboratory for accelerator mass spectrometry dating. Radiocarbon dates were calibrated using the ShCal13 calibration curve via OxCal.

Fig. 3. Prehistoric dog (kurī) coprolites from the Masonic Tavern site, Auckland, New Zealand. Specimen numbers are as follows: A, AD30; B, AD33; C, AD29; D, AD34; E, AD34 (transverse section); F, AD31. Colours relate the coprolites to other figures in this paper. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3. Results 3.1. Microscopic analyses Pollen and fern spores were observed in four of the coprolites examined, but at very low abundances (ranging from 5 to 17 grains per pollen subsample). The low abundance of pollen in the coprolites probably reflects indirect ingestion, e.g. via drinking water or adhering to food, rather than direct consumption of vegetation. However, the composition of the pollen and spore assemblages (Fig. 4) provides clear insights into the contemporaneous vegetation community around the site. The dominant components of the pollen/spore assemblages are monolete fern (representing a range of ground fern taxa) and bracken (Pteridium esculentum), with a background of podocarp trees including rimu (Dacrydium cupressinum), matai (Prumnopitys taxifolia) and miro (Prumnopitys ferruginea). Together, these elements indicate that the dogs inhabited a locally disturbed site within a wider landscape dominated by tall podocarp forest. Arbuscular mycorrhizal spores, identical to those of the genus Glomus, were relatively abundant in the coprolites. Fine charcoal fragments were also abundant on the pollen slides, and large fragments of charcoal were visible in some of the coprolite specimens (Fig. 3). No parasite eggs or larvae were observed on slides prepared from the coprolites. The larger fragments comprising the coprolites were dominated by fish remains, including bone fragments, teeth, otoliths and scales (Fig. 5). Other items present in the coprolites included marine bivalve shell fragments, large charcoal and burnt bone fragments, and land snails (Fig. 5). 3.2. Ancient DNA analyses A PCR product was amplified from the extraction control using the 18S rDNA primers, but not with the trnL primers. The control sequences were dominated by the stramenopile families Chromulinaceae and Ochromonadaceae, and the bird family Phasianidae. Of these, just one

assignment of Chromulinaceae was made in the sequences obtained from the coprolite extracts. Analysis of the closest blast hits to the Phasianidae sequences suggested they were likely to be domestic chicken (Gallus gallus domesticus), the DNA of which is suspected to be a common contaminant of molecular reagents (Thomson et al., 2014; Leonard et al., 2007). Other taxa identified in the extraction control sequences included Leporidae (rabbits/hares), Muridae (mice/rats), Hominidae (human), Canidae (dog) and Suidae (pig), all of which can occur as sporadic reagent contaminants (e.g. Leonard et al., 2007; Zheng et al., 2011; Boessenkool et al., 2012). However, the overall 18S rDNA taxonomic profile of the extraction control (Fig. 6) was distinct enough from those of the coprolite extracts (Fig. 7) to assume that our results were not affected by reagent contamination. Within the 18S rDNA sequences obtained from the coprolite extracts, dog was the most frequently assigned identity, occurring in four of the samples (Fig. 7). The remaining coprolite (AD29) yielded DNA from relatively few taxa (Fig. 7) and was noticeably damper than the other samples (Table 1) suggesting poorer preservation. However, it was inferred to also be from dog due to its similar size, shape (Fig. 3) and pollen composition (Fig. 4) compared with the other specimens that yielded dog DNA. Bony-fish (Euteleosteomorpha) was the second most frequently assigned identity, occurring in two coprolites (Fig. 7). One species that was frequently returned as the nearest match to sequences from the coprolites was the Australasian snapper, which is common in the sea around the Masonic Tavern site (and dominates the faunal remains from the site) and frequently occurs in Māori middens in the North Island and northern South Island. Several different fish taxa were assigned at mid-level taxonomic ranks (4 families: Sciaenidae, Sparidae, Cichlidae and Salmonidae), yet most of these do not occur in New Zealand waters. There are two possible reasons for this finding. It may hint at some diversity in the fish taxa present in the coprolites, but that our ability to place identities on them is limited by a paucity of 18S rDNA sequences available for New Zealand fish taxa. Alternatively, errors, either in the sequences themselves or in the sequence identities in the reference database, may have resulted in

Fig. 4. Pollen and spore assemblages in prehistoric dog (kurī) coprolites from the Masonic Tavern site, Auckland, New Zealand.

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Fig. 5. Microscopic fragments from prehistoric dog (kurī) coprolites from the Masonic Tavern site, Auckland, New Zealand: A) Charopid land snail; B) fish teeth; C) fish scale; D) fish bone; E) marine fish otolith; F) mouth plate; G) fish bone; H) marine shell; I) fish vertebra; J) burnt bone; K) wood charcoal.

erroneous assignment of the closest matching taxa. In most instances the latter explanation appears most likely, as Pagrus auratus still appears within the top 50 matches for sequences assigned to these other fish families. Several plant taxa of interest were also identified in the DNA sequences from the coprolite extracts (Fig. 7; Table 2). Cucurbitaceae was detected in three coprolites (AD31 for 18S rDNA, and AD30 and AD33 for trnL). Based on the nearest matches, and which Cucurbitaceae were known to be present in New Zealand prior to European arrival (Table 2) these sequences most likely represent cultivated commensal

bottle gourd (Lagenaria siceraria). Sequences attributed to Moraceae (AD29 for trnL) provide evidence for another cultivated commensal plant, the paper mulberry (Broussonetia papyrifera) (Table 2). Likely native plant taxa identified from the coprolite DNA sequences include Cyperaceae (nearest match to Carex spp.), Juncaceae (nearest match to Juncus spp.), Potamogetonaceae (nearest match to Potamogeton), Nothofagaceae (nearest native match to Fuscospora truncata), Liliaceae (nearest native match to Freycinetia and Cordyline), Plantaginaceae (nearest match to Veronica), Piperaceae (nearest match to Macropiper) and Poaceae (Table 2). The identify of other plant DNA sequences in

Fig. 6. Taxonomic profile assigned by MEGAN based on 18S rDNA sequences amplified from the extraction control. The size of the circles is proportional to the square root of the number of sequences assigned to that taxon.

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Fig. 7. Taxonomic profiles assigned by MEGAN based on (A) 18S rDNA sequences, and (B) trnL sequences amplified from prehistoric dog (kurī) coprolites from the Masonic Tavern site, Auckland, New Zealand. The size of the circles is proportional to the square root of the number of sequences assigned to that taxon. * denotes taxa also detected in extraction controls. Pictured taxa are discussed in the text: snapper (Euteleosteomorpha); dog (Canidae); gourd (Cucurbitaceae) and paper mulberry (Moraceae).

the coprolites is less clear. For example, sequences attributed to Tapisciaceae are just 1 nucleotide different from several native plant taxa (Polygonum, Coriaria arborea, C. sarmentosa, Dracophyllum longifolium and Epacris) (Table 2). These likely erroneous assignments (including Burmanniaceae, Campynemataceae, Cordiaceae, Fabaceae,

Malvaceae and Xanthoceroceae) occur exclusively in the 18S rDNA sequences (Table 2), and probably reflect less sequence variability between plant taxa for this locus compared with trnL. They may actually represent plant taxa identified using trnL, for example Lagenaria siceraria was a near match to 18S rDNA sequences assigned to

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Table 2 Assessment of taxonomic assignments of plant DNA sequences from prehistoric dog (kurī) coprolites from the Masonic Tavern site, Auckland, New Zealand. Taxon assigned by MEGAN v.5.7.0 (Fig. 6)

Marker Closest match using BLAST against GenBank sequences, and sequence similarity and score in parentheses

Closest matching prehistoric New Zealand genera using BLAST against GenBank sequences, and sequence similarity and score in parentheses

Burmanniaceae 18S Campynemataceae 18S

Gymnosiphon longistylus (100%; 243) Campynema lineare (100%; 243)

Cordiaceae Cucurbitaceae

18S 18S

Cordia (2 spp.) (99%; 237) Cucumis sativus (100%; 243)

Cyperaceae Fabaceae

trnL trnL 18S

Momordica (2spp.), Telfairia pedata (98%; 217) Carex (many spp.) (100%; 333) Bauhinia tomentosa (100%; 243)

Juncaceae Liliaceae

trnL trnL 18S

Malvaceae

18S

Moraceae Nothofagaceae Pinaceae Piperaceae Plantaginaceae Poaceae

trnL trnL trnL trnL trnL 18S

Glycine (8 spp.) (99%; 268) Juncus (many spp.) (97%; 250) Wisteria floribunda, Colchicum autumnale, Lilium (3 spp.) (98%; 231) Verbena hastata, Verbascum thapsus, Penstemon (2 spp.), Corchorus (6 spp.), Cristaria insularis (99%; 237) Morus mongolica (100%; 272) Nothofagus gunnii, N. truncata (100%; 289) Picea (many spp.) (100%; 281) Piper cenocladum (100%; 257) Veronica planopetiolata (100% for 96% coverage; 254) Oryza (7 spp.), Secale cereale, Sesleria tenerrima, Helictotrichon convolutum, Zea mays, Lolium multiflorum (100%; 243) Potamogeton octandrus (96%; 294) Rehmannia glutinosa (98%; 231)

Potamogetonaceae trnL Rehmanniaceae 18S Solanaceae

18S

Tapisciaceae

18S

Solanum (6 spp.), Nicotiana benthamiana, Lycium barbarum, Lycopersicon esculentum (100%; 243) Tapiscia sinensis (99%; 237)

Xanthoceroceae

18S

Xanthoceras sorbifolium (98%; 231)

a

Dioscorea alata (95%; 204) Lagenaria siceraria, Ripogonum, Veronica, Plantago, Ileostylus micranthus (99%; 237) Solanum, Ipomoea (98%) (231) Dracophyllum longifolium, Coriaria arborea, Coriaria sarmentosa, Ileostylus micranthus, Polygonum, Plantago, Veronica, Lagenaria siceraria (98%; 211) Lagenaria siceraria (98%; 211) Carex (100%; 333) Polygonum, Coriaria arborea, C. sarmentosa, Dracophyllum longifolium, Epacris (99%; 237) None – possible contamination Juncus (97%; 250) Freycinetia, Cordyline australis (98%; 226) Solanum (98%; 231) Broussonetia papyrifera (97% for 99%; 248) N. truncata (100%; 289) None – possible contamination Macropiper excelsuma (100%) Veronica Poa (99%; 237) Potamogeton (96%; 294) Plantago, Veronica, Lagenaria siceraria, Ileostylus micranthus, Utricularia (98%; 226) Ipomoea, Solanum (98%; 231) Polygonum, Coriaria arborea, C. sarmentosa, Dracophyllum longifolium, Epacris (98%; 231) Dracophyllum longifolium, Coriaria arborea, C. sarmentosa, Ileostylus micranthus, Polygonum, Veronica, Lagenaria siceraria (96%; 215)

Denotes unpublished reference sequence from authors not on GenBank.

Campynemataceae (Table 2). Several 18S rDNA sequences assigned to different families were near matches to genera that include the food plants poroporo (Solanum) and kūmara (Ipomoea batatas) (Table 2), but the presence of these was not confirmed by trnL sequences.

3.3. Radiocarbon dating The radiocarbon dated charcoal fragments ranged in age from 632 ± 20 14C years BP to 178 ± 20 14C years BP suggesting a relatively long period of Maori occupation at the site, spanning from at least the 14th to 19th centuries (Table 3). The presence of butchered moa (at least 11 individuals) in the Stage 1 faunal assemblage is supporting evidence for early (pre-1450 CE) human occupation of this site.

4. Discussion 4.1. Kurī diet Microscopic and molecular analyses of the prehistoric coprolites from the Masonic Tavern archaeological site confirmed their identity as being from kurī, and reveal that these dogs had an omnivorous diet that included fish and cultivated plants. The fish remains may have been derived from scraps scavenged from fire pits, as evidenced by the abundant charcoal fragments in the coprolites. The presence of mycorrhizal Glomeraceae spores in some of the coprolites could reflect post-depositional infiltration by fine plant roots, yet relatively high abundances of subterranean mycorrhizal spores were also noted in putative kurī coprolites by Horrocks et al., (2002). He concluded that while

Table 3 AMS radiocarbon dates on charcoal from the Masonic Tavern site, Auckland, North Island, New Zealand. Sample code

Lab number

Radiocarbon age

Calibrated age AD (95.4% confidence range)

S1118, feature 153

Wk-42,637

303 ± 20

S1152, feature 155

Wk-42,638

178 ± 20

S5293, feature 281

Wk-42,639

632 ± 20

S5300, feature 294

Wk-42,640

313 ± 21

S5304, feature 293

Wk-42,641

393 ± 20

1510–1577 (28.6%) 1621–1665 (66.8%) 1670–1748 (40.1%) 1756–1783 (5.6%) 1795–1817 (10.8%) 1829–1894 (25.4%) 1921–1950 (13.5%) 1316–1356 (61.6%) 1381–1408 (33.8%) 1508–1583 (46.7%) 1620–1657 (48.7%) 1458–1516 (41.8%) 1540–1625 (53.6%)

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they could be indicative of foraging on the ground and in soil, their high abundance more likely reflected the direct consumption of root crops. In support of this, some of the plant taxa identified by aDNA and pollen analysis of the coprolites have edible roots or rhizomes that were known to have been eaten by early Māori (e.g. Cordyline, Pteridium). The presence of freshwater protozoa (Oikomonadaceae), algae (Cryptomonadaceae and Hydruraceae), diatoms (Naviculaceae), and aquatic plants such as Cyperaceae, Juncaceae and Potamogetonaceae, as detected by our aDNA analysis (Fig. 6), is indicative of wetlands or lake margins having been used as a source of drinking water. Our results support the idea that kurī in northern New Zealand consumed a high proportion of plant matter, including cultivated commensal species. Strong evidence for two of the six commensal cultivated plant species brought to New Zealand by the first settlers (gourd and paper mulberry [which has edible fruits]) was obtained from aDNA analysis of the coprolites. The gourd DNA sequences from the coprolites provide an interesting question of identity. Although the bottle gourd (Lagenaria siceraria), which is known to have been brought to New Zealand during the prehistoric era, was 97.6% similar, the bitter gourd (Momordica charantia) was a 98.4% match (a single nucleotide nearer to the coprolite sequences). Although this could be due to sequencing error or miscoding lesions, it would be worthy of further study. Although it is not known to have ever been brought to New Zealand during the prehistoric era, the bitter gourd appears to have been a minor commensal species in the Pacific. The species was obtained in Tahiti by the first Cook expedition (Smith, 1981) and was also grown during the prehistoric period in Hawaii (Ladefoged et al., 2003). There is also evidence to suggest the presence of further commensal plants within the coprolites. aDNA sequences from two coprolites were attributed to Burmanniaceae (Fig. 6), the only New Zealand member of this family being the unusual Thismia rodwayi, a small nonchlorophyllous plant that associates with saprophytic fungi on the roots of plants. Comparison of the coprolite sequences with reference sequences for this species show just an 89% similarity. However, Burmanniaceae is placed within the order containing yams (Dioscoreales), and a comparison of the sequence with the purple yam (Dioscorea alata) (another of the cultivated plants brought to New Zealand by Polynesian settlers) shows a 95% similarity (Table 2). Considering the potential for sequencing errors and miscoding lesions, which are prevalent in ancient DNA (Hofreiter et al., 2001), and the fact there are no other native Dioscoreales in New Zealand, this raises the possibility that the dogs from the Masonic Tavern site may also have been consuming yams. Possible DNA sequences attributed to Solanum were also present in two of the coprolites (Fig. 6; Table 2). Irwin et al. (2004) reported finding a seed of Solanum nodiflorum (now synonymised with S. americanum) in a putative kurī coprolite from New Zealand's North Island. Unfortunately no reference DNA sequences were available for S. americanum that covered the entire region of the 18SrDNA gene that we sequenced from the coprolite. The native range of S. americanum is unclear, and it has been suggested that it may have been spread throughout the Pacific by humans (Prebble, 2008), and therefore it may represent another commensal plant species in the Masonic Tavern coprolites. Alternatively, Māori also have many traditional uses for native species of Solanum (Ngā Tipu Whakaoranga database, http://maoriplantuse.landcareresearch.co.nz, record number 1100, accessed 29 Sep. 2015) and S. aviculare, found in northern New Zealand, may also therefore be considered a candidate for the Masonic Tavern coprolite DNA sequences. A range of native plant taxa were identified from aDNA analysis of the coprolites. Some, such as Cordyline or Macropiper, are known to have been used by Māori and so may have been directly consumed by kurī. Others, such as Fuscospora and Veronica, may have originated from drinking water or incidental ingestion of soil containing the DNA, or from wood/charcoal fragments incidentally ingested while scavenging in middens. Both Veronica (formerly Hebe) and Fuscospora (formerly Nothofagus) are known to have been used for firewood by early

Māori (New Zealand Radiocarbon Database; http://www.waikato.ac. nz/nzcd/intro.html, accessed October 2015). Overall, the dietary information from the Masonic Tavern archaeological site appears to support previous inferences about the diets of prehistoric kurī in northern New Zealand during the later (post1500 CE) settlement period. The diet seemed to be dominated by marine (fish, shellfish) components (perhaps scavenged from middens) and plant matter, strongly overlapping with the diets of Māori inhabiting northern coastal areas at this time. No bone fragments or DNA attributable to birds, reptiles or marine mammals were identified from the coprolites. Although most of the radiocarbon dates were from this later settlement period (Table 3), a radiocarbon date of 632 ± 20 from one of the fire features (Table 3), and the presence of moa bones in the faunal assemblage of the site, suggest that the early settlement era (pre-1450 CE) is also represented in the site. Further analysis of kurī coprolites from the Masonic Tavern archaeological site may therefore provide insights into dietary changes of dogs between these time periods. 4.2. Identity of other New Zealand archaeological coprolites Using aDNA analysis we have identified the depositor of coprolites from a New Zealand archaeological site for the first time, by demonstrating that the coprolites from the Masonic Tavern site were deposited by kurī. Interestingly, the content of the Masonic Bay coprolites have strong similarities to those of coprolites previously described from other New Zealand archaeological sites, but for which the identity of the depositing species could not be definitively assigned. This suggests that they too, were likely deposited by dogs. For example, putative dog coprolites from the Kohika site near Whakatane on the North Island also contained fish bone, charcoal, and fragments of marine shell, and plant material including Cyperaceae and Solanum (Irwin et al., 2004). Coprolites from Great Barrier Island, whose origins were debated as being either dog or human, also contained bottle gourd (as pollen), high abundances of monolete fern spores and truffle spores, as well as palynological indicators of the use of wetlands for drinking water (e.g. Myriophyllum and Cyperaceae) (Horrocks et al., 2002). However, in the absence of diagnostic bone fragments (such as the fish bone fragments seen here and at Kohika) there may be little chance of being able to distinguish dog and human coprolites using microscopy, given the apparent overlap in diets (especially in regards to cultivated plant foods). Here, we have shown the potential of aDNA profiling to identify kurī coprolites. Further application of this methodology to archaeological coprolites from New Zealand will continue to provide new insights into the diets and husbandary of kurī in the pre-contact era. Acknowledgments We thank N. Bolstridge for preparation of the palynology slides and D. Park and T. Lawrence for assistance with DNA sequencing. References Anderson, A., 1981. Pre-European hunting dogs in the South Island, New Zealand. N. Z. J. Archaeol. 3, 15–20. Anderson, A., 2005. Subpolar settlement in South Polynesia. Antiquity 79, 791–800. Anderson, A.J., Clark, G.R., 2001. Advances in New Zealand mammlogy 1990–2000: Polynesian dog or kuri. J. R. Soc. N. Z. 31, 161–163. Birks, H.J.B., Birks, H.J.B., 2016. How have studies of ancient DNA from sediments contributed to the reconstruction of Quaternary floras? New Phytologist 209, 499–506. Boessenkool, S., Epp, L.S., Haile, J., Bellemain, E., Edwards, M., Coissac, E., Willerslev, E., Brochmann, C., 2012. Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA. Mol. Ecol. 21, 1806–1815. Bon, C., Berthonaud, V., Maksud, F., Labadie, K., Poulain, J., Artiguenave, F., Wincker, P., Aury, J., Elalouf, J., 2012. Coprolites as a source of information on the genome and diet of the cave hyena. Proceedings of the Royal Society of London B 279, pp. 2825–2830. Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Huntley, J., Fierer, N., Owens, S.M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J.A., Smith, G., Knight, R.,

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