1. Introduction
The islands of New Zealand (NZ) are exceptional for their long oceanic isolation from the supercontinent Gondwana (>52 Ma) [
1], large size (>260,000 km²), and absence of mammals (except bats) among the recent native land-fauna. NZ’s native biodiversity is rich in phylogenetically distinct lineages, and extraordinary examples of gigantism and flightlessness among the avifauna. Important examples include the nine species of megaherbivore ratite moa, weighing up to 250 kg (Dinornithiformes) [
2,
3], four of the world’s five known flightless songbirds [
4], the world’s heaviest parrot (also flightless) [
5], and the largest-known eagle (
Aquila moorei) [
6]. However, widespread extinction (>40% of endemic bird species) [
3,
7] following human settlement in NZ around 1270 AD [
8] means that phylogenetic studies on many NZ taxa have been dependent on advances in ancient DNA (aDNA) research [
9]. As a result, the evolutionary histories of many of NZ’s unique animal taxa, including the adzebill (
Aptornis: Aptornithidae), remain unresolved or inconclusive.
Adzebills were large, specialised, flightless birds endemic to NZ, which have been a taxonomic and ecological enigma since they were first described by Richard Owen in 1844 [
10,
11,
12,
13]. The genus
Aptornis includes two recently extinct species: the North Island Adzebill (
A. otidiformis), typically reaching around 16 kg; and the larger South Island Adzebill (
A. defossor), typically reaching around 19 kg (though a maximum size of 25 kg has been suggested) [
2]. In addition, fragments of a fossil adzebill (
A. proasciarostratus), strikingly similar to recent adzebills, have also been described from the Early Miocene (16–19 Ma) deposits near St Bathans in the southern South Island, suggesting a long history of the group in NZ [
14]. Both recent species were nearly wingless and had a robust and specialised morphology. The massive beak and skull, as well as the neck vertebrae, were heavily reinforced and likely supported powerful muscles. Adzebills also had robust legs and feet, which may have been used with the massive bill to excavate or immobilize food or prey. Stable isotopes from adzebill bone specimens confirm that adzebills had a high trophic niche and were likely predators or scavengers, although their exact feeding strategy remains unknown [
2,
15]. Archaeological deposits confirm that adzebills were hunted by early Maori [
16,
17,
18], who also rapidly cleared the dry, lowland podocarp forests in eastern NZ [
19] which were the birds’ main habitat during the Holocene [
2,
15]. Both activities led to the extinction of the recent adzebill species around the same time as the moa, circa 1500 CE [
2,
20].
The highly derived morphology of adzebills has long complicated their classification, though they have usually been placed in Gruiformes along with several other bird families found in former fragments of Gondwana (especially New Caledonia, Madagascar, and South America). It has often been proposed that the ancestor of these “gruiform” bird families, including adzebills, inhabited Gondwana prior to its break up and that their present biogeography reflects continental vicariance [
21]. Specifically, morphological analyses have suggested adzebills are most closely related to the New Caledonian Kagu (
Rhynochetos jubatus) [
21,
22,
23,
24], consistent with the possible existence of emergent land connecting New Zealand and New Caledonia during the Paleogene [
25,
26]. However, recent phylogenetic studies have found that most traditional gruiforms do not form a monophyletic group, except for two superfamilies (each comprising three extant families): the Gruoidea (cranes and allies) and Ralloidea (rails and allies) [
27,
28,
29]. For example, the Kagu and the South American Sunbittern
Eurypyga helias (together comprising Eurypygiformes) appear to form the sister-taxon to tropicbirds (Phaethontiformes) [
24,
29,
30]. Previous analyses of a 673 bp fragment of the mitochondrial 12S rRNA gene from the South Island Adzebill suggested that adzebills were members of the Ralloidea in the Gruiformes and unrelated to kagu [
31], although insufficient data and limited taxon sampling prevented confident identification of the adzebills’ precise phylogenetic affinities. These alternative and incompatible hypotheses for the classification of adzebills—as members of either Eurypygiformes or Gruiformes—have widely disparate implications for their biogeographic and temporal origins.
If adzebills are ralloids, and not eurypygiforms, then the question becomes: “to what living ralloids are they most closely related?” Any attempts to answer this question require a robust phylogenetic framework for Ralloidea, which historically contains an uncertain number of families (with varying taxonomic contents). For example, the flufftails (
Sarothrura spp.) were previously considered members of the ralloid family Rallidae (the rails) but are now recognised as comprising their own family (Sarothruridae) [
32] more closely related to the finfoots (Heliornithidae) [
24,
28]. Similarly, the Madagascan wood rails (
Mentocrex kioloides sspp.), which are presently classified as Rallidae [
32], were recently suggested to be closer relatives of Sarothruridae [
33].
In this study, we present the first near-complete mitochondrial genome sequences from both recent species of adzebill. We also present new genetic data from several key gruiform lineages of uncertain affinity, including members of the Rallidae. We analyse these new sequences alongside all available mitochondrial data for ralloids, as well as the kagu, to confidently resolve the phylogenetic position of the adzebills and estimate the timeline for their evolution.
4. Discussion
Our phylogenetic analyses unambiguously demonstrated that adzebills (
Aptornis; Aptornithidae) are crown gruiforms (
Figure 1,
Figure 2 and
Figure 3), and we reject the hypothesis that adzebills are close relatives of the Kagu (
Rhynochetos jubatus; Eurypygiformes) [
21,
22,
23,
24]. Instead, adzebills were found to form an early branch (~39.6 Ma) within Ralloidea as sister-taxon to the Sarothruridae, which we suggest comprises both the flufftails (
Sarothrura) and the Madagascan wood rails (
Mentocrex). The Aptornithidae-Sarothruridae clade was suggested by our analysis to form a clade with the finfoots (Heliornithidae), comprising the sister-group of the “true” rails (Rallidae) (
Figure 1,
Figure 2 and
Figure 3). This unexpectedly close relationship between some of the largest and smallest ralloids—adzebills and flufftails, respectively—is not the only instance of discordance between morphological and molecular based phylogenies of Ralloidea [
22,
33,
55,
75,
88]. For example,
Sarothrura was long considered a member of Rallidae, and
Sarothrura ayresi was originally described in the Rallidae genus
Coturnicops [
22,
88,
89]. In contrast, our analyses confirm previous suggestions that the African Grey-throated Rail (
Canirallus oculeus) and the Madagascan wood rails (
Mentocrex kioloides sspp.) are not closely related [
86,
89,
90], with the former in fact being a divergent member of Rallidae while the latter was closely related to
Sarothrura. The presence of species with “rail-like” morphology in both Rallidae and among heliornithoids (the name we give to the Aptornithidae-Sarothruridae-Heliornithidae clade) suggests that this represents the ancestral state for ralloids, including the ancestors of adzebills.
If the ancestors of heliornithoids resembled archetypal rails, this makes the morphological evolution of adzebills and flufftails, as well as the related finfoots (which are highly-specialised for foot-propelled diving) [
91], all the more remarkable. It is also notable that heliornithoids are arguably more morphologically and ecologically varied than the related Rallidae, despite a much lower species diversity [
22,
90]. All flufftails are diminutive, weighing only ~25–50 g [
89], whereas Madagascan wood rails may weigh up to ~280 g [
92], a more typical size for a rail. By comparison, the three species of finfoot range between 120–879 g [
92]. In contrast, adult adzebills are estimated to have weighed 16–19 kg (depending on the species), perhaps reaching up to 25 kg [
2,
3]. The fossil adzebill
A. proasciarostratus from the Miocene St Bathans deposits of New Zealand (16–19 Ma, the only major terrestrial Cenozoic fossil site in New Zealand outside of the Late Quaternary) was only slightly smaller than the two recent species [
14]. Thus, if Madagascan wood rails are used as a proxy for the size of the ancestor of adzebills, this suggests that the mass of adzebills increased >50-fold in ~20–24 Ma (the time between their estimated common ancestor with Sarothruridae—39.6 Ma—and the age of the St Bathans Fauna). If flighted ancestors of adzebills only arrived in New Zealand following the marine inundation of New Zealand during the Oligocene (peaking during the Waitakian 22–25 Ma) [
93] then the temporal window for their size increase would be even further compressed. Similarly rapid evolution appears to have resulted in the presence of flightless rails on numerous oceanic islands (especially taxa in the
Gallirallus-Hypotaenidia complex, or in the genera
Fulica, Gallinula,
Gallirallus, and
Porphyrio), many of which became quite large (e.g. the largest Quaternary Rallidae species—NZ’s extant
Porphyrio hochstetteri and extinct
P. mantelli—exceed or exceeded 3–4 kg) [
76,
81,
94,
95]. However, none of these rails rival the adzebills for size suggesting either that the adzebill ancestors may have exploited some unique circumstances or that insufficient time has elapsed for extant flightless taxa to reach comparable size (as many extant flightless lineages originated only during the Pliocene or Pleistocene) [
76,
81,
94,
95].
We had difficulty identifying morphological features that reliably distinguished heliornithoids from ‘true’ rails, which is perhaps unsurprising if the more derived lineages—such as the enormous adzebills and the aquatic finfoots—evolved independently from rail-like ancestors. Further, using morphological data to establish the sister taxon of adzebills is problematic due to the antiquity of their flightlessness and their unique feeding specialization, resulting in an “extreme autapomorphy” [
22]. By enforcing the
Aptornis-Mentocrex-Sarothrura clade in our reanalysis of Livezey’s [
22] morphological dataset, we showed that no extant rails were recovered as likely “heliornithoids” (including
Canirallus). However, it is plausible that other “rail” taxa may yet be revealed as heliornithoids rather than rallids. Genera currently lacking molecular data include
Gymnocrex (East Indonesia and New Guinea) and
Rougetius (East Africa), both of which have been considered early-diverging members of the Rallidae based on phylogenetic analyses of morphological data [
22,
88,
90]. In addition,
Rallicula from New Guinea, which also lacks molecular data, has been simultaneously considered a close relative of
Sarothrura and
Rallina (incompatible with molecular phylogenies that suggest
Sarothrura and
Rallina are only distantly related). All other unsequenced extant or recently extinct rail genera are island forms which are likely to have recent dispersal origins (e.g.
Aphanapteryx,
Cyanolimnas, Mundia, and
Pareudiastes) [
32]. Our results suggest that substantial work remains to refine the phylogeny of ralloids and that the automatic assignment of species to Rallidae based on “rail-like” morphology is questionable. The latter unfortunately means it may be impossible to correctly discern fossil rails from fossil heliornithoids, which complicates efforts to determine the early biogeography of Ralloidea and especially the geographical source of the adzebill lineage.
Our study reveals a major discordance between the phylogeny and biogeography of the adzebills and their relatives. Unless unrecognised representatives of Sarothruridae occur among extant “rails”, the closest living relatives of adzebills are exclusively Afro-Madagascan. Interestingly, this disjunct relationship echoes the sister-taxon relationship observed between the kiwi (Apterygiformes) and Madagascan elephant birds (Aepyornithiformes) [
41] and also between the Madagascan Teal (
Anas bernieri) and the New Zealand teals (
A. aucklandica, A. chlorotis, A. nesiotis, and
A. chathamica) [
42]. Like these examples, the relationship between the adzebills and Sarothruridae is unlikely to result from continental vicariance, since both the age of the divergence between Aptornithidae and Sarothruridae and the divergence between their common ancestor and Heliornithidae are much too recent (25.5–53.8 and 34.35–62.2 Ma, respectively), versus the separation of Africa and Madagascar from Gondwana >100 Ma [
96]. Instead, adzebills likely descended from an ancestral heliornithoid that dispersed to New Zealand overwater from another Gondwanan landmass. It is possible that this ancient dispersal occurred via Antarctica, as geological and palaeontological evidence suggest that at least some coastal regions of Antarctica (and nearby offshore islands) were unglaciated and experienced a temperate climate that supported southern beech (Nothofagaceae) forests until the end of the Eocene (reviewed by Askin and Spicer [
95]) or perhaps even the Early Oligocene (see Cantrill [
97]). The ancestors of kiwi and moa may also have arrived in New Zealand via Antarctica, as molecular dating results suggest they diverged from their respective nearest living relatives during the Eocene (e.g. Mitchell et al. [
41]). In any case, it is likely that “heliornithoids” were formerly more widespread and have subsequently become extinct across much of their former range (with the possible exception of the finfoot lineage, which presently has a pantropical distribution). For example, the Miocene ralloid
Paraortygometra porzanoides from France was suggested to have close affinities to
Sarothrura [
82]. It is possible that the highly derived morphology (and concomitant specialised evolutionary niche) of the adzebills may have contributed to its long persistence, preserving it from whatever environmental conditions promoted the high turnover of more “typical” ralloid lineages observed in the NZ fossil record [
98].
We estimated that the North Island and South Island adzebills only diverged relatively recently (0.2–2.3 Ma, with a mean estimate of 1.1 Ma). As the geological precursors of New Zealand’s North and South Islands were separated by the Manawatu Strait between ~30 Ma and ~2 Ma [
99], the age of the North and South Island adzebill lineages is incompatible with long-term endemism on both islands. Conversely, the divergence between the two recent adzebill species coincides closely with the formation of an isthmus (1.5–2 Ma) across the Manawatu Strait, which persisted until the modern Cook Strait formed around ~0.45 Ma [
99,
100]. Thus, it is likely that adzebills survived in the larger South Island of New Zealand—from which the fossil adzebill
A. proasciarostratus has been described (16–19 Ma)—and only dispersed into the North Island after the Manawatu land-bridge formed. A similar model has been suggested for several moa species, with the Pleistocene opening and closing of land connection between the North and South Island driving the divergence (1.45 ± 0.8 Ma) between North Island Giant Moa and South Island Giant Moa (
Dinornis novaezealandiae and
D. robustus, respectively), and between the North Island Mantell’s Moa and South Island Heavy-footed Moa (
Pachyornis geranoides and
P. elephantopus, respectively). As for moa, it is possible that any adzebill lineages endemic to the North Island during the Oligocene subsequently became extinct during the marine transgression (peaking around 23 Ma) that heavily reduced the land area of what would eventually become the North Island [
99]. South Island adzebills were slightly larger on average than North Island adzebills, a pattern common in other New Zealand birds with North/South Island sister-species (such as geese in
Cnemiornis or moa in
Pachyornis) and is consistent with Bergmann’s rule [
22]. Otherwise, both species were physically near-identical. It is therefore likely that adzebills occupied the same niche in the North Island as they did in the South Island, and apart from a slight change in mass, little to no macroevolution occurred since the species diverged.
In conclusion, our molecular data revealed adzebills to be crown ralloids, contrary to a long-standing hypothesis that they were close relatives of the New Caledonian kagu. The resolution of this centuries-old taxonomic issue implies that adzebills were not of Gondwanan vicariant origin, but instead descended from a rail-like bird that arrived in New Zealand by long-distance overwater dispersal during the latest Eocene. Consequently, the ancestors of the adzebill almost certainly became flightless after their arrival in New Zealand. Our new results—confidently placing the giant adzebills within ralloids—further reinforce Ralloidea as an ideal clade for studying the genomic drivers and consequences of flightlessness. The independent loss of flight in adzebills over 16 million years ago stands in stark contrast to the more recent (Pliocene/Pleistocene) losses of flight in extant island rail species.
Data Availability
Mitochondrial genome consensus sequences are available on GenBank (MK434259-MK434265). Unmapped sequencing reads and phylogenetic analysis files associated with this study are available on figshare (DOI:
https://doi.org/10.25909/5c5293c2ef984).