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Do temporal size differences influence species identification of archaeological albatross remains when using modern reference samples?
Journal of Archaeological Science 33 (2006) 349e359 http://www.elsevier.com/locate/jas
Do temporal size differences influence species identification of archaeological albatross remains when using modern reference samples? Masaki Eda a,*, Yoshiyuki Baba b, Hiroko Koike b, Hiroyoshi Higuchi a a b
School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo, 113-8657, Japan Graduate School of Social and Cultural Studies, Kyushu University, Ropponmatsu 4-2-1, Chuo-ku, Fukuoka City, Fukuoka, 810-8560, Japan Received 4 October 2004; received in revised form 14 June 2005; accepted 27 July 2005
Abstract Zooarchaeological remains have been identified to species, using identification criteria based on specific morphological variations among modern specimens. However, temporal size changes in bones, due to micro-evolution and/or phenotypic plasticity, could distort identification of archaeological remains according to these criteria. We developed species identification criteria for North Pacific albatrosses (Short-tailed, Laysan and Black-footed Albatrosses) using both mensural- and DNA-based analysis and actually identified many archaeological remains from a site using these criteria. Our mensural-based criteria could accurately discriminate the modern Short-tailed Albatross from modern Laysan and Black-footed Albatrosses and indicated that the archaeological remains included both Short-tailed and Laysan or Black-footed Albatrosses. DNA-based criteria, however, suggested that all remains were Short-tailed Albatross. The most plausible explanation for this inconsistency would be misidentification using mensural-based analysis, due to temporal size changes in bones or existence of birds from extinct population(s) or breeding region(s) with mensurally different bones from recent birds. This is the first study that suggests temporal size changes in bones may distort the species identification of archaeological remains according to modern size variations. Further studies are required to judge if this pattern is unusual or not. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Species identification; Ancient DNA; Short-tailed Albatross; Microevolution; North Pacific; Avian bone; Zooarcheology
1. Introduction Identification of archaeological remains is the basis of zooarchaeology. Referring to identified animals and their ecology, the seasonality of site formation and the foraging areas of inhabitants have been discussed [9,10,32]. Generally, archaeological remains have been * Corresponding author. Graduate School of Social and Cultural Studies, Kyushu University, Ropponmatsu 4-2-1, Chuo-ku, Fukuoka City, Fukuoka, 810-8560, Japan. Tel.: C81 92 726 4671; fax: C81 92 726 4847. E-mail address: [email protected] (M. Eda). 0305-4403/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jas.2005.07.017
identified to the species level, using identification criteria based on the specific morphological variation among modern specimens. These identification criteria tacitly assume that morphological characters have been temporally invariable, or never jumped across species ranges even if some changes occurred (e.g. [11]). On the other hand, it has been revealed that the bone morphology, especially size, of some terrestrial mammals changed due to micro-evolution and/or phenotypic plasticity since the beginning of the Holocene or the late Pleistocene [6,13,31,35]. These temporal bone size variations have been considered as resulting from variations in environment, such as fluctuations in temperature, habitat
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fragmentation and/or depression of the food supply. Therefore, species identification criteria that enable us to analyse archaeological remains independent of morphology are required. Techniques for ancient DNA analysis, developed during the last decade, have been used effectively for species identification of archaeological remains that were difficult to classify using morphology-based identification [1,2,22,25,41]. For example, Butler and Bowers [2] suggested that Oncorhynchus nerka whose bones are morphologically indistinguishable from other Oncorhynchus species were included in archaeological remains. Newman et al. [25] showed that even highly fragmented and morphologically unidentifiable archaeological bones could be classified into sheep and goats. Furthermore, Loreille et al. [22] and Bar-Gal et al. [1] compared morphology-based identification for sheep and goats with DNA-based identification, and suggested that morphology-based identification criteria were almost correct. In this study, we identified albatross remains by ancient DNA analysis and compared the results with species identification based on morphological variations in modern specimens. Many albatross (Diomedeidae) remains have been found at Holocene archaeological sites in the northern Pacific coastal regions of the Japanese Archipelago (reviewed in [7]), the Aleutian Islands (e.g. [21,42]), and the west coast of North America (e.g. [4,12,30]). They are easily identified to family and genus levels. This is because the wing and leg bones are characterized by a much longer and flatter shape than those of other bird families [9], and there is only one genus (Phoebastria) in this region [5,36]. However, species identification within the genus, namely of Short-tailed (P. albatrus), Laysan (P. immutabilis) and Black-footed Albatrosses (P. nigripes), has not been elucidated, since no research has been conducted on the appropriate identification criteria using sufficient numbers of osteological specimens, especially for the Short-tailed individuals (see also [27e 29,42]). Correct species identification of these albatross remains would be useful for understanding human foraging areas. This is because they included a species that was mostly observed in the continental shelf region (Short-tailed Albatross [36]) and two species that were abundant over the outer continental shelf (Laysan and Black-footed Albatrosses [39,40]). Therefore, the proportion of Laysan and Black-footed Albatross bones in the archaeological remains would be expected to increase with increased foraging over the outer continental shelf. At the same time, correct species identification is also required to understand albatross ecology, since numerous sites at which albatross remains have been found are located in coastal regions of the Seas of Japan and Okhotsk where few albatrosses have been distributed recently (reviewed in [7]).
In this study, we examined whether temporal size changes in bones distort the species identification of albatross remains according to size variations in modern specimens. First, we developed identification criteria based on modern specimens using two approaches: traditional caliper measurements, and DNA analysis that enables us to identify the materials independent of their morphological characters. Second, we classified albatross remains according to the two identification criteria. Finally, we compared the results with each other.
2. Materials One-hundred and five carpometacarpi of recent albatrosses (13 Short-tailed, 60 Laysan and 32 Black-footed Albatrosses) were collected to provide identification criteria using caliper measurements. These were provided by the American Museum of Natural History; the Smithsonian Institution (USNM); the Museum of Vertebrate Zoology, at the University of California Berkeley (MVZ); the National Science Museum, Tokyo (NSMT); the Yamashina Institute for Ornithology; the University of Alaska Museum (UAM); the Peabody Museum, Yale University; the Natural History Museum, Tring, and the personal collections of H. Hasegawa, K. Kawakami, M. Moriguchi, Y. Osa, and one of the authors (M. E., Table 1). Three of the carpometacarpi (one of each of the species) exhibited more or less incomplete ossification at the proximal joint of the Os metacarpale alurare and majus. We defined this character as a landmark for ‘‘juveniles’’, since the character was observed in a juvenile Short-tailed Albatross (USNM018224) and not observed for any other measured known age carpometacarpi including specimens described as immature Short-tailed Albatross (USNM567025) and three year old Blackfooted Albatross (NSMT388). Those ‘‘juvenile’’ specimens were excluded from the following analysis to avoid the influence of age related size difference. No osteological characters differentiating the albatross species were found on the element, although Yesner [42] indicated that the tuberosity of metacarpal II, facets for fused digits II/III, and ligamental attachment of the pisiform process of the Short-tailed Albatross were more developed than those of the Laysan Albatross based on a small sample of only one Short-tailed and four Laysan Albatross specimens. DNA sequences were compared to those reported by Nunn et al. [26]. Thirty-five left-side intact carpometacarpi that were classified as Diomedeidae were collected from the Hamanaka 2 site on Rebun Island, Hokkaido, northern Japan (Fig. 1). The reasons we selected this bone element were that it usually represented the minimum number of individuals and was preserved more intact than other elements in the site [8]. All of these bones were determined to be about 1000 years old, because they were
Table 1 Range, mean, standard deviation (SD) and coefficient of variance (V) for proximal breadth (Bp), distal diagonal (Did) and the greatest length (GL), of North Pacific modern albatross carpometacarpi Species
Sex
Short-tailed
All F U
Black-footed
All F
M
U
Laysan
All F
M
U
Sampling location
Non-breeding area Torishima Island, Izu Islands Minami-kojima Island, Senkaku Islands Non-breeding area
N
12 1 6 1
Bp
Did Mean
SD
V
Range
Mean
SD
V
Range
Mean
SD
V
19.9e23.2
21.3 21.0 21.6 21.9
1.0
4.6
12.7e14.9
0.7
5.0
107.2e126.7
4.7
13.2e14.7
0.7
111.7e126.7
115.6 107.2 118.0 113.9
5.4
1.1
14.0 13.9 14.1 14.0
12.7e14.9
13.8
1.0
110.6e120.1
114.5
4.3
11.6e13.5
0.5
98.6e113.7
3.9
20.4e23.2
19.9e21.8
20.9
0.9
31 1 5
18.2e20.7 19.5e20.0
19.39 19.1 19.6
0.6
Hawaii Islands Northwestern Hawaii Islands Non-breeding area Northwestern Hawaii Islands
1 5
19.2e20.7
19.3 20.0
Non-breeding area Northwestern Hawaii Islands Ogasawara Islands Torishima Island, Izu Islands Non-breeding area or unknown
3 1 3 7 5
59 1 9
Non-breeding area Hawaii Island, Hawaii Islands Northwestern Hawaii Islands
2 1 7
Non-breeding area Northwestern Hawaii Islands
2 4
Mukojima Island, Ogasawara Islands Non-breeding area or unknown
1 32
Included specimens
Range
4
Hawaii Island, Hawaii Islands Northwestern Hawaii Islands
GL
19.5e20.2 19.0e19.9 18.2e19.7 18.6e20.6
17.7e20.5 18.3e19.4 18.0e18.4
0.2
12.7e13.2
12.6 12.3 12.8
0.6
12.8e13.5
12.3 13.1
19.7 19.2 19.5 18.9 19.2
0.4
11.8e13.1
0.5 0.6 0.8
12.2e12.9 11.6e12.6 12.2e13.1
18.9 18.9 18.7
0.6 0.4
19.0e20.5
18.2 18.9 19.3
0.5
18.6e19.3 18.0e19.5
18.9 18.7
0.6
3.2
2.9
10.3e13.1 11.4e12.1 11.9e12.0
19.0
103.7e109.1
0.3
110.0e113.7
106.5 111.6
12.6 12.1 12.6 12.1 12.5
0.7
107.1e111.4
0.4 0.4 0.4
102.6e110.5 98.6e108.4 101.6e110.2
12.1 11.8 11.8
0.5
11.9e13.1
11.9 12.3 12.6
0.4
12.2e12.3 11.7e12.6
12.2 12.1
0.4
18.6 17.7.e20.0
0.2
106.7 103.6 107.2
0.2
4.0
4.2
94.2e109.6 94.2e101.9 96.8e98.2
0.6
10.3e13.0
12.0
108.8 106.4 106.6 102.6 106.4
2.3
100.3 96.7 97.4
3.2 2.6
2.2
99.1e102.0 96.4e105.1
100.5 100.3
3.8
100.9
ANM25837 USNM289170, 289171, 488173, 488176, 497964 MVZ175960 USNM289168, 289169, 488172, 488174, 488175 MVZ72294; USNM622480; YIO-60004 USNM497920 KP12-1, 12-2; NSMT388 EP166-169; NSMT390, 391; YIO-60005 OPbfa001; USNM19740, 432184; YIO-60001, 60002
4.0 3.4 4.0
100.0e107.2
96.1e109.6
3.7
1.5
98.4 0.6
MVZ28980; UAM5723; USNM018225; YPM106517
2.1
97.5 98.8 102.8
11.9
USNM567025 EP097, 170, 171; NSMT283, 285, 389 HP001
5.5
3.2
3.2 ANM25839 MVZ142647; USNM289156, 289158, 289161, 488178-488181, 498067 EP130, 145 ANM25838 USNM289160, 488177, 497965, 498137-498139, 500859 ANM18657; MVZ177560 MVZ142648; BMNH S/1967.8.3; USNM289155, 498120 KP11-1 ANM9837-9839, 9841-9844, 9846, 9847, 10123, 10125-10138; EP022, 098, 151; KP11-4; MP001, 002; OPlya001; YIO-60003
Abbreviated designations are: female (F); male (M); sex unknown (U); the American Museum of Natural History (ANM); the Smithsonian Institution (USNM); the Museum of Vertebrate Zoology, at the University of California Berkeley (MVZ); the National Science Museum, Tokyo (NSMT); the Yamashina Institute for Ornithology (YIO); the University of Alaska Museum (UAM); the Peabody Museum, Yale University (YPM); the Natural History Museum, Tring, UK (BMNH); personal collections of H. Hasegawa (HP); K. Kawakami (KP); M. Moriguchi (MP); Y. Osa (OP); M.E (EP).
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Hamanaka 2 site
0
2.0
(km)
Rebun Is. Russia
China Hokkaido
Honshu Shikoku Kyushu
Fig. 1. Location of the Hamanaka 2 study site, Hokkaido, northern Japan.
M. Eda et al. / Journal of Archaeological Science 33 (2006) 349e359
found with pottery and stone tools from the Okhotsk period, between the 7th and 12th centuries AD [24]. Judging from our morphological criterion, 12 of them were ‘‘juvenile’’ and excluded from the following analysis.
3. Methods 3.1. Caliper measurements Three caliper distances were measured for each recent specimen following von den Dreisch [37]: greatest length (GL), proximal breadth (Bp), and distal diagonal (Did). Most measurements were conducted by M.E., while measurements for specimens were provided by Becky Williams and Dr Carla Cicero of the MVZ and Dr Kevin Winker of the UAM. Measurement values for each specimen were classified into groups by species, sex, and sampling location (specific breeding site or nonbreeding area and unknown) to calculate mean, range (for groups including more than one specimen), standard deviation (for groups including more than two specimen), and coefficient of variance (for each species). Homogeneity test of covariance matrices and discriminant function analysis were performed using SYSTAT ver. 8.0 (SPSS Inc.). Discriminant function analysis was performed using the linear discriminant function, since covariance matrices were not significantly different among species (see Section 4). The leave-one-out classification was performed to test the robustness of the identification criteria. Each archaeological albatross specimen was classified into a particular species according to scores calculated by the classification function. The caliper distances for each specimen were substituted into the classification function for each species, and they were placed into the species whose classification function score was the highest. 3.2. DNA analysis We designed a primer (Lcyt.246.dio: 5#-CCTCCAC GCAAACGGAG-3#) for the mitochondrial cytochrome b region and paired it with H15149.uni (5#-CGGGG AGTCTTACTATAAACAGGAGT-3# [19]) to amplify a 143 bp fragment which allows the distinction of all albatross species, referring to the sequence for modern albatrosses [26]. All DNA extraction and subsequent first polymerase chain reaction (PCR) amplification preparations were performed in the laboratory of H.K. at Kyushu University where no modern molecular albatross studies have previously been conducted. This laboratory was separated from the post-PCR amplification facility. The archaeological albatross remains were cleaned and ground into about 300 mg of powder using a dental-drill sterilized with HCl and by UV. We ground one sample per day at a UV sterilized clean bench.
353
We extracted DNA using the Geneclean kit for Ancient DNA (Q-BIO Gene) with over night preincubation (5 ml of ethylendiaminetetraacetic acid 0.5 M pH 8.0, 200 ml of 10% sodium dodecyl sulphate, 200 ml of 20 mg/ml proteinase K). Blank extractions containing all buffers but lacking any sample were carried out to detect any contamination in buffers and solutions. PCR amplifications were carried out in 25 ml reaction volumes consisting of 1e5 ml of the DNA extract, 0.2 mM of each nucleotide, 0.1e0.2 mM of each primer, 0.2e2 mg/ml of bovine serum albumin and 1.25e2.5 units of Taq polymerase (Takara) in a 1! concentration of the supplied reaction buffer. PCR cycling consisted of the following parameters; step 1, 94 C for 1 min; step 2, 94 C for 30 s, 50e60 C for 45 s, 72 C for 45 s (cycled 40 times) and a final extension at 72 C for 1 min. One microlitre was re-amplified (30 cycles) in a volume of 25 ml with the same primers and parameters. An amplification blank was included in each PCR reaction batch. DNA sequencing of PCR products was carried out in both directions on a CEQ 2000 DNA Analysis System (Beckman Coulter) using the same primers as used for PCR, following purification of the template with a PCR Product Pre-sequencing Kit (USB). Amplification and sequencing were independently replicated for each sample that gave positive amplification. Extraction was also independently replicated for each sample that gave positive amplification and did not have a unique sequence at the mtDNA control region (unpublished data). This was due to finding contamination among DNA samples and amplification errors by Taq polymerase. A neighbour joining (NJ) tree [34] was constructed using Kimura’s two-parameter model [18]. The reliability of specific grouping in the tree was assessed using 1000 bootstrap replicates. The construction of the NJ tree and bootstrap replicates were conducted using MEGA ver. 2.1 [20]. Species identifications were made by comparison of their sequences and positions in the NJ tree to a set of modern sequences.
4. Results 4.1. Caliper measurement On average, the Short-tailed Albatross was the largest; it was followed by the Black-footed and the Laysan Albatrosses, although ranges for each measurement overlapped among the three species (Table 1). The range of the coefficient of variance was from 2.9 to 5.8. There was no significant difference among the covariance matrices for three species (c2 Z 15.57, df Z 12, p O 0.05). The three albatross species differed significantly in their measurements (Pillai’s trace Z 0.84, F Z 23.69, df Z 6, 196, p ! 0.001) and discriminant functions could be calculated (Table 2). In the leave-one-out classification,
354
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Table 2 Unstandardized discriminant functions and group means for three North Pacific albatross species Measurement
Discriminant function coefficients
Bp Did GL Constant
Species
Function 1
Function 2
0.344 0.456 0.180 ÿ31.077
1.812 0.115 ÿ0.278 ÿ7.611
Group means
Short-tailed Black-footed Laysan
Function 1
Function 2
3.458 0.547 ÿ0.991
0.563 0.217 0.563
Bp, proximal breadth; GL, greatest length; Did, distal diagonal.
4.2. DNA analysis DNA samples from 18 of the 23 (78%) Diomedeidae archaeological carpometacarpi were successfully amplified and provided target sequences (143 bp) in the mtDNA cytochrome b region. They included nine each of the specimens identified as Short-tailed and Blackfooted Albatrosses in the mensural-based identification. The replicated mtDNA sequences derived from each specimen were consistent for all cases. The other five samples (22%) could not be amplified despite more than five trials for each sample. No negative controls yielded any PCR products when the PCR included any sequenced samples. The eighteen 143 bp albatross specimen sequences had one C-T transition site that was a third codon position silent substitution (Table 4). Six sequences had T at the site and the others had C. The former was identical Table 3 Percentage correct classification of modern specimens in leave-one-out classification
Short-tailed Black-footed Laysan Total
Short-tailed
Black-footed
Laysan
%correct
11 3 0 14
1 19 8 28
0 9 51 60
90.9 61.3 86.4 79.4
Columns and rows show the classified and the known species name, respectively.
to the Short-tailed Albatross reference sequence (U48952). The NJ tree showed that all sequences deriving from specimens were monophylic with the reference sequence for modern Short-tailed Albatross, with 68% bootstrap support (Fig. 3).
5. Discussion Using three caliper measurements, 79% of 102 carpometacarpi of modern North Pacific albatrosses were accurately classified into Short-tailed, Black-footed and Laysan Albatrosses. While species identifications between Black-footed and Laysan Albatrosses were confounded, more than 95% of modern albatross specimens were correctly classified into Short-tailed and Blackfooted or Laysan Albatrosses. The criteria would be useful to understand human foraging areas, since Short-tailed and Black-footed or Laysan Albatrosses were observed in different region of the ocean. The criteria were applied to 23 archaeological specimens from the Hamanaka 2 site, and 12 of them were identified as Short-tailed Albatross while the other 11 were Black-footed or Laysan
4
Canonical score 2
79% of all specimens were correctly identified, and Short-tailed, Black-footed and Laysan Albatross specimens were correctly identified with 91%, 61% and 86% accuracy, respectively (Table 3). Most of the misidentifications, 81% of 21 cases, were observed between Black-footed and Laysan Albatrosses, while only 19% were between Black-footed and the Short-tailed Albatross. In the scatter plot of canonical scores for each specimen, most Short-tailed Albatrosses separated from Laysan and Black-footed Albatrosses but the latter two highly overlapped each other (Fig. 2). Twenty-three zooarchaeological albatross remains were classified based on classification function with 12 and 11 specimens being identified as Short-tailed and Black-footed Albatrosses, respectively.
0
-4 -4
4
0
8
Canonical score 1 Fig. 2. A scatter plot of canonical score 1 vs. canonical score 2 for the carpometacarpi of Short-tailed (B), Black-footed (6), and Laysan Albatrosses (>) in discriminant function analysis. Archaeological remains from the Hamanaka 2 site were also included: yield DNA (C) and did not yield DNA (!). Note that all archaeological specimens that provided successful DNA analyses were identified as Short-tailed Albatross.
355
M. Eda et al. / Journal of Archaeological Science 33 (2006) 349e359 Table 4 Variable sites for part of the mitochondrial cytochrome b region for all albatross species and archaeological bones Species/sample Variable sites name 2 6 4 Short-tailed A HM-02 $ HM-13 $ HM-18 $ HM-22 $ HM-28 $ HM-35 $ HM-01 $ HM-05 $ HM-06 $ HM-10 $ HM-11 $ HM-16 $ HM-17 $ HM-21 $ HM-24 $ HM-26 $ HM-34 $ HM-39 $ Laysan $ Black-footed $ Waved $ Wandering $ Amsterdam $ Royal $ Grey-headed $ Black-browed $ Yellow-nosed $ Buller’s $ Shy $ Light-mantled $ Sooty Dark-mantled $ Sooty Grey Petrel C
2 7 0 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T $ $ $
2 7 3 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
2 8 2 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T T T T $ $
2 8 5 T $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ C C C C C C C C C C C C
Genbank accession no. 2 8 8 T $ $ $ $ $ $ C C C C C C C C C C C C $ $ $ C C $ C C C C C C
2 9 1 T $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ C $ $ $ $ C $ $ $ $ $ C
2 9 7 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
3 0 0 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T
3 0 5 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A $ T T T T T T
3 0 8 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ G $ C $ $ $ $ $ $ $
3 1 4 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T
3 2 0 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T $ T
3 2 4 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ G C G G $ G
3 2 6 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T T T T $
3 2 7 G $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A A $ $ $ $ $ $ A
3 2 9 T $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ C A A A A A $
3 3 5 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T $ $ $ $ $ $
3 3 6 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ G G $ $ $ $ $ $ $ $ $
3 3 8 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ G $ $ $ $ $ $ $ $ $
3 4 4 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T
3 5 1 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T $ $ $ $ $ $ $ $ $
3 5 3 G $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A A A A A $
3 5 4 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
3 5 6 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A $ $ $ $ $ $ $ $
3 5 9 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ G $ $
3 6 0 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
3 6 6 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
3 6 8 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T T T $ $ $ $ $ $
3 7 1 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T T T T T $ $
3 8 0 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T $ $ $ $ $ $ $ $ $ $
3 8 3 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T T T T T T
3 9 2 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T
3 9 5 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A G A A A A
3 9 8 T $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ C C $ $ $
3 9 9 T $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ C C C $ $ $ C C C C $ C
4 0 1 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
4 0 4 G $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A A A A A A A A A
4 0 5 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
U48952
U48949 U48950 U48951 U48947 U48948 U48946 U48954 U48955 U48944 U48945 U48953 U48943
$ T $ C C C $ T T $ T T G $ A $ $ $ $ T $ $ $ $ $ $ $ $ $ $ T T A $ C $ A $ U48942 $ $ T $ C $ T A A $ $ $ $ $ $ $ $ G $ $ $ $ T A T T T $ $ $ T $ A C C T A C U48940
Numbering is based on the published complete cytochrome b region sequence [26] and dots indicate identity with sequence.
Albatrosses. In contrast, all cytochrome b sequences from the albatross specimens were monophylic with the reference sequence of the modern Short-tailed Albatross. This suggests that these specimens were from the Shorttailed Albatross. Nine of the 18 specimens were classified as Black-footed or Laysan Albatrosses using the mensural-based identification criterion. Results of species identifications based on the two identification approaches could have been inconsistent in three ways: (a) remains of Black-footed or Laysan Albatrosses were classified with the Short-tailed Albatross in the DNA analysis; (b) remains of Short-tailed Albatrosses were classified with the Black-footed or the Laysan Albatrosses in the mensural analysis; and (c) remains included many individuals that were hybrids of the Short-tailed and the Black-footed or the Laysan Albatrosses. The last case (c) is unlikely to explain all of the
inconsistent results, since no such hybrid birds have been identified in more than 25 years on Torishima Island, the largest breeding ground for the Short-tailed Albatross (H. Hasegawa, personal communication). If misidentifications occurred in the DNA-based classification, a serious level of contamination must be supposed. On the other hand, if misidentifications occurred in the mensural-based classification, the size of the bones may have changed and obscured species differences over the last millennium. It is unlikely that misidentification occurred in the DNA-based identification, i.e. case (a), although the authenticity of deduced sequences has been the most controversial problem in ancient DNA studies [16,23]. We believe that the results in this study are fully reliable. This is because (1) DNA extraction and first PCR preparation were performed in a laboratory where no
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M. Eda et al. / Journal of Archaeological Science 33 (2006) 349e359
HM-01 HM-05 HM-06 HM-10 HM-11 HM-16 HM-17 61 HM-21 HM-24 HM-26 HM-34
68
HM-39 Short-tailed (U48952) HM-04 HM-30 53
HM-38 HM-04
71
HM-30 HM-38 Laysan (U48949) Black-footed (U48950) 75
Waved (U48951) Royal (U48946) Wandering (U48947)
75 71
Amsterdam (U48948) Gray-headed (U48954) Buller’s (U48945)
68
55
Yellow-nosed (U48944) Black-browed (U48955)
64
Shy (U48953) Light mantled Sooty (U48943) 99
Dark-mantled Sooty (U48942) Gray petrel (U48940)
0.02 Fig. 3. Neighbour-joining tree of the 143 bp of mitochondrial cytochrome b region sequence for all albatross species and archaeological bones. The sequences for modern albatross species are from Nunn et al. [26]. Numbers in parentheses represent the accession numbers in GenBank. Bootstrap values over 50% obtained from 1000 resamplings are shown.
modern molecular albatross studies have been conducted; (2) only one sample was ground to powder in one day; (3) no negative controls yielded any PCR products when extraction and PCR included any sequenced samples; (4) amplification and sequencing were independently replicated for all positive samples and identical results were obtained; (5) the phylogenetic relationships of all samples in the mtDNA cytochrome-b region sequence were completely consistent with the relationships
of the mtDNA control region sequence (unpublished data); and (6) authenticity of the determined sequences was also supported by replicated extractions or uniqueness of the control region sequences. Therefore, it is very unlikely that a serious level of contamination occurred and that the inconsistent results came from misidentification in DNA analysis. The misidentifications in mensural-based classifications, or case (b), seem to be the most plausible
M. Eda et al. / Journal of Archaeological Science 33 (2006) 349e359
explanation for the inconsistent identification of archaeological remains. Although this could result from the underestimation of the size range of modern species due to the insufficient number of referred samples, especially for the Short-tailed Albatross, the effect is likely minimal. This is because (1) there was no significant difference among covariance matrices for three species; and (2) coefficient of variance for the Short-tailed is not smaller than for the other two species. The misidentifications, therefore, would result from the temporal bone size variations in the Short-tailed Albatross. Temporal bone size variations during the Holocene and the late Pleistocene were recorded for some terrestrial mammals, such as fox [6], wild boar [35], deer [31,35], and pocket gopher [13]. Temporal bone size variations were considered to reflect temporal body size variations owing to variations in environment, such as fluctuation in temperature, habitat fragmentation and/or depression in the food supply, although it was difficult to determine what the actual cause was. The Short-tailed Albatross may have experienced such kinds of events that might have changed their body size. It may also be due to excessive hunting on their breeding islands in the late 19th and the early 20th centuries. Although it is estimated that there were about six million individuals and more than 14 breeding areas in the late 19th century, the recent number of birds and breeding areas are about 1500 and two, respectively [14,15]. Such a catastrophic population reduction might have affected the body size through relaxed intraspecific competition for food. This inference is consistent with the fact that many of the Short-tailed Albatross remains were much smaller in size than modern specimens of the species. Alternatively, most of the archaeological remains from the Hamanaka 2 site might be from extinct population(s) or breeding region(s) with carpometacarpi morphologically different from birds of Torishima Island where at least half of the Short-tailed Albatross specimens in this study were obtained. Intraspecific morphological variations have been reported for many avian species, even if there was no genetic difference among breeding regions [17,33,38]. For example, the Greyheaded Albatross (Thalassarche chrysostoma) showed no genetic difference in their mtDNA control region among breeding islands, but their body size significantly decreased in direct correlation to distance travelled between their colonies and foraging areas [3,38]. Further studies of Short-tailed Albatross remains and recent breeding colonies are required to examine this possibility. It is important to note that the understanding of human foraging areas and albatross ecology will drastically change depending upon the results of species identification based on mensural- and DNA-based criteria. For the understanding of human foraging areas, the proportion of Laysan and Black-footed Albatross bones
357
in the albatross remains will be important, since these species have been frequently observed in offshore areas [39,40]. In the mensural-based identifications, the Laysan and Black-footed Albatross bones provided approximately half of the albatross remains at the Hamanaka 2 site, and this suggests that human inhabitants of this site often hunted in offshore areas. On the contrary, the DNA-based identification did not suggest offshore hunting, since there were no or, at most, few Laysan and Black-footed Albatross bones. From the perspective of albatross ecology, we [7] suggest that coastal areas along the Seas of Japan and Okhotsk have rarely been used by albatrosses in modern times, though formerly there were many albatrosses. We proposed two explanations for the shrinkage of their distribution range: (1) excessive hunting in the breeding areas; and (2) distributional changes of prey for albatrosses. Knowing which albatross species are and were distributed through the Seas of Japan and Okhotsk will be important to narrow down these two hypotheses, although further studies of other archaeological sites are required to judge whether only the Shorttailed Albatross was found in the Seas of Japan and Okhotsk. In conclusion, this study clearly suggests that temporal size changes in bones distort the species identification of archaeological remains when based on modern reference sample size variations. Further studies are required to judge if this pattern is unusual or not. At the moment, we would like to recommend careful species identification for genera in which some species experienced drastic reductions of demography and distribution. This study also suggests that combining ancient DNA and mensural analyses will be useful to reveal changes in ecological conditions for many species of animals.
Acknowledgements We are very grateful to Dr Ushio Maeda, who gave us the opportunity to study albatross remains from Hamanaka 2 site. We would like to express our gratitude to Drs Kenichi Shinoda and Shin Nishida who greatly supported DNA analysis. Becky Williams and Dr Carla Cicero of the Museum of Vertebrate Zoology, at the University of California Berkeley and Dr Kevin Winker of the University of Alaska Museum provided caliper measurement data. Judith Porcasi gave us valuable information on albatross studies on the west coast of the USA. Hiroshi Hasegawa, Kazuto Kawakami, Mitsuru Moriguchi and Yuichi Osa kindly allowed us to measure the modern specimens. We are indebted to the institutions and curators who allowed us to examine their specimens: the American Museum of Natural History; the Smithsonian Institution; the National Science Museum, Tokyo; the Yamashina Institute for Ornithology; the Peabody Museum, Yale University; and the
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Natural History Museum, Tring, UK. Dr Brian Chisholm and Anthony Hemmens spent time correcting our English draft. Comments from two anonymous reviewers made the paper more valuable. This study was financially supported in part by the Sasakawa Scientific Research Grant from The Japan Science Society for M.E. Finally, we pray for the repose of the souls of many Short-tailed Albatrosses, which played such a very important role in our study.
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[20]
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Do temporal size differences influence species identification of archaeological albatross remains when using modern reference samples? Masaki Eda a,*, Yoshiyuki Baba b, Hiroko Koike b, Hiroyoshi Higuchi a a b
School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo, 113-8657, Japan Graduate School of Social and Cultural Studies, Kyushu University, Ropponmatsu 4-2-1, Chuo-ku, Fukuoka City, Fukuoka, 810-8560, Japan Received 4 October 2004; received in revised form 14 June 2005; accepted 27 July 2005
Abstract Zooarchaeological remains have been identified to species, using identification criteria based on specific morphological variations among modern specimens. However, temporal size changes in bones, due to micro-evolution and/or phenotypic plasticity, could distort identification of archaeological remains according to these criteria. We developed species identification criteria for North Pacific albatrosses (Short-tailed, Laysan and Black-footed Albatrosses) using both mensural- and DNA-based analysis and actually identified many archaeological remains from a site using these criteria. Our mensural-based criteria could accurately discriminate the modern Short-tailed Albatross from modern Laysan and Black-footed Albatrosses and indicated that the archaeological remains included both Short-tailed and Laysan or Black-footed Albatrosses. DNA-based criteria, however, suggested that all remains were Short-tailed Albatross. The most plausible explanation for this inconsistency would be misidentification using mensural-based analysis, due to temporal size changes in bones or existence of birds from extinct population(s) or breeding region(s) with mensurally different bones from recent birds. This is the first study that suggests temporal size changes in bones may distort the species identification of archaeological remains according to modern size variations. Further studies are required to judge if this pattern is unusual or not. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Species identification; Ancient DNA; Short-tailed Albatross; Microevolution; North Pacific; Avian bone; Zooarcheology
1. Introduction Identification of archaeological remains is the basis of zooarchaeology. Referring to identified animals and their ecology, the seasonality of site formation and the foraging areas of inhabitants have been discussed [9,10,32]. Generally, archaeological remains have been * Corresponding author. Graduate School of Social and Cultural Studies, Kyushu University, Ropponmatsu 4-2-1, Chuo-ku, Fukuoka City, Fukuoka, 810-8560, Japan. Tel.: C81 92 726 4671; fax: C81 92 726 4847. E-mail address: [email protected] (M. Eda). 0305-4403/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jas.2005.07.017
identified to the species level, using identification criteria based on the specific morphological variation among modern specimens. These identification criteria tacitly assume that morphological characters have been temporally invariable, or never jumped across species ranges even if some changes occurred (e.g. [11]). On the other hand, it has been revealed that the bone morphology, especially size, of some terrestrial mammals changed due to micro-evolution and/or phenotypic plasticity since the beginning of the Holocene or the late Pleistocene [6,13,31,35]. These temporal bone size variations have been considered as resulting from variations in environment, such as fluctuations in temperature, habitat
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fragmentation and/or depression of the food supply. Therefore, species identification criteria that enable us to analyse archaeological remains independent of morphology are required. Techniques for ancient DNA analysis, developed during the last decade, have been used effectively for species identification of archaeological remains that were difficult to classify using morphology-based identification [1,2,22,25,41]. For example, Butler and Bowers [2] suggested that Oncorhynchus nerka whose bones are morphologically indistinguishable from other Oncorhynchus species were included in archaeological remains. Newman et al. [25] showed that even highly fragmented and morphologically unidentifiable archaeological bones could be classified into sheep and goats. Furthermore, Loreille et al. [22] and Bar-Gal et al. [1] compared morphology-based identification for sheep and goats with DNA-based identification, and suggested that morphology-based identification criteria were almost correct. In this study, we identified albatross remains by ancient DNA analysis and compared the results with species identification based on morphological variations in modern specimens. Many albatross (Diomedeidae) remains have been found at Holocene archaeological sites in the northern Pacific coastal regions of the Japanese Archipelago (reviewed in [7]), the Aleutian Islands (e.g. [21,42]), and the west coast of North America (e.g. [4,12,30]). They are easily identified to family and genus levels. This is because the wing and leg bones are characterized by a much longer and flatter shape than those of other bird families [9], and there is only one genus (Phoebastria) in this region [5,36]. However, species identification within the genus, namely of Short-tailed (P. albatrus), Laysan (P. immutabilis) and Black-footed Albatrosses (P. nigripes), has not been elucidated, since no research has been conducted on the appropriate identification criteria using sufficient numbers of osteological specimens, especially for the Short-tailed individuals (see also [27e 29,42]). Correct species identification of these albatross remains would be useful for understanding human foraging areas. This is because they included a species that was mostly observed in the continental shelf region (Short-tailed Albatross [36]) and two species that were abundant over the outer continental shelf (Laysan and Black-footed Albatrosses [39,40]). Therefore, the proportion of Laysan and Black-footed Albatross bones in the archaeological remains would be expected to increase with increased foraging over the outer continental shelf. At the same time, correct species identification is also required to understand albatross ecology, since numerous sites at which albatross remains have been found are located in coastal regions of the Seas of Japan and Okhotsk where few albatrosses have been distributed recently (reviewed in [7]).
In this study, we examined whether temporal size changes in bones distort the species identification of albatross remains according to size variations in modern specimens. First, we developed identification criteria based on modern specimens using two approaches: traditional caliper measurements, and DNA analysis that enables us to identify the materials independent of their morphological characters. Second, we classified albatross remains according to the two identification criteria. Finally, we compared the results with each other.
2. Materials One-hundred and five carpometacarpi of recent albatrosses (13 Short-tailed, 60 Laysan and 32 Black-footed Albatrosses) were collected to provide identification criteria using caliper measurements. These were provided by the American Museum of Natural History; the Smithsonian Institution (USNM); the Museum of Vertebrate Zoology, at the University of California Berkeley (MVZ); the National Science Museum, Tokyo (NSMT); the Yamashina Institute for Ornithology; the University of Alaska Museum (UAM); the Peabody Museum, Yale University; the Natural History Museum, Tring, and the personal collections of H. Hasegawa, K. Kawakami, M. Moriguchi, Y. Osa, and one of the authors (M. E., Table 1). Three of the carpometacarpi (one of each of the species) exhibited more or less incomplete ossification at the proximal joint of the Os metacarpale alurare and majus. We defined this character as a landmark for ‘‘juveniles’’, since the character was observed in a juvenile Short-tailed Albatross (USNM018224) and not observed for any other measured known age carpometacarpi including specimens described as immature Short-tailed Albatross (USNM567025) and three year old Blackfooted Albatross (NSMT388). Those ‘‘juvenile’’ specimens were excluded from the following analysis to avoid the influence of age related size difference. No osteological characters differentiating the albatross species were found on the element, although Yesner [42] indicated that the tuberosity of metacarpal II, facets for fused digits II/III, and ligamental attachment of the pisiform process of the Short-tailed Albatross were more developed than those of the Laysan Albatross based on a small sample of only one Short-tailed and four Laysan Albatross specimens. DNA sequences were compared to those reported by Nunn et al. [26]. Thirty-five left-side intact carpometacarpi that were classified as Diomedeidae were collected from the Hamanaka 2 site on Rebun Island, Hokkaido, northern Japan (Fig. 1). The reasons we selected this bone element were that it usually represented the minimum number of individuals and was preserved more intact than other elements in the site [8]. All of these bones were determined to be about 1000 years old, because they were
Table 1 Range, mean, standard deviation (SD) and coefficient of variance (V) for proximal breadth (Bp), distal diagonal (Did) and the greatest length (GL), of North Pacific modern albatross carpometacarpi Species
Sex
Short-tailed
All F U
Black-footed
All F
M
U
Laysan
All F
M
U
Sampling location
Non-breeding area Torishima Island, Izu Islands Minami-kojima Island, Senkaku Islands Non-breeding area
N
12 1 6 1
Bp
Did Mean
SD
V
Range
Mean
SD
V
Range
Mean
SD
V
19.9e23.2
21.3 21.0 21.6 21.9
1.0
4.6
12.7e14.9
0.7
5.0
107.2e126.7
4.7
13.2e14.7
0.7
111.7e126.7
115.6 107.2 118.0 113.9
5.4
1.1
14.0 13.9 14.1 14.0
12.7e14.9
13.8
1.0
110.6e120.1
114.5
4.3
11.6e13.5
0.5
98.6e113.7
3.9
20.4e23.2
19.9e21.8
20.9
0.9
31 1 5
18.2e20.7 19.5e20.0
19.39 19.1 19.6
0.6
Hawaii Islands Northwestern Hawaii Islands Non-breeding area Northwestern Hawaii Islands
1 5
19.2e20.7
19.3 20.0
Non-breeding area Northwestern Hawaii Islands Ogasawara Islands Torishima Island, Izu Islands Non-breeding area or unknown
3 1 3 7 5
59 1 9
Non-breeding area Hawaii Island, Hawaii Islands Northwestern Hawaii Islands
2 1 7
Non-breeding area Northwestern Hawaii Islands
2 4
Mukojima Island, Ogasawara Islands Non-breeding area or unknown
1 32
Included specimens
Range
4
Hawaii Island, Hawaii Islands Northwestern Hawaii Islands
GL
19.5e20.2 19.0e19.9 18.2e19.7 18.6e20.6
17.7e20.5 18.3e19.4 18.0e18.4
0.2
12.7e13.2
12.6 12.3 12.8
0.6
12.8e13.5
12.3 13.1
19.7 19.2 19.5 18.9 19.2
0.4
11.8e13.1
0.5 0.6 0.8
12.2e12.9 11.6e12.6 12.2e13.1
18.9 18.9 18.7
0.6 0.4
19.0e20.5
18.2 18.9 19.3
0.5
18.6e19.3 18.0e19.5
18.9 18.7
0.6
3.2
2.9
10.3e13.1 11.4e12.1 11.9e12.0
19.0
103.7e109.1
0.3
110.0e113.7
106.5 111.6
12.6 12.1 12.6 12.1 12.5
0.7
107.1e111.4
0.4 0.4 0.4
102.6e110.5 98.6e108.4 101.6e110.2
12.1 11.8 11.8
0.5
11.9e13.1
11.9 12.3 12.6
0.4
12.2e12.3 11.7e12.6
12.2 12.1
0.4
18.6 17.7.e20.0
0.2
106.7 103.6 107.2
0.2
4.0
4.2
94.2e109.6 94.2e101.9 96.8e98.2
0.6
10.3e13.0
12.0
108.8 106.4 106.6 102.6 106.4
2.3
100.3 96.7 97.4
3.2 2.6
2.2
99.1e102.0 96.4e105.1
100.5 100.3
3.8
100.9
ANM25837 USNM289170, 289171, 488173, 488176, 497964 MVZ175960 USNM289168, 289169, 488172, 488174, 488175 MVZ72294; USNM622480; YIO-60004 USNM497920 KP12-1, 12-2; NSMT388 EP166-169; NSMT390, 391; YIO-60005 OPbfa001; USNM19740, 432184; YIO-60001, 60002
4.0 3.4 4.0
100.0e107.2
96.1e109.6
3.7
1.5
98.4 0.6
MVZ28980; UAM5723; USNM018225; YPM106517
2.1
97.5 98.8 102.8
11.9
USNM567025 EP097, 170, 171; NSMT283, 285, 389 HP001
5.5
3.2
3.2 ANM25839 MVZ142647; USNM289156, 289158, 289161, 488178-488181, 498067 EP130, 145 ANM25838 USNM289160, 488177, 497965, 498137-498139, 500859 ANM18657; MVZ177560 MVZ142648; BMNH S/1967.8.3; USNM289155, 498120 KP11-1 ANM9837-9839, 9841-9844, 9846, 9847, 10123, 10125-10138; EP022, 098, 151; KP11-4; MP001, 002; OPlya001; YIO-60003
Abbreviated designations are: female (F); male (M); sex unknown (U); the American Museum of Natural History (ANM); the Smithsonian Institution (USNM); the Museum of Vertebrate Zoology, at the University of California Berkeley (MVZ); the National Science Museum, Tokyo (NSMT); the Yamashina Institute for Ornithology (YIO); the University of Alaska Museum (UAM); the Peabody Museum, Yale University (YPM); the Natural History Museum, Tring, UK (BMNH); personal collections of H. Hasegawa (HP); K. Kawakami (KP); M. Moriguchi (MP); Y. Osa (OP); M.E (EP).
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Hamanaka 2 site
0
2.0
(km)
Rebun Is. Russia
China Hokkaido
Honshu Shikoku Kyushu
Fig. 1. Location of the Hamanaka 2 study site, Hokkaido, northern Japan.
M. Eda et al. / Journal of Archaeological Science 33 (2006) 349e359
found with pottery and stone tools from the Okhotsk period, between the 7th and 12th centuries AD [24]. Judging from our morphological criterion, 12 of them were ‘‘juvenile’’ and excluded from the following analysis.
3. Methods 3.1. Caliper measurements Three caliper distances were measured for each recent specimen following von den Dreisch [37]: greatest length (GL), proximal breadth (Bp), and distal diagonal (Did). Most measurements were conducted by M.E., while measurements for specimens were provided by Becky Williams and Dr Carla Cicero of the MVZ and Dr Kevin Winker of the UAM. Measurement values for each specimen were classified into groups by species, sex, and sampling location (specific breeding site or nonbreeding area and unknown) to calculate mean, range (for groups including more than one specimen), standard deviation (for groups including more than two specimen), and coefficient of variance (for each species). Homogeneity test of covariance matrices and discriminant function analysis were performed using SYSTAT ver. 8.0 (SPSS Inc.). Discriminant function analysis was performed using the linear discriminant function, since covariance matrices were not significantly different among species (see Section 4). The leave-one-out classification was performed to test the robustness of the identification criteria. Each archaeological albatross specimen was classified into a particular species according to scores calculated by the classification function. The caliper distances for each specimen were substituted into the classification function for each species, and they were placed into the species whose classification function score was the highest. 3.2. DNA analysis We designed a primer (Lcyt.246.dio: 5#-CCTCCAC GCAAACGGAG-3#) for the mitochondrial cytochrome b region and paired it with H15149.uni (5#-CGGGG AGTCTTACTATAAACAGGAGT-3# [19]) to amplify a 143 bp fragment which allows the distinction of all albatross species, referring to the sequence for modern albatrosses [26]. All DNA extraction and subsequent first polymerase chain reaction (PCR) amplification preparations were performed in the laboratory of H.K. at Kyushu University where no modern molecular albatross studies have previously been conducted. This laboratory was separated from the post-PCR amplification facility. The archaeological albatross remains were cleaned and ground into about 300 mg of powder using a dental-drill sterilized with HCl and by UV. We ground one sample per day at a UV sterilized clean bench.
353
We extracted DNA using the Geneclean kit for Ancient DNA (Q-BIO Gene) with over night preincubation (5 ml of ethylendiaminetetraacetic acid 0.5 M pH 8.0, 200 ml of 10% sodium dodecyl sulphate, 200 ml of 20 mg/ml proteinase K). Blank extractions containing all buffers but lacking any sample were carried out to detect any contamination in buffers and solutions. PCR amplifications were carried out in 25 ml reaction volumes consisting of 1e5 ml of the DNA extract, 0.2 mM of each nucleotide, 0.1e0.2 mM of each primer, 0.2e2 mg/ml of bovine serum albumin and 1.25e2.5 units of Taq polymerase (Takara) in a 1! concentration of the supplied reaction buffer. PCR cycling consisted of the following parameters; step 1, 94 C for 1 min; step 2, 94 C for 30 s, 50e60 C for 45 s, 72 C for 45 s (cycled 40 times) and a final extension at 72 C for 1 min. One microlitre was re-amplified (30 cycles) in a volume of 25 ml with the same primers and parameters. An amplification blank was included in each PCR reaction batch. DNA sequencing of PCR products was carried out in both directions on a CEQ 2000 DNA Analysis System (Beckman Coulter) using the same primers as used for PCR, following purification of the template with a PCR Product Pre-sequencing Kit (USB). Amplification and sequencing were independently replicated for each sample that gave positive amplification. Extraction was also independently replicated for each sample that gave positive amplification and did not have a unique sequence at the mtDNA control region (unpublished data). This was due to finding contamination among DNA samples and amplification errors by Taq polymerase. A neighbour joining (NJ) tree [34] was constructed using Kimura’s two-parameter model [18]. The reliability of specific grouping in the tree was assessed using 1000 bootstrap replicates. The construction of the NJ tree and bootstrap replicates were conducted using MEGA ver. 2.1 [20]. Species identifications were made by comparison of their sequences and positions in the NJ tree to a set of modern sequences.
4. Results 4.1. Caliper measurement On average, the Short-tailed Albatross was the largest; it was followed by the Black-footed and the Laysan Albatrosses, although ranges for each measurement overlapped among the three species (Table 1). The range of the coefficient of variance was from 2.9 to 5.8. There was no significant difference among the covariance matrices for three species (c2 Z 15.57, df Z 12, p O 0.05). The three albatross species differed significantly in their measurements (Pillai’s trace Z 0.84, F Z 23.69, df Z 6, 196, p ! 0.001) and discriminant functions could be calculated (Table 2). In the leave-one-out classification,
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Table 2 Unstandardized discriminant functions and group means for three North Pacific albatross species Measurement
Discriminant function coefficients
Bp Did GL Constant
Species
Function 1
Function 2
0.344 0.456 0.180 ÿ31.077
1.812 0.115 ÿ0.278 ÿ7.611
Group means
Short-tailed Black-footed Laysan
Function 1
Function 2
3.458 0.547 ÿ0.991
0.563 0.217 0.563
Bp, proximal breadth; GL, greatest length; Did, distal diagonal.
4.2. DNA analysis DNA samples from 18 of the 23 (78%) Diomedeidae archaeological carpometacarpi were successfully amplified and provided target sequences (143 bp) in the mtDNA cytochrome b region. They included nine each of the specimens identified as Short-tailed and Blackfooted Albatrosses in the mensural-based identification. The replicated mtDNA sequences derived from each specimen were consistent for all cases. The other five samples (22%) could not be amplified despite more than five trials for each sample. No negative controls yielded any PCR products when the PCR included any sequenced samples. The eighteen 143 bp albatross specimen sequences had one C-T transition site that was a third codon position silent substitution (Table 4). Six sequences had T at the site and the others had C. The former was identical Table 3 Percentage correct classification of modern specimens in leave-one-out classification
Short-tailed Black-footed Laysan Total
Short-tailed
Black-footed
Laysan
%correct
11 3 0 14
1 19 8 28
0 9 51 60
90.9 61.3 86.4 79.4
Columns and rows show the classified and the known species name, respectively.
to the Short-tailed Albatross reference sequence (U48952). The NJ tree showed that all sequences deriving from specimens were monophylic with the reference sequence for modern Short-tailed Albatross, with 68% bootstrap support (Fig. 3).
5. Discussion Using three caliper measurements, 79% of 102 carpometacarpi of modern North Pacific albatrosses were accurately classified into Short-tailed, Black-footed and Laysan Albatrosses. While species identifications between Black-footed and Laysan Albatrosses were confounded, more than 95% of modern albatross specimens were correctly classified into Short-tailed and Blackfooted or Laysan Albatrosses. The criteria would be useful to understand human foraging areas, since Short-tailed and Black-footed or Laysan Albatrosses were observed in different region of the ocean. The criteria were applied to 23 archaeological specimens from the Hamanaka 2 site, and 12 of them were identified as Short-tailed Albatross while the other 11 were Black-footed or Laysan
4
Canonical score 2
79% of all specimens were correctly identified, and Short-tailed, Black-footed and Laysan Albatross specimens were correctly identified with 91%, 61% and 86% accuracy, respectively (Table 3). Most of the misidentifications, 81% of 21 cases, were observed between Black-footed and Laysan Albatrosses, while only 19% were between Black-footed and the Short-tailed Albatross. In the scatter plot of canonical scores for each specimen, most Short-tailed Albatrosses separated from Laysan and Black-footed Albatrosses but the latter two highly overlapped each other (Fig. 2). Twenty-three zooarchaeological albatross remains were classified based on classification function with 12 and 11 specimens being identified as Short-tailed and Black-footed Albatrosses, respectively.
0
-4 -4
4
0
8
Canonical score 1 Fig. 2. A scatter plot of canonical score 1 vs. canonical score 2 for the carpometacarpi of Short-tailed (B), Black-footed (6), and Laysan Albatrosses (>) in discriminant function analysis. Archaeological remains from the Hamanaka 2 site were also included: yield DNA (C) and did not yield DNA (!). Note that all archaeological specimens that provided successful DNA analyses were identified as Short-tailed Albatross.
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M. Eda et al. / Journal of Archaeological Science 33 (2006) 349e359 Table 4 Variable sites for part of the mitochondrial cytochrome b region for all albatross species and archaeological bones Species/sample Variable sites name 2 6 4 Short-tailed A HM-02 $ HM-13 $ HM-18 $ HM-22 $ HM-28 $ HM-35 $ HM-01 $ HM-05 $ HM-06 $ HM-10 $ HM-11 $ HM-16 $ HM-17 $ HM-21 $ HM-24 $ HM-26 $ HM-34 $ HM-39 $ Laysan $ Black-footed $ Waved $ Wandering $ Amsterdam $ Royal $ Grey-headed $ Black-browed $ Yellow-nosed $ Buller’s $ Shy $ Light-mantled $ Sooty Dark-mantled $ Sooty Grey Petrel C
2 7 0 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T $ $ $
2 7 3 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
2 8 2 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T T T T $ $
2 8 5 T $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ C C C C C C C C C C C C
Genbank accession no. 2 8 8 T $ $ $ $ $ $ C C C C C C C C C C C C $ $ $ C C $ C C C C C C
2 9 1 T $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ C $ $ $ $ C $ $ $ $ $ C
2 9 7 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
3 0 0 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T
3 0 5 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A $ T T T T T T
3 0 8 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ G $ C $ $ $ $ $ $ $
3 1 4 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T
3 2 0 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T $ T
3 2 4 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ G C G G $ G
3 2 6 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T T T T $
3 2 7 G $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A A $ $ $ $ $ $ A
3 2 9 T $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ C A A A A A $
3 3 5 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T $ $ $ $ $ $
3 3 6 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ G G $ $ $ $ $ $ $ $ $
3 3 8 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ G $ $ $ $ $ $ $ $ $
3 4 4 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T
3 5 1 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T $ $ $ $ $ $ $ $ $
3 5 3 G $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A A A A A $
3 5 4 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
3 5 6 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A $ $ $ $ $ $ $ $
3 5 9 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ G $ $
3 6 0 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
3 6 6 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
3 6 8 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T T T $ $ $ $ $ $
3 7 1 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T T T T T $ $
3 8 0 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T $ $ $ $ $ $ $ $ $ $
3 8 3 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T T T T T T
3 9 2 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ T
3 9 5 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A G A A A A
3 9 8 T $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ C C $ $ $
3 9 9 T $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ C C C $ $ $ C C C C $ C
4 0 1 C $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
4 0 4 G $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ A A A A A A A A A
4 0 5 A $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $
U48952
U48949 U48950 U48951 U48947 U48948 U48946 U48954 U48955 U48944 U48945 U48953 U48943
$ T $ C C C $ T T $ T T G $ A $ $ $ $ T $ $ $ $ $ $ $ $ $ $ T T A $ C $ A $ U48942 $ $ T $ C $ T A A $ $ $ $ $ $ $ $ G $ $ $ $ T A T T T $ $ $ T $ A C C T A C U48940
Numbering is based on the published complete cytochrome b region sequence [26] and dots indicate identity with sequence.
Albatrosses. In contrast, all cytochrome b sequences from the albatross specimens were monophylic with the reference sequence of the modern Short-tailed Albatross. This suggests that these specimens were from the Shorttailed Albatross. Nine of the 18 specimens were classified as Black-footed or Laysan Albatrosses using the mensural-based identification criterion. Results of species identifications based on the two identification approaches could have been inconsistent in three ways: (a) remains of Black-footed or Laysan Albatrosses were classified with the Short-tailed Albatross in the DNA analysis; (b) remains of Short-tailed Albatrosses were classified with the Black-footed or the Laysan Albatrosses in the mensural analysis; and (c) remains included many individuals that were hybrids of the Short-tailed and the Black-footed or the Laysan Albatrosses. The last case (c) is unlikely to explain all of the
inconsistent results, since no such hybrid birds have been identified in more than 25 years on Torishima Island, the largest breeding ground for the Short-tailed Albatross (H. Hasegawa, personal communication). If misidentifications occurred in the DNA-based classification, a serious level of contamination must be supposed. On the other hand, if misidentifications occurred in the mensural-based classification, the size of the bones may have changed and obscured species differences over the last millennium. It is unlikely that misidentification occurred in the DNA-based identification, i.e. case (a), although the authenticity of deduced sequences has been the most controversial problem in ancient DNA studies [16,23]. We believe that the results in this study are fully reliable. This is because (1) DNA extraction and first PCR preparation were performed in a laboratory where no
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HM-01 HM-05 HM-06 HM-10 HM-11 HM-16 HM-17 61 HM-21 HM-24 HM-26 HM-34
68
HM-39 Short-tailed (U48952) HM-04 HM-30 53
HM-38 HM-04
71
HM-30 HM-38 Laysan (U48949) Black-footed (U48950) 75
Waved (U48951) Royal (U48946) Wandering (U48947)
75 71
Amsterdam (U48948) Gray-headed (U48954) Buller’s (U48945)
68
55
Yellow-nosed (U48944) Black-browed (U48955)
64
Shy (U48953) Light mantled Sooty (U48943) 99
Dark-mantled Sooty (U48942) Gray petrel (U48940)
0.02 Fig. 3. Neighbour-joining tree of the 143 bp of mitochondrial cytochrome b region sequence for all albatross species and archaeological bones. The sequences for modern albatross species are from Nunn et al. [26]. Numbers in parentheses represent the accession numbers in GenBank. Bootstrap values over 50% obtained from 1000 resamplings are shown.
modern molecular albatross studies have been conducted; (2) only one sample was ground to powder in one day; (3) no negative controls yielded any PCR products when extraction and PCR included any sequenced samples; (4) amplification and sequencing were independently replicated for all positive samples and identical results were obtained; (5) the phylogenetic relationships of all samples in the mtDNA cytochrome-b region sequence were completely consistent with the relationships
of the mtDNA control region sequence (unpublished data); and (6) authenticity of the determined sequences was also supported by replicated extractions or uniqueness of the control region sequences. Therefore, it is very unlikely that a serious level of contamination occurred and that the inconsistent results came from misidentification in DNA analysis. The misidentifications in mensural-based classifications, or case (b), seem to be the most plausible
M. Eda et al. / Journal of Archaeological Science 33 (2006) 349e359
explanation for the inconsistent identification of archaeological remains. Although this could result from the underestimation of the size range of modern species due to the insufficient number of referred samples, especially for the Short-tailed Albatross, the effect is likely minimal. This is because (1) there was no significant difference among covariance matrices for three species; and (2) coefficient of variance for the Short-tailed is not smaller than for the other two species. The misidentifications, therefore, would result from the temporal bone size variations in the Short-tailed Albatross. Temporal bone size variations during the Holocene and the late Pleistocene were recorded for some terrestrial mammals, such as fox [6], wild boar [35], deer [31,35], and pocket gopher [13]. Temporal bone size variations were considered to reflect temporal body size variations owing to variations in environment, such as fluctuation in temperature, habitat fragmentation and/or depression in the food supply, although it was difficult to determine what the actual cause was. The Short-tailed Albatross may have experienced such kinds of events that might have changed their body size. It may also be due to excessive hunting on their breeding islands in the late 19th and the early 20th centuries. Although it is estimated that there were about six million individuals and more than 14 breeding areas in the late 19th century, the recent number of birds and breeding areas are about 1500 and two, respectively [14,15]. Such a catastrophic population reduction might have affected the body size through relaxed intraspecific competition for food. This inference is consistent with the fact that many of the Short-tailed Albatross remains were much smaller in size than modern specimens of the species. Alternatively, most of the archaeological remains from the Hamanaka 2 site might be from extinct population(s) or breeding region(s) with carpometacarpi morphologically different from birds of Torishima Island where at least half of the Short-tailed Albatross specimens in this study were obtained. Intraspecific morphological variations have been reported for many avian species, even if there was no genetic difference among breeding regions [17,33,38]. For example, the Greyheaded Albatross (Thalassarche chrysostoma) showed no genetic difference in their mtDNA control region among breeding islands, but their body size significantly decreased in direct correlation to distance travelled between their colonies and foraging areas [3,38]. Further studies of Short-tailed Albatross remains and recent breeding colonies are required to examine this possibility. It is important to note that the understanding of human foraging areas and albatross ecology will drastically change depending upon the results of species identification based on mensural- and DNA-based criteria. For the understanding of human foraging areas, the proportion of Laysan and Black-footed Albatross bones
357
in the albatross remains will be important, since these species have been frequently observed in offshore areas [39,40]. In the mensural-based identifications, the Laysan and Black-footed Albatross bones provided approximately half of the albatross remains at the Hamanaka 2 site, and this suggests that human inhabitants of this site often hunted in offshore areas. On the contrary, the DNA-based identification did not suggest offshore hunting, since there were no or, at most, few Laysan and Black-footed Albatross bones. From the perspective of albatross ecology, we [7] suggest that coastal areas along the Seas of Japan and Okhotsk have rarely been used by albatrosses in modern times, though formerly there were many albatrosses. We proposed two explanations for the shrinkage of their distribution range: (1) excessive hunting in the breeding areas; and (2) distributional changes of prey for albatrosses. Knowing which albatross species are and were distributed through the Seas of Japan and Okhotsk will be important to narrow down these two hypotheses, although further studies of other archaeological sites are required to judge whether only the Shorttailed Albatross was found in the Seas of Japan and Okhotsk. In conclusion, this study clearly suggests that temporal size changes in bones distort the species identification of archaeological remains when based on modern reference sample size variations. Further studies are required to judge if this pattern is unusual or not. At the moment, we would like to recommend careful species identification for genera in which some species experienced drastic reductions of demography and distribution. This study also suggests that combining ancient DNA and mensural analyses will be useful to reveal changes in ecological conditions for many species of animals.
Acknowledgements We are very grateful to Dr Ushio Maeda, who gave us the opportunity to study albatross remains from Hamanaka 2 site. We would like to express our gratitude to Drs Kenichi Shinoda and Shin Nishida who greatly supported DNA analysis. Becky Williams and Dr Carla Cicero of the Museum of Vertebrate Zoology, at the University of California Berkeley and Dr Kevin Winker of the University of Alaska Museum provided caliper measurement data. Judith Porcasi gave us valuable information on albatross studies on the west coast of the USA. Hiroshi Hasegawa, Kazuto Kawakami, Mitsuru Moriguchi and Yuichi Osa kindly allowed us to measure the modern specimens. We are indebted to the institutions and curators who allowed us to examine their specimens: the American Museum of Natural History; the Smithsonian Institution; the National Science Museum, Tokyo; the Yamashina Institute for Ornithology; the Peabody Museum, Yale University; and the
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Natural History Museum, Tring, UK. Dr Brian Chisholm and Anthony Hemmens spent time correcting our English draft. Comments from two anonymous reviewers made the paper more valuable. This study was financially supported in part by the Sasakawa Scientific Research Grant from The Japan Science Society for M.E. Finally, we pray for the repose of the souls of many Short-tailed Albatrosses, which played such a very important role in our study.
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