How To Classify The Animal Called A Moa

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Peer-reviewed

Research Article

Identification, Nomenclature, and Growth of Moa Chicks (Aves: Dinornithiformes) from the Genus Euryapteryx

• Leon Huynen,
• Brian J. Gill,
• Anthony Doyle,
• Craig D. Millar,
• David M. Lambert

10

• Published: June 12, 2014
• https://doi.org/10.1371/periodical.pone.0099929

Figures

Abstruse

Background

The analysis of growth in extinct organisms is difficult. The general lack of skeletal cloth from a range of developmental states precludes determination of growth characteristics. For New Zealand's extinct moa nosotros take available to us a choice of rare femora at different developmental stages that accept allowed a preliminary determination of the early on growth of this giant flightless bird. We use a combination of femora morphometrics, ancient DNA, and isotope analysis to provide information on the identification, classification, and growth of extinct moa from the genus Euryapteryx.

Results

Using ancient Deoxyribonucleic acid, nosotros identify a number of moa chick bones for the species Euryapteryx curtus, Dinornis novaezealandiae, and Anomalopteryx didiformis, and the beginning chick bone for Pachyornis geranoides. Isotope assay shows that ∂¹⁵Northward levels vary between the two known size classes of Euryapteryx, with the larger size class having reduced levels of ∂¹⁵Northward. A growth series for femora of the two size classes of Euryapteryx shows that early femora growth characteristics for both classes are near identical. Morphometric, isotopic, and radiographic analysis of the smallest Euryapteryx bones suggests that i of these femora is from a freshly hatched moa at a very early stage of development.

Decision

Using morphometric, isotopic, and ancient Dna analyses take allowed the determination of a number of characteristics of rare moa chick femora. For Euryapteryx the analyses advise that the smaller sized class II Euryapteryx is identical in size and growth to the extant Darwin'due south rhea.

Commendation: Huynen L, Gill BJ, Doyle A, Millar CD, Lambert DM (2014) Identification, Classification, and Growth of Moa Chicks (Aves: Dinornithiformes) from the Genus Euryapteryx. PLoS I ix(6): e99929. https://doi.org/x.1371/journal.pone.0099929

Editor: Tom Gilbert, Natural History Museum of Denmark, Denmark

Received: Feb 18, 2014; Accepted: May twenty, 2014; Published: June 12, 2014

Copyright: © 2014 Huynen et al. This is an open-access article distributed nether the terms of the Artistic Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The authors thank the Australian Research Quango for support (Grant number DP110101364; The molecular evolution of wings in ratites). The funders had no role in study pattern, data collection and analysis, decision to publish, or grooming of the manuscript.

Competing interests: The authors accept alleged that no competing interests exist.

Background

In depth analysis of growth in aboriginal animals is often limited due to the scarcity and degraded nature of skeletal material or tissues of unlike ages. Similarly, the rare occurrence of different anile basic for New Zealand's extinct ratite moa (Aves: Dinornithiformes) has made any analysis of moa growth difficult [1], [2], [3].

Adult moa ranged in size from less than 20 kg for the small-scale coastal moa Euryapteryx curtus curtus to over 200 kg for the South Island giant moa Dinornis robustus [two]. The identification of species within the Euryapteryx genus has been specially difficult. Latest data suggest the being of a small subspecies (E. curtus curtus) limited to New Zealand'southward North Island, and a larger subspecies (E. curtus gravis) institute only in New Zealand's S Isle [4].

How moa grew is largely unknown with about published work comparing moa to the growth characteristics of their extant relatives [two], [3]. Relatively recent work analysing cortical growth marks in moa limb basic suggest that, dissimilar their modernistic relatives, moa had a particularly long pre-adult growth menses [5].

We analyse moa growth using a number of rare moa chick femora, currently housed at New Zealand'due south Auckland Museum and kindly fabricated available to the states. The museum houses a significant number of samples of moa chick bones from sand-dune sites in New Zealand's upper North Isle, particularly from the Karikari Peninsula/Doubtless Bay area, including Tokerau Beach. Adult basic from these sites have been attributed to three moa species with most being derived from E. curtus curtus [2], [six], [7].

To date, but i embryonic moa has been identified to species, where basic associated with an egg were shown to belong to the heavy-footed moa Pachyornis elephantopus [2], [8]. Equally basic of developing chicks, which often lack identifying characters, are particularly hard to place [1], [two] we use a minimally subversive technique to genetically assign differently sized immature moa femora to the species level. We then use bone morphometrics to present a growth serial of chick femora for Euryapteryx. In addition, we present isotope and radiographic data for the smallest moa femora to decide whether these may take derived from unhatched eggs. The isotope data has also allowed us to further explore the status of two subspecies proposed for moa from the genus Euryapteryx [9].

Results

We successfully amplified a relatively short (∼70 bp) hypervariable mitochondrial Deoxyribonucleic acid fragment from 29 of 32 immature bones sampled from various locations in New Zealand (Tables 1,two; Figures 1,2). Femur LB6261c was identified as belonging to Dinornis novaezealandiae and is 72 mm long (Table 2, Figure 2). Bones of a late-term embryonic moa (identified every bit Pachyornis elephantopus) were recovered from within an egg in 1866 [2], [eight]. The egg was 226 mm long and 155 mm wide with the embryonic femur beingness approximately 48 mm long (with ends restored). An egg institute at Kaikoura and attributed to Dinornis was shown to exist 240 mm long, and by proportion, its embryo (if at the same phase as the Pachyornis egg) would take had a femur approximately 51 mm long. Therefore the size of LB6261c suggests information technology was from a recent hatchling. Further prove for the extreme immaturity of this femur is the lack of caudal tuberosities on the femur shaft, a feature of the femur that separates Dinornis from the emeid moas [one], [2]. Turvey and Holdaway (2005) [3] described 'postnatal' bones of Dinornis and showed that femora at growth-stage 1 began at 156 mm in length with their youngest stage 1 individual having an estimated weight of fifteen.eight kg. Thus their sample included only well-grown chicks and did not include hatchlings. The unmarried Pachyornis geranoides femur: At 82 mm long, LB7976 (Table two) is likely to be from a well-developed chick, since this moa species is relatively small. Although difficult to determine due to erosion, an excavation at the proximal stop of the bone may exist the pneumatic fossa that characterises this species [one], [2]. LB7976 is the only known chick bone of this species. Four Anomalopteryx didiformis femora were identified; AIM LB6666c, AIM LB6261a-b, and AIM LB6285a (Tabular array 2), all derive from Doubtless Bay, and are the first chick basic to be identified (by DNA) for this species, as well as existence the first record of this species from Doubtless Bay. For Euryapteryx curtus 22 femora specimens were identified by Deoxyribonucleic acid, and include the 11 smallest femora (come across Figure 2). These form the kickoff big sample of chick bones attributable with certainty to this species, and were used for detailed morphological, radiographic, and isotopic analyses. The sequence targeted allows discrimination of all moa species and identifies two distinct genetic haplotypes for Euryapteryx. The haplotypes can be separated by a single SNP that assembly class I Euryapteryx with thick eggshells (0.98 mm - 1.60 mm) and class II Euryapteryx with sparse eggshells (0.74 mm–0.98 mm) [6]. The distribution of the two Euryapteryx classes (I and Ii) closely mimics the distribution proposed for subspecies East. curtus gravis and East. curtus curtus respectively [four].

Figure 2. Examples of moa chick femora.

From left to right: LB5990, LB8295, LB6070, LB6261d, LB6284, LB6261c, LB12961, LB6285b, LB6657, LB6071, LB6069. All are left femora except the ii at far right. DNA analysis suggests all are from Euryapteryx except for LB6261c which was identified every bit Dinornis. Gridlines are at 10 mm intervals.

https://doi.org/x.1371/journal.pone.0099929.g002

The length and width of each femur was recorded for both Euryapteryx classes and compared to obtain a growth serial (Figure 3). No deviation could be found in early on femora growth characteristics betwixt the two Euryapteryx classes. Morphometric and mass calculations were carried out on the smallest Euryapteryx femur (LB12960; belonging to class II Euryapteryx) to determine whether this femur may take been from an unhatched egg. Two femur-based equations exist that are commonly used to determine avian mass; one using least shaft circumference provided past [15] and i using total femur length [11] (see methods). Although both methods are relatively accurate for the conclusion of mass of an avian developed, mass calculation using femur length has been shown to be more accurate for developing birds [16]. Using femur length, estimated at c. 47 mm (measured at 44 mm only adding three mm to allow for missing ends) the mass of the LB12960 individual was calculated to be only 470 g. For Darwin'due south rhea, very similar to grade 2 Euryapteryx in adult size (15–28.6 kg) and eggshell thickness (0.73–1.1 mm) [17] newly hatched individuals range in weight from 0.327–0.491 kg with an average weight of 0.426 kg [18]. This suggests that femur LB12960 may have derived from a moa embryo, but is probable to be from a very young newly hatched chick.

Figure 3. Femoral growth serial for chick bones from Euryapteryx curtus.

Graph showing femur length vs width (mm). Blueish - Euryapteryx class I, eI; Red - Euryapteryx course Ii, eII; For comparison, chick bones from other moa species identified are likewise shown. Green - D. novaezealandiae, Dn; Purple - A. didiformis, Ad; Dark-brown - P. geranoides, Pg. A line of best fit is shown in grey.

https://doi.org/10.1371/journal.pone.0099929.g003

Further assay of the developmental stage of the smallest Eurypateryx femora was carried out by radiography. 6 bones were analysed, of which four had essentially intact bone of variable thickness at the ends (Table 3). These ends stand for to the metaphyses of the femora adjacent to the growth plates, the epiphyses having been lost or separated. The ends of intact bones suggest that the cartilage 'cones' described by [19] had already migrated away from the bone ends and growth plates. This is a feature found in their study of rheas only later on the birds were of 3 weeks maturity or more. It is therefore reasonable to assume that these iv chicks were a few weeks former at to the lowest degree. Only two of the moa chick bones had consummate defects in the metaphyses, identifying them as neonatal or embryonic (Figure 4). Ane of these (LB12960) has a slightly larger defect than the other and subjectively has less trabecular bone overall than whatever of the others. These data farther suggest that this bone may take come from a late phase embryo or newly hatched chick, in concordance with the nitrogen isotope (below) and morphometric findings (Tabular array 2).

Figure iv. Radiographic analysis of Euryapteryx class Two femora.

A. Chick LB13978: more mature form with intact bone finish on correct, consistent with chick of a few weeks historic period where cartilage cone has migrated down shaft. B. Chick LB12960: immature bone with open finish on correct, consequent with cartilage cone present up to growth plate as occurs with embryo and neonate.

https://doi.org/10.1371/journal.pone.0099929.g004

Additional analyses were carried out on the smallest femora using isotope counts in the hope that femora from unhatched individuals, by feeding on yolk, take different ∂¹⁵North and/or ∂¹³C levels to femora from hatched chicks feeding on insects. To account for habitat or species-specific biases, isotope levels were determined for several moa species from dissimilar environments including sand-dune, cave, and swamp (Table 4, Figure 5). No species-specific differences in isotope levels were plant among the few samples that were measured. All the same, moa bone samples from caves had very low ∂¹⁵N values (−0.1–two.90 parts per one thousand thousand (^o/oo)), while those from swamps had ∂¹⁵North levels of 4.ii–eight.half dozen ^o/oo and sand-dune samples ranged in ∂^fifteenNorthward from 3.1–5.v ^o/oo, values that are likely to exist indicative of the specific environment (Table four, Figure 5). Isotopic analysis showed relatively low values of ∂¹³C (18.iii–23.one ^o/o) in all Euryapteryx bones suggesting that individuals of this moa species preferred more open habitats and, as expected for New Zealand, a C3-based nutrition [20].

Figure 5. Isotope assay of selected moa bones.

Moa bone samples from unlike species (Table three) and environments were subjected to isotopic analyses. Light blue - cave samples; Orange - Dune samples; Green - Swamp samples; Bluish - Euryapteryx form I, eI; Crimson - Euryapteryx course II, eII; The isolated eII sample is the possible newly hatched Euryapteryx course II individual.

https://doi.org/10.1371/journal.pone.0099929.g005

Equally almost all Euryapteryx chick bones in this study were obtained from the same area, climate and habitat differences are unlikely to be a source of isotope variation. Interestingly, ∂¹⁵N levels between the two Euryapteryx classes showed form I ∂¹⁵N values were generally low and ranged from 2.2–3.half dozen ^o/oo (due north = 3, mean  = 2.83, SD = 0.58) while class II ∂¹⁵N values ranged from 3.5–v.5 ^o/oo (northward = half-dozen, mean  = four.86, SD = 0.71). Although the sample numbers are depression, the class I and class Two∂¹⁵N values proved to significantly different; p = 0.0115, Table 4, Effigy 5). An unusually high ∂¹⁵N level (x.3 ^o/oo) was found for the smallest Euryapteryx bone, AIM LB12960 (Tabular array 4, Figure 5).

Give-and-take

Morphometrics of femora from each variant showed very similar growth curves at the early stages of development. For the closely related emu and ostrich it has been shown that cortical bone thickness remains constant for the offset ii months later hatching, with the highest radial growth charge per unit occurring 7–fourteen days posthatch [21]. Similarly for Euryapteryx, extrapolation of the growth bend to 0 suggests that at the very early stages of development, femur width increased proportionately more than than femur length (Figure 3). For course II Euryapteryx, femur growth is linear until femur length reaches approximately 150 mm.

Isotope analysis gives an indication of diet, climate, and habitat. Carbon 13 (^thirteenC) levels in bone give an indication of whether the plants ingested utilized a C3 or C4 photosynthetic pathway, while Nitrogen xv (¹⁵N) levels provide data on the organisms trophic level [22]. In general, bone ∂¹³C values become more than negative when animals feed in open shrubland, as opposed to vegetation from closed canopy or forested areas [two], [xx], [23]. ∂^fifteenDue north levels are an indication of not only trophic level but tin can likewise be indicative of habitat, climate, and salinity, with loftier ∂¹⁵N values existence more characteristic of individuals found in more saline, hot, dry environments [24]. The Isotope data nosotros obtained provides further testify that the two genetic variants of Euryapteryx curtus are likely to represent subspecies. One of these was likely to be a small sized moa that was associated with thin eggshells. The other was probably a larger sized moa associated with thick eggshells [8]. The smallest Euryapteryx course II bone sample (LB12960) returned a very high ∂¹⁵N value. Loftier ∂^fifteenN values are mostly associated with high trophic levels. It has been plant, for sharks at least, that embryos take elevated ∂¹⁵N levels compared to their mothers [25]. Furthermore ∂^fifteenN values similar to those establish in sample LB12960 take been obtained for egg yolk from insect-fed (high protein) chickens [26] suggesting, in accord with the morphometric and radiographic data, that this femur may have come from within an egg or from a newly hatched individual.

Newly hatched ostrich and emu chicks increase in body mass by at least 50% per month, and attain adult size within ane year [21]. For Euryapteryx, this process is likely to exist much slower with the smaller Euryapteryx curtus curtus (North Island subspecies; adult weight ∼twenty kg; [2] reaching maturity afterward 4 years and the larger Euryapteryx curtus gravis (mainly Southward Island subspecies; adult weight ∼80 kg; [2]) not reaching adulthood for over 9 years [4]. Assuming Eastward. c. curtus and E. c. gravis neonates average 0.43 kg [18] and 0.95 kg respectively (latter weight based on average ostrich hatch weight; [27]), this would suggest a growth charge per unit for Eu. curtus curtus of ∼x% per month and Eu. curtus gravis a very ho-hum ∼5% per calendar month. For this reason, moa chicks were likely to exist highly dependent on developed(southward) back up for a significant period of time. This is somewhat unusual for ratites where extant ratites such as the kiwi, emu, ostrich, and rhea are considered precocious and require piffling developed support across the initial growth stages [17]. In direct contrast to emu, ostrich, and rhea which are subject to predation by goannas, hyenas, and large cats respectively [28] information technology is perhaps fortunate that prior to the arrival of humans, moa had very few natural predators [2]. This scarcity of natural predators may have allowed moa to survive its protracted early stages of evolution.

Nosotros use a variety of analytical methods to gain information on rare chick basic from New Zealand'due south moa. The analyses have allowed the identification of the first chick bone for P. geranoides, the determination of early growth for moa from the genus Euryapteryx, the characterization of a very rare newly hatched Euryapteryx individual, and accept also provided futher evidence for the being of possible subspecies within this genus.

Materials and Methods

Moa bone samples

All moa bone samples were kindly loaned by the Auckland War Memorial Museum (AIM) and Canterbury Museum (CM). Chick bones were identified from the Auckland War Memorial Museum (AIM) moa bone collection by their reduced size in relation to adult bones. Well preserved specimens with known collection locality were selected for sampling. Where possible the left femur was chosen. Permission to sample moa specimens was obtained from the respective museum curators. No permits were required for the described report, which complied with all relevant regulations.

Morphometrics

Maximum femur length was adamant past measuring parallel to the long axis of shaft, equally shown in Figure 4Al of [nine]. Femur width was measured at mid-point forth the shaft, at right angles to the inductive-posterior airplane with the femur head to one side as shown in Figure v.III.D of [10]. Immature femora were measured irrespective of incompleteness due to a lack of fused epiphyses. Measurements were made to the nearest 0.1 mm using Vernier calipers. Moa weight was determined using total femur length where body mass (kg)  =  (femur length (mm)/61.64)^2.7855 according to [11].

Radiography

The smallest moa basic were subjected to computed tomography at high resolution using a multi-detector scanner (Siemens Emotion sixteen, Siemens Medical, Erlangen, Germany). Helical thin slices (0.6 mm, 90 mm field of view, 130 kilovolt peak, 60 milliampere seconds, 512×512 matrix, bone algorithm) were generated and reconstructed into 0.6 mm contiguous slices along and at right angles to the bone. These were viewed and measured on a PC using Siemens software (Syngo fastView, Siemens Berlin, 2009).

Isotope analysis

Isotope analysis was carried out on selected bones at the Australian Rivers Constitute, Griffith University, Commonwealth of australia using the following standards: Primary; N - Ambient air IAEA-305a, C - ANU sucrose, Elemental; Acetanilide Working; 'Prawn'. Mass Spectrophotometry was carried out on a GV Isoprime spectrophotometer (Manchester UK) using a Eurovector EA 3000 inlet. Samples were taken from the outer (radial) layer where possible [12].

DNA extraction and distension

Dna was extracted, amplified, and sequenced using the mitochondrial primers mcrshFF and mcrshRR as outlined in [13]. Deoxyribonucleic acid was extracted from approximately 50 mg of os shavings past incubation overnight at 56°C with proteinase 1000, and so purified by phenol:chloroform extraction and silica bed binding using a Qiagen DNeasy Blood & Tissue Kit. DNA was eluted from the silica column and subjected to PCR using the mitochondrial control region primers mcrshFF and mcrshRR equally outlined in [13] and visualised by agarose gel electrophoresis and ethidium bromide staining. Positive amplifications were sequenced using primer F6t as described in [thirteen]. Deoxyribonucleic acid results were obtained blind, where bone identity remained unknown until sequences were obtained.

Ancient Dna procedures

DNA was extracted and amplified according to the criteria proposed past [14]. Aboriginal DNA was extracted in a defended ancient Deoxyribonucleic acid laboratory at Griffith University, Australia, and amplified at a dissever isolated modernistic lab facility. For sequence verification, several samples were replicated independently at the Massey University Ancient DNA facility, Albany, Auckland, New Zealand.

Author Contributions

Conceived and designed the experiments: LH BJG CDM DML. Performed the experiments: LH BJG AD. Analyzed the data: LH BJG Advert. Contributed reagents/materials/analysis tools: AD BJG DML. Wrote the paper: LH BJG Advertising DML.

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