Early evolution of small body size in Homo floresiensis

Domain: earth_science
Published: 2024-08-06
Editorial outcome: minor_revision

Abstract
Recent discoveries of Homo floresiensis and H. luzonensis raise questions regarding how extreme body size reduction occurred in some extinct Homo species in insular environments. Previous investigations at Mata Menge, Flores Island, Indonesia, suggested that the early Middle Pleistocene ancestors of H. floresiensis had even smaller jaws and teeth. Here, we report additional hominin fossils from the same deposits at Mata Menge. An adult humerus is estimated to be 9−16% shorter and thinner than the type specimen of H. floresiensis dated to ~60,000 years ago, and is smaller than any other Plio-Pleistocene adult hominin humeri hitherto reported. The newly recovered teeth are both exceptionally small; one of them bears closer morphological similarities to early Javanese H. erectus. The H. floresiensis lineage most likely evolved from early Asian H. erectus and was a long-lasting lineage on Flores with markedly diminutive body sizes since at least ~700,000 years ago.

Introduction
The So’a Basin in central Flores, Indonesia, is a key region for elucidating the origin and evolution of H. floresiensis, a diminutive hominin species known from the Late Pleistocene at Liang Bua, a limestone cave in western Flores1,2. As with another small-bodied Homo discovered in Luzon3, the evolutionary history of this insular hominin species has been the subject of protracted debate4. Previous field studies of the Early to Middle Pleistocene (Calabrian–Chibanian) sequence of the So’a Basin have recovered fossil remains of endemic fauna (dwarfed Stegodon, Komodo monitor, giant rat, birds, crocodiles and tortoises)5,6, technologically simple stone artefacts (the oldest of which date back to at least 1.02±0.02 million years ago [Ma])7,8, and, importantly, a fragmentary mandible and six isolated teeth of a small-sized hominin9. These hominin fossils were excavated from a sandstone layer of fluvial origin (Layer II) of the upper fossil-bearing interval at the Mata Menge site, which is dated to between 0.65 and 0.773 Ma5,6. These fossils exhibit general morphological affinities to the type series of H. floresiensis from Liang Bua (0.1–0.06 Ma)10 and to early H. erectus from Java (1.1–0.8 Ma)11, but lack the unique molar specializations characterizing the former and were substantially smaller than the latter9.

Overall, the Mata Menge fossils suggest that they represent an ancestral segment of the Liang Bua H. floresiensis lineage, and that the Flores hominins are dwarfed descendants of large-bodied early Asian H. erectus9. Some cladistic/phylogenetic analyzes, however, support a direct evolutionary link between H. floresiensis and smaller-bodied basal Homo such as H. habilis or even Australopithecus12–14. It is important to resolve this controversy in order to elucidate the pattern and timing of body size evolution in the Flores hominins.

Notably, the Mata Menge mandible and teeth are slightly smaller than the type specimens of H. floresiensis from Liang Bua. This suggests that drastic dentognathic reduction had occurred on Flores by the early Middle Pleistocene epoch, more than 600,000 years before the earliest fossil evidence for H. floresiensis at Liang Bua. Until now, however, the lack of postcranial elements in the Mata Menge assemblage had limited our understanding of body size evolution on Flores.

In this paper, we report the discovery and morphology of a hominin postcranial fossil from Mata Menge, an extremely small distal humeral shaft (SOA-MM9) (Fig. 1). This specimen and two small-sized teeth (SOA-MM10 and SOA-MM11) were recovered as additions to the existing hominin assemblage from Layer II at this site (Table 1). Our histomorphic examination confirms the adult status of the humerus. We also show that shaft morphology is more similar to small-bodied Homo (e.g., LB1 and H. naledi) than to Australopithecus (e.g., A.L. 288-1), and a molar crown (SOA-MM11) bears closer shape similarities to early Javanese H. erectus than to early African Homo. The increased Mata Menge fossil sample supports its classification to an early representative of H. floresiensis, which probably experienced drastic body size reduction from large-bodied Asian H. erectus sometime between ~1.0 and 0.7 Ma.

Results
Context and geological age
All hominin fossils originated from the top of a ribbon-shaped, indurated pebbly sandstone layer (Layer II), which was deposited in a small stream channel on a volcaniclastic alluvial fan5 ~0.7 Ma ago. This age estimate is based on the identification of the Brunhes-Matuyama boundary15 dated at 0.773 Ma by palaeomagnetic measurements combined with a series of fission track dates on zircons in tuffaceous strata stratigraphically 16.5 m below Layer II5,6. A minimum age of 0.65±0.02 Ma for Layer II is provided by a 40Ar/39Ar date on single hornblende crystals from an airfall tephra (PGT-2) occurring stratigraphically at 14 m above Layer II.

Layer II, with a maximum thickness of 50 cm, overlies a reddish paleosol (Layer III) with an undulating erosional contact. A series of massive, tuffaceous clay-rich mudflow layers (Layers Ia-f) sealed off Layers II and III subsequently (Fig. 2; Supplementary Note 1). The humerus fragment SOA-MM9 was retrieved in several pieces within one week of opening Excavation 32A in 2013, but was recognized as such only in 2015 after reconstruction in the laboratory. The specimen was damaged in the process of excavating it from the extremely compact sandstone of Layer II. A maxillary deciduous canine (dc: SOA-MM10) was excavated in 2015 at ~5 cm below the boundary between Layers I and II, while a mandibular third molar (M3: SOA-MM11) was excavated in 2016 at ~15 cm below the top of Layer II. All hominin fossils are concentrated in the upper part of Layer II, while fossils of other fauna tend to be distributed more evenly in this unit. There is evidence for fluvial transportation of the fossils prior to burial, with many (but not all) specimens fractured (apart from excavation damage), weathered and/or rounded to some extent16. However, the three hominin fossils described here show minimal/no evidence of abrasion. Taphonomic and sedimentological observations suggest that the hominin fossils were deposited during a moderate to low-energy flow regime in the stream, following a relatively brief period on the surface during which the remains were disarticulated (Supplementary Note 1). Shortly after incorporation of the fossils in the streambed the entire stream valley was filled with a 6.5 m thick sequence of mudflows. Succeeding field excavations in 2017–2019 and 2023 have yielded no more hominin fossils from this site.

Developmental age of the humerus (SOA-MM9)
This specimen is an undistorted, distal half of the right humeral shaft that measures 88 mm in maximum preserved length (Fig. 1; Supplementary Note 2). Despite its small size, cortical bone histomorphology of SOA-MM9 clearly indicates its adult status. We examined its development stage based on age-associated increase of osteons and related structures, a method widely utilized for age estimates of extant and fossil hominins17–21.

Histological sections were examined for cortical samples taken at the mid-posterior shafts of SOA-MM9 (‘HS’ in Fig. 1) and from a modern human sample (N=20, see Supplementary Data 1). To allow for regional variation in osteon formation within each bone area21, data were collected from two additional (nearby) sites in the midshaft for all the modern human (H. sapiens) specimens (Fig. 3a). In two parameters indicative of bone maturity, Osteon Population Density (OPD)21 and Haversian Canal Index (HCI), SOA-MM9 was found to exhibit distinctly greater values (OPD=16.5, HCI=0.85) than in any of the modern human subadult humeri (0.0–8.9 and 0.0–0.63, respectively) (Fig. 3b, Supplementary Data 1). The values for the Mata Menge humerus are also greater than the means of our modern human adult samples (13.6 and 0.78, respectively), indicating that the SOA-MM9 individual was well within adulthood at time of death. Although the external cortical surfaces of SOA-MM9 exhibit microscopic damages that might have reduced one of the marginal osteons to half (~100 microns) (Fig. 3c), such post-depositional alterations would have limited impact on our age estimation. Even if we assume surface abrasion of 200 microns, the OPD value for SOA-MM9 would drop only slightly to ~15.8. Furthermore, the dominance of secondary osteons in the outer cortex (Fig. 3c) indicates that subperiosteal bone growth during the growth period had already been terminated in this individual22.

No evidence of pathology was found in SOA-MM9. Cortical bone thinning and woven bone would be pathognomic of some metabolic disorders17, but these features are not evident in SOA-MM9. The relative cortical bone thickness of SOA-MM9 (0.07: the ratio of cortical bone thickness relative to the humeral shaft circumference) (Supplementary Data 1) is almost identical to the mean for the modern human adult sample (0.069). Patients with osteogenesis imperfecta, which may lead to short stature, exhibit subnormal OPD values19, a tendency that is in opposition to the SOA-MM9 condition. Additionally, the weak but distinct development of the lateral supracondylar ridge of SOA-MM9 (Fig. 3d) indicates normal development of the extensor carpi radialis longus muscle.

Humeral size
In all available dimensions of shaft diameter/circumference and length, SOA-MM9 is smaller than LB1 (H. floresiensis) and any other adult individuals of small-bodied fossil hominins (Australopithecus and H. naledi: Supplementary Data 2). Its minimum circumference (46 mm) is less than U.W. 101-283 (47.5 mm), BOU-12/1 (52 mm), and the smallest humeri in our prehistoric modern human sample (46.5 mm, N=1050, see Supplementary Data 2). Centroid size at the ~19% level cross-section is also the smallest compared to any sampled adult specimens of Australopithecus, Paranthropus, and Homo including H. naledi and Liang Bua H. floresiensis (Fig. 4, Supplementary Data 3). The distal shaft length measured between the NF (nutrient foramen) and hOF (superior margin of the hollow leading to the olecranon fossa) points of SOA-MM9 (58 mm) is distinctly shorter than the other hominin fossils, including LB1 (64 mm) (Supplementary Data 2), although the vertical position of NF is variable in human humeri23.

The fragmentary nature of SOA-MM9 precludes a precise reconstruction of its original length, but it can be estimated as follows. First, the preserved proximo-posterior end of SOA-MM9 is very close to the 50% level because this portion exhibits the following suite of features characteristic of hominin humeral midshafts: 1) The radial sulcus (spiral groove) is present not on the lateral surface but on the posterolateral aspect, seen as a flattened area in CT slices no. 1900 and 2000 (‘RS’ in Fig. 5); 2) in lateral view, the anterior margin exhibits a slight concavity around no. 1900 (‘AM’ in Fig. 5, see the surface rendered image on the left side), indicating that this part, which is ~13 mm below the preserved proximo-posterior end, leads to the deltoid tuberosity proximally. The distal margin of the deltoid tuberosity is situated on the anterolateral surface at the 48.4% level in our modern human sample mean (N=366, range: 43−53%), 51% in LB124, and 48% in KNM-WT1500025; 3) NF is present 21 mm distal to the preserved proximo-posterior end of SOA-MM9. The projected distance along the shaft from the 50% level and the lower margin of the NF is 23 mm in LB1, 1 mm in KNM-WT15000, 15 mm in MH2 (Au. sediba)26, and 21.2 mm in our modern human sample mean (N=366, SD=10.0 mm, range=–2 to 59 mm). Each of the above three characters shows substantial variation, but their simultaneous expression at the preserved proximal shaft strongly suggests that SOA-MM9’s proximo-dorsal end was very close to the original 50% level. This positioning is consistent with other shaft morphologies exhibited by SOA-MM9 (Supplementary Note 3, Fig. 5, Supplementary Figs. 1 and 2, and Supplementary Data 4).

Next, distally, the CT section no. 250 of SOA-MM9 (Fig. 5), which is sliced at the hOF point, corresponds to the 12.5–14% level (or 11.5–15% more broadly) of maximum length (as explained below). Because of the observed allometric relationships that shorter modern human humeri tend to have relatively large distal epiphyses (Supplementary Note 3), we referred to the following two samples to draw upon the above figures. One is the short-statured prehistoric Holocene population from Tanegashima Island, Japan (N=13, maximum humeral length: 245–292 mm), and the other is a subset of short humeri from the prehistoric Jomon population from Japan (N=10, maximum humeral length: 240–250 mm). The means of the hOF levels in these samples were 12.5% and 14%, respectively, with their ranges 11.5−13.5% and 13−15%, respectively. The equivalent values in short hominin fossil humeri are 13% in both LB1 and A.L. 288-1.

Based on the above evaluations of SOA-MM9 humeral shaft preservation (proximal, 50% level; distal, 12.5−14% or 11.5−15% level), the original maximum humeral length of SOA-MM9 is estimated to be 211–220 mm or 206–226 mm, respectively. Alternatively, if we apply the mean ratios between the NF-hOF length and the maximum humeral length in our modern human male and female samples (0.29 to 0.30, Supplementary Data 2), the estimated maximum length of SOA-MM9 is 194–200 mm, but we surmise that this is less reliable given the weak correlation between the two measurements (r=0.37).

Comparative humeral morphology
SOA-MM9 lacks characteristic features of Australopithecus distal humeri, such as a prominent flange-like lateral supracondylar ridge, a projecting medial supracondylar crest, and marked curvature in the sagittal plane, although expression of these traits tend to be weak in comparatively gracile specimens of this genus26–29. With respect to cross-sectional shape (distal shaft 19% level), SOA-MM9 is similar to small-bodied Homo (H. naledi and H. floresiensis) in having a mediolaterally narrow profile that is unusual among the comparative groups (Fig. 6). It is different, however, from small-bodied Australopithecus individuals such as A.L. 288-1, whose humerus is only slightly longer than estimated for SOA-MM9 (Fig. 6). Procrustes distances support the cross-section shape variation summarized by the PCA results. Whereas the distances of SOA-MM9 to group mean shapes (Supplementary Fig. 4a) or individual specimens (Supplementary Fig. 4b) of H. naledi and H. floresiensis do not exceed the degree of within-species variation represented by our modern human sample, the same distances to the other fossil taxa are much greater.

Maxillary deciduous canine (dc: SOA-MM10)
This right tooth preserves a complete crown and a broken, short segment of the root (Fig. 1j). The crown is extremely small, situated well below the reported range of H. sapiens (Fig. 7a, and Supplementary Table 1), in a similar way to the previously reported Mata Menge dc (Fig. 7b). The specimen has a primitive, relatively low distal shoulder that resembles Australopithecus and Sangiran H. erectus homologs, although PCAs based on four or five linear measurements indicate that this morphology is marginally within the large variation seen in H. sapiens (Supplementary Fig. 5). Occlusal wear exposes a small dentine patch on the cusp tip and a thin line of dentine on the elongated distal incisal margin. The latter suggests the presence of a primitive, tall mesial cusp configuration on its occluding deciduous first molar (dm1), as known for an early Javanese H. erectus dm1 from Sangiran, S7–6730.

Mandibular third molar (M3: SOA-MM11)
The preserved left crown has reduced distal cusp areas, distally protruding hypoconulid, and no distal interproximal facet (Fig. 1k). Wear has flattened much of the occlusal surface, except for the metaconid that remains relatively high. Crown diameters are comparable to those of the smaller Liang Bua individual (LB6/1) and are marginally within the large variation exhibited by our global H. sapiens sample (Fig. 7c). It has five principal cusps arranged in a ‘+’ pattern and is different from the mandibular third molars of Liang Bua H. floresiensis that exhibit a derived, four-cusped morphology (LB1, LB6/1)31,32. Occlusal crown contour examined by normalized Elliptic Fourier Analysis shows that it clusters firmly with Sangiran H. erectus and marginally with H. ergaster, having a mesiodistally short crown. It is outside the range of variation exhibited by H. habilis sensu lato, which is primarily characterized by a mesiodistally elongated and distally tapered crown (Fig. 7d, Fig. 8, Supplementary Note 4, Supplementary Fig. 6) with a tendency for better developed hypoconulids and accessory cusps33–35.

Comparison of size with the Liang Bua H. floresiensis
In all available measurements of the mandibular body, postcanine teeth (P3, M1/2 and M3) and distal humerus, the Mata Menge fossils reported here or previously9 are smaller than the Liang Bua H. floresiensis remains (LB1 and LB6/1) by 1–21% (Table 2). Stature estimates, based on humeral length of SOA-MM9 (211−220 mm) and LB1 (243 mm), are 103–108 cm and 121 cm, respectively, using the human pygmy model36; or 93–96 cm and 102 cm, respectively, using the ape model37.

Methods
Permission to undertake excavations at Mata Menge was granted by the Indonesian State Ministry of Research and Technology (RISTEK permits 300/SIP/FRP/SM/VIII/2013 and 2183/FRP/SM/X/2015), the provincial government of East Nusa Tenggara in Kupang, and the Ngada District Administration. CT scan of SOA-MM9 was conducted in Tokyo with a permission issued in 2015 from the Geological Agency, Bandung.

CT scan and measurements
A micro-CT scan of SOA-MM9 was taken by using TXS320-ACTIS (Tesco Co.) at the National Museum of Nature and Science, Tokyo, with the following scanning parameters: 205 kV and 0.2 mA with a 0.5 mm thick copper plate prefilter, a 1024×1024 matrix, 0.04 mm pixel size and 0.04 mm slice interval (0.043 mm slice thickness). Micro-CT scans (voxel size = 0.156 mm) of 88 adult prehistoric Japanese (Holocene hunter-gatherer-fishers from the Jomon period) were also obtained for the length estimation of SOA-MM9 (see below). Linear measurements were taken using a spreading digital caliper (to the nearest 0.1 mm), an osteometric board (to the nearest 1.0 or 0.5 mm) and measuring tape (to the nearest 0.5 mm). Cross sectional properties were calculated based on CT scans at 15–50% vertical level of the shaft, using the software CT-Rugle (ver. 1.2, Medic Engineering Inc., Japan) and ImageJ (ver. 1.53f51, National Institutes of Health, USA).

Humeral analyzes
Comparative samples. To characterize its humeral morphology as a specialized insular hominin group, SOA-MM9 was compared with a wide variety of Pliocene and Pleistocene Afro-Asian hominin humeri (Ardipithecus, Australopithecus, Paranthropus, African early Homo, Dmanisi Homo, H. erectus/ergaster and H. naledi), as well as H. floresiensis from Liang Bua (LB1) and a series of modern human samples including some short-statured populations. The individual specimens included for linear metric comparison and geometric morphometric analysis are shown in Supplementary Data 2 and 3, respectively. The modern human samples used for the linear metric analysis are in Supplementary Data 2, while the modern human sample for the geometric morphometric analysis (Supplementary Data 3) is a mixed-sex sample of adults collected (by J. M. Plavcan) at the Smithsonian National Museum of Natural History (Washington, DC)44. As for the geometric morphometric analysis, the fossil sample was divided into six morphological groups, five of which have been established by previous studies29,44–46, while a sixth group consists of five specimens attributed to Homo naledi. We also collected outline data from a scan of the humerus of the LB1 skeleton (i.e., LB1/50) attributed to H. floresiensis1. The adult/subadult status for each specimen was determined by the epiphysial fusion of the proximal and/or distal ends, or other information if available (e.g., dental development). The linear metric comparisons focus on the adult samples, while the geometric morphometric analysis contains some subadult specimens, as noted in Fig. 4 and Supplementary Data 3. See Supplementary Note 7 for additional information about the H. naledi sample.

Key landmarks. SOA-MM9 lacks most of the widely used osteometric landmarks, but the following points are usable:
Nutrient foramen (NF): the distal margin of the nutrient foramen on the midshaft.
hOF point: the proximal margin of the hollow leading to the olecranon fossa.

Developmental age. Human bones undergo substantial histomorphic changes during development and much of adulthood. In a limb bone shaft, periosteal cortical bone growth occurs as deposition of circumferential lamellar bone and primary osteons with non-Haversian canals. The proportion of these primary structures decreases as secondary osteons (Haversian canal surrounded by concentric rings of lamellar bone) appear and increase through the bone remodeling. In late adulthood, the cortical bone is dominated by secondary osteons17,47. This process is numerically demonstrated by the count or density of the elements of secondary osteon (intact osteons, fragmentary osteons, Haversian canals, resorption bay, etc.)20,48–50. However, because the rate of such histomorphic change varies considerably depending on the locus in a bone, regional differences must be considered in histomorphological age estimation21,51. Because no histomorphometric data was available in the literature for human humeral midshaft, we collected our referential data using modern Japanese humeri. Our sample, which consists of 10 adults, 6 adolescents, and 4 child individuals, was unearthed from cemeteries of the Edo period (17th-19th centuries A.D.) in Tokyo City and is stored at the National Museum of Nature and Science, Japan. Based on the standard ossification procedure52, we categorized those humeri with completely fused epiphyses to ‘adult,’ those with unfused proximal epiphysis and completely fused distal epiphysis to ‘adolescent,’ and those with separate epiphyses as ‘child.’ The adolescent category was further subdivided into ‘early adolescent’ (fusion at distal epiphysis only, N=2), ‘mid adolescent’ (fusion at distal epiphysis and medial epicondyle, N=2), and ‘late adolescent’ (proximal epiphysis partially fused, N=2). We (J.S.) cut out a small piece of the bone from the mid-posterior shaft of the SOA-MM9 humerus, to prepare a sectional sample (‘HS’ in Fig. 1c). The location of this section is 6.5 mm distal to the preserved proximal edge, and 14.5 mm proximal to the distal margin of the nutrient foramen, and is assumed to be slightly distal to the missing deltoid tuberosity. The obtained section covers a full thickness from its outer (periosteal) to inner (intrathecal) surfaces. After embedding in resin, we prepared a polished surface to observe with an ultra-high-definition microscope (VHX-7000, Keyence). From each of the modern human humeri, we prepared a cortical section at the posterior surface 10 mm distal to the lower end of the deltoid tuberosity. First, we cut out a small piece of the bone using a diamond cutter. After embedding in resin, a transverse section of 70 μm-thickness was made by a microtome (SP-1600, Leica) to observe under an ordinary light microscope (Imager A1, Leica). We focused on the outer one-third of the cortical bone, because the periosteal region is an active bone growth field and is useful for histomorphometric growth studies20. To allow for regional variation mentioned above, we examined three adjacent loci: one on the midsagittal line (middle field), and the others on either side of it (lateral and medial fields) as illustrated in Fig. 3. The section prepared for SOA-MM9 corresponds to the lateral field. In each observation field, we counted the numbers of intact secondary osteons (N.On), osteon fragments (N.Fr), resorption bays (N.Re), Haversian canals of the secondary osteons/osteon fragments (N.Ca), and non-Haversian canals (N.nCa), as defined elsewhere20,53. An ‘osteon fragment’ is a secondary osteon eroded by later-formed osteons. A structure straddling the border of the observation field was counted only if more than half of it was inside. We use the following three size-free parameters as measures of cortical bone growth and pathology.
1) Osteon Population Density (OPD): This widely used index, calculated here as (N.On+N.Fr+N.Re) per area (mm2)21, monitors the increase of secondary made structures. A greater OPD value reflects advanced growth stage.
2) Haversian Canal Index (HCI): This is a ratio of the secondary made canals. It is calculated as (N.Ca/(N.Ca+N.nCa)), and increases from 0 to 1 with bone growth.
3) Relative Cortical Bone thickness (rCBt): We define this index as the mean cortical bone thickness divided by the minimum circumference of each humeral shaft. The former is the average of the three cortical bone thicknesses at the medial, middle and lateral fields in Fig. 3, which we measured using a public-domain ImageJ (U.S. National Institutes of Health, available at https://imagej.nih.gov/ij/).

Length estimation. The extant and fossil hominin humeri exhibit a uniform pattern of transition in cross-sectional shape (i.e., flatness, angle of the long axis, ratio between the cortical and total areas, and other features) from the mid- to the distal shaft levels (Supplementary Figs. 1 and 2). We (S.M. and Y.K.) refer to this information to reconstruct the original maximum humeral length (Martin no. 1a) of SOA-MM9.

Cross-sectional properties of the shaft. We (S.M.) used CT-Rugle 1.2 (Medic Engineering Co.) to calculate the cross-sectional properties of the humeral shaft.

Cross-sectional geometry of the shaft. Previous studies of fossil hominin humeri have demonstrated the taxonomic utility of the cross-sectional shape of the distal diaphysis sampled at ~19% of total (biomechanical) humerus length from the distal end29,44,46,54–56. The ~19% level of SOA-MM9 was located by Y.K. based on our estimate of its maximum length (211–220 mm), which was converted to the biomechanical humerus length using the ratio between the two (the former is 1.08% longer on average in our mixed-sex, prehistoric modern human (Jomon) sample: N=88). The 19% level thus located is within the CT slice nos. 573–639. Therefore, we chose three slices, nos. 573, 607 (best estimate), and 639 for the present analysis. Two-dimensional coordinates were collected by M.L. from all three sections of SOA-M9 following the procedure described previously44 (i.e., two Type 2 landmarks on the medial and lateral extremes of the specimen and 58 sliding semilandmarks on the anterior and posterior surfaces). Raw landmark configurations were superimposed into the same shape space using orthogonal least-squares generalized Procrustes (GPA) superimposition57, GPA was performed using tpsRelw software58 and semilandmarks were allowed to slide along the diaphyseal outline using the criterion of minimized bending energy59. Subsequent to GPA, morphometric relationships were assessed with the use of Procrustes distances (Dp) as a measure of shape dissimilarity60 and principal component analysis (PCA) as a means of visual summary (via ordination) of shape variation among the individual specimens.

Dental analysis
SOA-MM10 (maxillary deciduous canine: dc) was compared with the available sample of fossil hominins (Australopithecus and Early Pleistocene Homo), as well as a sample of H. sapiens (Supplementary Table 2) by Y.K. Unfortunately, there is no deciduous teeth in the existing H. floresiensis assemblage from Liang Bua. SOA-MM11 (mandibular third molar: M3) was compared with Liang Bua H. floresiensis and its claimed two major ancestral candidates, H. habilis and early Javanese H. erectus (Supplementary Table 2). The early Javanese H. erectus dental sample examined in this report is from Sangiran, Central Java. We divided this sample into two chronological subsamples, Sangiran Lower and Sangiran Upper, following the previous report that demonstrated significant morphological differences in tooth size, mandible features, cranial capacity, etc.61,62. Linear measurements were taken based on the original specimens or high-quality casts by Y.K. using a digital caliper (Mitsutoyo Inc.) or otherwise collected from the literature (Supplementary Table 2). Occlusal crown contours of SOA-MM11 was further analyzed by normalized elliptic Fourier analysis (size-standardized EFA), using the comparative samples shown in Supplementary Table 2 and based on the methods detailed elsewhere37. In brief, the occlusal contour of each specimen was obtained from a photograph of the original specimen or high-quality cast, in a way which minimizes the error derived from parallax effect and orientation of the tooth or scale. Images were uploaded into CanvasX software (ACD Systems) to extract the occlusal contour and, for a worn tooth, to reconstruct small parts of the crown lost by interproximal wear. Then, normalized elliptic Fourier analysis was conducted using the software SHAPE 1.363, after each crown contour was aligned along its mesiodistal axis. We did not assess sex for these materials because of the small sample and the reported low sexual dimorphism in modern human deciduous teeth64.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Kaifu et al. reported three new hominin fossils recovered from SOA-MM and assigned them,along with previously found materials,to Homo floresiensis.The more primitive traits and Sangiran affinity directly support the hypothesis that these dwarfed hominins evolved from H. erectus,which arrived here in the early Middle Pleistocene, or perhaps even earlier,as hinted by the evidence of artifacts. Have the authors considered whether the dwarfing process was gradual or a punctuational event,or if it relates to some environmental change? Regarding the cross-sectional geometry,the authors would better provide the original dataset instead of just figures.This will facilitate future comparisons.", "reviewer_2": "In this paper, the authors present new finds form Mata Menge, Flores, Indonesia. These add to the previously described material assigned to Homo floresiensis, dating to around 700 ka ago, significantly earlier than the eponymous material from Liang Bua (about 60ka). The most important specimen is an adult humerus, which is smaller and more gracile than that of the holotype, LB1. In addition to the humerus, two teeth were also recovered, a maxillary deciduous canine and a lower third molar, both of these are very small. Based on these new discoveries, the authors propose that floresiensis was present on Flores since about 700,000 years ago and probably evolved from earlier Asian Homo erectus.\n\nCongratulations to the authors on such a well executed an interesting study. These discoveries contribute significantly to our understanding of Homo floresiensis, and especially to how and when the remarkably small body size in this species evolved. The authors have provided detailed descriptions of the specimens and of their analyses, and in general, this paper clearly warrants publication due to the importance of the material described within, and the very detailed analyses. There are a few issues though that I think if dealt with would further strengthen the paper:\n1. I found it rather confusing and hard to understand which comparative sample was used where in the humeral analyses. Even though SI Table 2 lists a very large number of humeri used in the comparative analyses, only a subset of the modern comparative sample was used in the distal humeral GMM analyses. It would be better to be more explicit about this, so the reader doesn’t have to hunt for this information in several different places.\n2. Linked to the previous, why was the modern comparative sample for the GMM analyses of the distal humerus so small (n=21, at least according to the Ext. data Fig 7 legend), when compared with the sample used for the cross sectional properties (n=88, according to the Ext Fig 6) or SI Table 2 (several hundred, even though unclear whether the separate male and female listed are included in the n=770 listed for the mixed sex sample)?\n3. I also recommend to include the modern human comparative sample in the PCA plot of humeral morphometrics (Fig. 2). Fig. 2 is in a somewhat unusual perspective as a partly rotated 3D scatterplot, which makes seeing the effects of the individual principle components difficult . I would recommend to include some regular, 2D scatterplots of PC1 vs. PC2 and PC1 vs. PC3 in the SI. This would also allow to visualize the effects of the individual principal components on the shape (similar to how this is in Fig 3 d), currently this is only done for PC1 in Fig. 2.\n4. I was surprised to see that the analyses of in the end very similar data, the outlines of the lower M3 and of the distal humeral cross sections were analyzed using completely different methods, procrustes superimposition and sliding semilandmarks in the case of the humerus, and elliptical Fourier analysis in the case of the molars. What is the reason for this? I am honestly not sure which of the two is more appropriate (and whether they would give significantly different results), but I am confused why the authors chose these different approaches.\nNone of these issues subtract from the importance of this paper though, and I don’t think they would make a difference with regards to the conclusion that H. floresiensis saw a reduction in body size early after its arrival on Flores, and then went through a long, relatively stable period without major changes in body size and only slight changes in dental morphology.\nMinor Issues:\nLine 430 slice -> slices\nLine 555 sizse -> size\nExtended data Fig 2.: portions c and d are very pixelated and hard (might be a conversion issue)\nExtended data Fig 5.: white line indicating strong axis can not be discerned (might be resolution issue)\nSupplementary table 2: Au. prometeus -> prometheus", "reviewer_3": "This paper reports on new fossils discovered from the site of Mata Menge dated to approximately 700kya. The importance of the paper is threefold: 1) The analyses reinforce the interpretation that, based on morphological characteristics, the Mata Menge remains can be assigned to H. floresiensis, 2) that the characteristic small body size is established in this lineage by ca. 700,000 years BP, and 3) the morphological affinities of H. floresiensis are most comparable to Javanese H. erectus rather than H. habilis. The fossils are important, and worthy of the detailed analysis and reporting presented here. I found the analyses of the humerus and new dental remains are thorough and supportive of the interpretations. The paper will make an important contribution to the literature, and I recommend publication with relatively minor revisions that I suggest below.\nPage 3 – The new fossils reported are from the upper part of Layer II, the same context as the previously described fossil remains form Mata Menge. While these have been reported before, it would be useful to provide a sentence or two on the dating of the fossils for the current publication, as the age is important to the interpretation of the ‘early’ reduction in body size. The details of course could be left to other publications, but there needs to be a bit more context on the dating presented here.\nPage 4 – line 99 – Developmental age and pathology of the humerus – The use of histomorphology for age determination of the humerus is a good choice. The analyses look convincing in demonstrating that the humerus belongs to a mature adult based on the proportion of secondary osteons, but the images provided in word doc or pdf (Extended data fig 2c and d) are of insufficient resolution for the reader to evaluate this. I assume high quality images will be provided for the publication, but as they appear in the review files the reader has to trust that the authors have identified the osteons and osteon fragments correctly. As I said, I have no reason to question the analysis, but the reader needs to see high resolution images.\nPage 4 – line 124 – ‘Other characteristics do not contradict the assumption that SOA-MM9 is from a non-pathological adult individual’ – Avoid double negatives, change to ‘support the interpretation’ or similar. Also, the adult status is not an assumption, it is an interpretation, and one that the authors have just quite convincingly demonstrated. Actually, as a further point, this paragraph really attempts to rule out pathological explanations of the small, bodied morphology, so I would consider the age as demonstrated and focus purely on the lack of evidence for pathology in this section. With regards to the section heading (Line 99) the wording “Developmental age and pathology of the humerus (SOA-MM9)” led me to believe that the authors had identified a pathological condition. Since they have not, I would recommend dropping the word ‘pathology’ or changing it to ‘health status’.\nPage 5 – Line 141 – How is 19% defined, and why choose 19% over the more standard 30 or 35% locations? I note there is a more detailed description of this on Page 14 lines 425-432, but I think it still requires some clarification. Usually on a complete humerus it is measured from the most distal point of the trochlea along the shaft, and defined by the total length, which I assume is the case here. At what percentage of length do the authors estimate the distal end of the humerus (as it is preserved) is situated? I recommend some revision to figure 4 below, but it would also be helpful to add some detail to the methods text here. The authors say that it was ‘located based on our estimate of it’s maximum length’, but I assume that 19% is also based on the estimated location of the most distal articular point? If so, what is the estimated error range in the location of 19% in mm rather than slices? Conversely, if it’s from the preserved distal end, how is the same location estimated for comparative fossil material? Sorry to be pedantic here, but the wording is not absolutely clear. Saying something is located based on an estimated length is one thing, but there is no preserved ‘end’ of the bone, so there is an additional estimate in the location of that end that also adds error to the positioning section locations and this needs to be absolutely clear in the text.\nPage 6 – line 185 – should be estimated rather than calculated\nExtended Data Fig. 3 – add 19% level cross-section to figure legend (should be intelligible without reference to text).\nExtended Data Fig. 4 – I’m not convinced that listing the slice numbers is useful for the reader. My first question when looking at Fig 4 was to ask where the 19% cross-section was that was used in the previous analysis. This is an unconventional location, so identification of its location would be useful, as would the other section locations mentioned in the text. Can the slice numbers be expressed as distances or percentages of length?\nPage 11 Line 325 – should read ‘A’ or ‘The’ micro-CT scan’?", "reviewer_4": "This paper reports new proto-H. floresiensis fossils from Mata Menge, Flores. The manuscript is clear, well written and scientifically robust (I am not however a palaeoanthropologist) and the findings are appropriate for the readership of this journal.\nThe findings are important as they represent the first post-cranial elements and afford new information regarding the likely origins of this hominin and taxonomic relationships. I recommend publication of this paper following attention to the minor comments made below.\nLine 54: ‘fluvial sandstone’ is not correct, change to sandstone of fluvial origin or some such would be more appropriate.\nLine 92–97: this is quite a critical observation and I wonder about the implications? Were the bodies interred more rapidly or at least prior to the loss of soft tissues? Were they buried in slackwater sediments that represented significantly lower flow energy? Perhaps if the human fossils are found in the upper part of Layer II this represents a period when flow was decreasing? It feels to me that what this paper is missing is some more detail around the depositional environments and potential taphonomic history of the assemblage. Are there additional data/observations of sediments characteristics in the area where the human fossils were found? If so then some additional contextual information would be appropriate."}, "author_response": "Manuscript title: Early evolution of extremely small body size in Homo floresiensis\nWe thank four reviewers for their supportive, encouraging, and helpful comments. Our point-by-point response to each comment is below (the reviewers’ comments in blue; our replies in black).\nReviewer #1 (Remarks to the Author):\nKaifu et al. reported three new hominin fossils recovered from SOA-MM and assigned them,along with previously found materials,to Homo floresiensis.The more primitive traits and Sangiran affinity directly support the hypothesis that these dwarfed hominins evolved from H. erectus,which arrived here in the early Middle Pleistocene, or perhaps even earlier,as hinted by the evidence of artifacts.\nHave the authors considered whether the dwarfing process was gradual or a punctuational event,or if it relates to some environmental change?\n>>Thank you for this important question. We could narrow down the time range for body size reduction by this study (between ~1.0 and 0.7 Ma) but are not yet ready to answer it. We hope more fossil materials will be discovered from earlier Flores sites to approach this question.\nRegarding the cross-sectional geometry, the authors would better provide the original dataset instead of just figures. This will facilitate future comparisons.\n>>This is true. New Supplementary Table 4 has been added to report the cross-sectional property data of the SOA-MM9 humerus.\nReviewer #2 (Remarks to the Author):\nIn this paper, the authors present new finds form Mata Menge, Flores, Indonesia. These add to the previously described material assigned to Homo floresiensis, dating to around 700 ka ago, significantly earlier than the eponymous material from Liang Bua (about 60ka). The most important specimen is an adult humerus, which is smaller and more gracile than that of the holotype, LB1. In addition to the humerus, two teeth were also recovered, a maxillary deciduous canine and a lower third molar, both of these are very small. Based on these new discoveries, the authors propose that floresiensis was present on Flores since about 700,000 years ago and probably evolved from earlier Asian Homo erectus.\nCongratulations to the authors on such a well executed an interesting study. These discoveries contribute significantly to our understanding of Homo floresiensis, and especially to how and when the remarkably small body size in this species evolved. The authors have provided detailed descriptions of the specimens and of their analyses, and in general, this paper clearly warrants publication due to the importance of the material described within, and the very detailed analyses. There are a few issues though that I think if dealt with would further strengthen the paper:\n>>Thank you.\n1. I found it rather confusing and hard to understand which comparative sample was used where in the humeral analyses. Even though SI Table 2 lists a very large number of humeri used in the comparative analyses, only a subset of the modern comparative sample was used in the distal humeral GMM analyses. It would be better to be more explicit about this, so the reader doesn’t have to hunt for this information in several different places.\n>> We added the following texts to resolve this issue.\nLine 339 (the Method section): “The modern human samples used for the linear metric analyses are in Supplementary Table 2, while the modern human sample for the geometric morphometric analysis is a mixed-sex sample of adults collected (by J.M. Plavcan) at the Cleveland Museum of Natural History and the Smithsonian National Museum of Natural History (Washington, DC)44”\nLine 100: “see Supplementary Table 1”\nLine 133: “see Supplementary Table 2”\n2. Linked to the previous, why was the modern comparative sample for the GMM analyses of the distal humerus so small (n=21, at least according to the Ext. data Fig 7 legend), when compared with the sample used for the cross sectional properties (n=88, according to the Ext Fig 6) or SI Table 2 (several hundred, even though unclear whether the separate male and female listed are included in the n=770 listed for the mixed sex sample)?\n>> This is because the original data sets differ between the GMM and other analyses.\n3. I also recommend to include the modern human comparative sample in the PCA plot of humeral morphometrics (Fig. 2). Fig. 2 is in a somewhat unusual perspective as a partly rotated 3D scatterplot, which makes seeing the effects of the individual principle components difficult . I would recommend to include some regular, 2D scatterplots of PC1 vs. PC2 and PC1 vs. PC3 in the SI. This would also allow to visualize the effects of the individual principal components on the shape (similar to how this is in Fig 3 d), currently this is only done for PC1 in Fig. 2.\n>> We did not include moder humans from this analysis because our focus was to examine morphological affinities among fossil taxa. For the same reason, we excluded modern humans in our shape analyses (PCA based on the elliptical flourier analysis) of the molar (Fig. 3). We have presented 2D scatter plots in the new Supplementary Fig. 3.\n4. I was surprised to see that the analyses of in the end very similar data, the outlines of the lower M3 and of the distal humeral cross sections were analyzed using completely different methods, procrustes superimposition and sliding semilandmarks in the case of the humerus, and elliptical Fourier analysis in the case of the molars. What is the reason for this? I am honestly not sure which of the two is more appropriate (and whether they would give significantly different results), but I am confused why the authors chose these different approaches.\n>> We employed Elliptical Fourier analysis for the molar due to difficulties in defining its landmarks. However, as this reviewer expects, both analyses should reach similar results as far as each specimen is appropriately oriented.\nNone of these issues subtract from the importance of this paper though, and I don’t think they would make a difference with regards to the conclusion that H. floresiensis saw a reduction in body size early after its arrival on Flores, and then went through a long, relatively stable period without major changes in body size and only slight changes in dental morphology.\nMinor Issues:\nLine 430 slice -> slices >> Corrected\nLine 555 sizse -> size >> Corrected\nExtended data Fig 2.: portions c and d are very pixelated and hard (might be a conversion issue) >> The images (now Fig. 3) have been replaced with a better one.\nExtended data Fig 5.: white line indicating strong axis can not be discerned (might be resolution issue) >> The image (now Supplementary Fig. 1) has been replaced with a better one.\nSupplementary table 2: Au. prometeus -> prometheus >> Corrected\nReviewer #3 (Remarks to the Author):\nThis paper reports on new fossils discovered from the site of Mata Menge dated to approximately 700kya. The importance of the paper is threefold: 1) The analyses reinforce the interpretation that, based on morphological characteristics, the Mata Menge remains can be assigned to H. floresiensis, 2) that the characteristic small body size is established in this lineage by ca. 700,000 years BP, and 3) the morphological affinities of H. floresiensis are most comparable to Javanese H. erectus rather than H. habilis. The fossils are important, and worthy of the detailed analysis and reporting presented here. I found the analyses of the humerus and new dental remains are thorough and supportive of the interpretations. The paper will make an important contribution to the literature, and I recommend publication with relatively minor revisions that I suggest below.\nPage 3 – The new fossils reported are from the upper part of Layer II, the same context as the previously described fossil remains form Mata Menge. While these have been reported before, it would be useful to provide a sentence or two on the dating of the fossils for the current publication, as the age is important to the interpretation of the ‘early’ reduction in body size. The details of course could be left to other publications, but there needs to be a bit more context on the dating presented here.\n>>We included this information in the revised “Context and geological age” section of the Results.\nPage 4 – line 99 – Developmental age and pathology of the humerus – The use of histomorphology for age determination of the humerus is a good choice. The analyses look convincing in demonstrating that the humerus belongs to a mature adult based on the proportion of secondary osteons, but the images provided in word doc or pdf (Extended data fig 2c and d) are of insufficient resolution for the reader to evaluate this. I assume high quality images will be provided for the publication, but as they appear in the review files the reader has to trust that the authors have identified the osteons and osteon fragments correctly. As I said, I have no reason to question the analysis, but the reader needs to see high resolution images.\n>>We apologize for the inconvenience. We submitted high-resolutions images.\nPage 4 – line 124 – ‘Other characteristics do not contradict the assumption that SOA-MM9 is from a non-pathological adult individual’ – Avoid double negatives, change to ‘support the interpretation’ or similar. Also, the adult status is not an assumption, it is an interpretation, and one that the authors have just quite convincingly demonstrated. Actually, as a further point, this paragraph really attempts to rule out pathological explanations of the small, bodied morphology, so I would consider the age as demonstrated and focus purely on the lack of evidence for pathology in this section. With regards to the section heading (Line 99) the wording “Developmental age and pathology of the humerus (SOA-MM9)” led me to believe that the authors had identified a pathological condition. Since they have not, I would recommend dropping the word ‘pathology’ or changing it to ‘health status’.\n>> We appreciate these helpful comments. Because we have not (cannot) examined the health status, we simplified the subheading as “Developmental age of the humerus (SOA-MM9)”.\nPage 5 – Line 141 – How is 19% defined, and why choose 19% over the more standard 30 or 35% locations? I note there is a more detailed description of this on Page 14 lines 425-432, but I think it still requires some clarification. Usually on a complete humerus it is measured from the most distal point of the trochlea along the shaft, and defined by the total length, which I assume is the case here. At what percentage of length do the authors estimate the distal end of the humerus (as it is preserved) is situated? I recommend some revision to figure 4 below, but it would also be helpful to add some detail to the methods text here. The authors say that it was ‘located based on our estimate of it’s maximum length’, but I assume that 19% is also based on the estimated location of the most distal articular point? If so, what is the estimated error range in the location of 19% in mm rather than slices? Conversely, if it’s from the preserved distal end, how is the same location estimated for comparative fossil material? Sorry to be pedantic here, but the wording is not absolutely clear. Saying something is located based on an estimated length is one thing, but there is no preserved ‘end’ of the bone, so there is an additional estimate in the location of that end that also adds error to the positioning section locations and this needs to be absolutely clear in the text.\n>>Previous studies of fossil hominin humeri have demonstrated taxonomic utility of the cross-sectional shape of the distal diaphysis sampled at ~19% of total (biomechanical) humerus length from the distal end29,44,46,54-56. The ~19% level of SOA-MM9 was located based on our estimate of its maximum length (211−220 mm), which was converted to the biomechanical humeral length using the ratio between the two (the former is 1.08% longer on average in our mixed-sex, prehistoric modern human (Jomon) sample: N = 88). The 19% level thus located is within the CT slice nos. 573−639. Therefore, we chose three slices, nos. 573, 607 (best estimate), and 639 for the present analysis. Two-dimensional coordinates were collected from all three sections of SOA-M9 following the procedure described by Lague44 (i.e., two Type 2 landmarks on the medial and lateral extremes of the specimen and 58 sliding semilandmarks on the anterior and posterior surfaces). Raw landmark configurations were superimposed into the same shape space using orthogonal least-squares generalized Procrustes (GPA) superimposition57. GPA was performed using tpsRelw software58 and semilandmarks were allowed to slide along the diaphyseal outline using the criterion of minimized bending energy59. Subsequent to GPA, morphometric relationships were assessed with the use of Procrustes distances (Dp) as a measure of shape dissimilarity60 and principal component analysis (PCA) as a means of visual summary (via ordination) of shape variation among the individual specimens.\nPage 6 – line 185 – should be estimated rather than calculated\n>> Corrected\nExtended Data Fig. 3 – add 19% level cross-section to figure legend (should be intelligible without reference to text).\n>> Corrected\nExtended Data Fig. 4 – I’m not convinced that listing the slice numbers is useful for the reader. My first question when looking at Fig 4 was to ask where the 19% cross-section was that was used in the previous analysis. This is an unconventional location, so identification of its location would be useful, as would the other section locations mentioned in the text. Can the slice numbers be expressed as distances or percentages of length?\n>> We added the level of our estimated 19% level in Fig. 5 (former Extended Data Fig. 4). These slice numbers are identification number for each CT slice. We do not replace them with distances or percentages of length because the latter are estimates.\nPage 11 Line 325 – should read ‘A’ or ‘The’ micro-CT scan’?\n>> Corrected\nReviewer #4 (Remarks to the Author):\nThis paper reports new proto-H. floresiensis fossils from Mata Menge, Flores. The manuscript is clear, well written and scientifically robust (I am not however a palaeoanthropologist) and the findings are appropriate for the readership of this journal.\nThe findings are important as they represent the first post-cranial elements and afford new information regarding the likely origins of this hominin and taxonomic relationships. I recommend publication of this paper following attention to the minor comments made below.\nLine 54: ‘fluvial sandstone’ is not correct, change to sandstone of fluvial origin or some such would be more appropriate.\n>>Corrected\nLine 92–97: this is quite a critical observation and I wonder about the implications? Were the bodies interred more rapidly or at least prior to the loss of soft tissues? Were they buried in slackwater sediments that represented significantly lower flow energy? Perhaps if the human fossils are found in the upper part of Layer II this represents a period when flow was decreasing? It feels to me that what this paper is missing is some more detail around the depositional environments and potential taphonomic history of the assemblage. Are there additional data/observations of sediments characteristics in the area where the human fossils were found? If so then some additional contextual information would be appropriate.\n>>To address what we can infer about the taphonomy of these fossils, we included brief sentences in the main text (at the end of the “Context and geological age”) and added the new Supplementary Note 1 in Supplementary Information."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors took all of my suggestions into account, and answered my questions in great detail. This is a great paper, and I am happy to see it published.\nBence Viola", "reviewer_3": "I have reviewed the revised manuscript and the revisions made in response to my previous recommendations. The authors have fully addressed the suggestions that I made previously, and the revised manuscript is polished and ready for publication.", "reviewer_4": "My feedback was limited to a few minor comments, all of which have been addressed satisfactorily, many thanks and congratulations."}, "author_response": ""}]

Decision letter