Gyps
Creators: name: Jeff A Johnson
name: Heather RL Lerner
name: Pamela C Rasmussen
name: David P Mindell
Publisher: name: Zenodo
Year: 2006
Subjects: Biodiversity
Taxonomy
Animalia
Chordata
Aves
Accipitriformes
Accipitridae
Gyps

description: Results [for Gyps]  
Sequence characteristics  
Among 60 representative individuals from the genus Gyps using complete mitochondrial (mt) cytochrome B (cytB) sequence data (1024 bp), 27 unique haplotypes were distinguished based on 81 variable sites (76 transitions and five transversions). Combined analysis of 2092 characters from mt cytB and NADH dehydrogenase subunit 2 (ND2), from a smaller set of individuals (n = 20), identified16 unique haplotypes based on 131 variable sites (121 transitions and 10 transversions) among ingroup taxa. For 400 bp of mt control region (CR), 15 unique haplotypes were identified for 20 individual Gyps vultures, including 29 variable sites (25 transitions, four transversions and one indel). When CR was combined with corresponding cytB and ND2 sequence data, 19 unique haplotypes based on 160 variable sites were observed among 20 individual Gyps vultures. Uncorrected percent sequence divergence between taxa was similar across loci with CR showing slightly higher divergence estimates; however, these differences were taxon specific with cytB or ND2 showing higher divergence estimates in some cases (Table 1).  
Nucleotide composition varied slightly between cytB and ND2 with both loci displaying lower levels of guanine (13 and 10%, respectively) and higher levels of cytosine (34 and 37%) nucleotides than expected by chance. CR also possessed lower levels of guanine (19%); however, it differed from cytB and ND2 in showing higher levels of thymine (32%) nucleotides. Tests for departure from homogeneity in base frequencies across taxa with and without uninformative mt characters were not significant for all three loci analyzed separately or combined (χ2, P> 0.05).  
Phylogenetic analyses  
The AIC identified the GTR+G model of sequence evolution[36] for analyses of both cytB and ND2. When partitioned by codon position, GTR+G, HKY+I, and HKY models were selected for each successive codon position (1st, 2nd, and 3rd, respectively) for cytB, and HKY+G, HKY+I, and GTR+I models were selected for each successive codon position for ND2. The CR was analyzed with equal weights among characters in all analyses. The same topology was found in both MP and Bayesian analyses  
irrespective of utilizing codon positions for the Bayesian cytB analyses and also for each of the two multi-locus datasets; however, the mixed models provided increased support indices at most nodes for all data sets, and therefore, only the support indices while utilizing codon partitions are shown for the Bayesian results (Figs. 2, 3).  
Regardless of dataset (single or multi-locus), monophyly of the genus Gyps and each species was strongly supported with high bootstrap support and posterior probabilities for each clade (Figs. 2, 3). The high number of nucleotide differences consistently observed between taxa further highlight these diagnostic relationships (Table 1). No geographic partitioning was observed within species or sub species possessing large samples sizes (i.e., G. bengalensis and G. f. fulvus; data not shown). However, within G. indicus, the Long-billed (G. i. indicus) and the Slender-billed (G. i. tenuirostris) vultures formed two separate monophyletic clades with high statistical support. Similarly, representative individuals of the two subspecies of Eurasian Vulture, G. f. fulvus and G. f. fulvescens were phylogenetically distinct; however, they were not placed as sister taxa. Both fulvescens samples clustered with the Himalayan Vulture (G. himalayensis; Figs. 2, 3). One of the two birds identified as G. f. fulvescens had an identical CR haplotype and differed by a single nucleotide from four of the six and all of the himalayensis haplotypes in cytB and ND2, respectively (Table 1; Additional file 1). DNA extractions for these taxa were conducted separately with multiple independent PCR amplifications to verify these results and to help rule out the possibility of contamination.  
There were a few differences in sister relationships among Gyps species when comparing results from different datasets(i.e., whether analyses were conducted for each locus separately or combined with others; Figs. 2, 3). The CR analysis identified monophyletic species similar to cytB and ND2; however, further resolution was limited with all species forming a single polytomy (tree not shown). When ND2 was analyzed separately (tree not shown), its topology was identical to that provided by the combined cytB and ND2 results (Fig. 3), while the topology for cytB alone differed from results given by the multi-locus datasets. In all analyses, the earliest divergence separated G. bengalensis from all other Gyps taxa; however, whether the next divergence is for G. africanus or G. himalayensis / G. f. fulvescens varies by dataset analyzed, with G. africanus divergence supported as the second divergence within Gyps by cytB and ND2 combined as well as the cytB, ND2 and CR combined dataset. All analyses supported a sister relationship between G. f. fulvus and G. rueppellii, with this clade sister to a clade consisting of G. i. indicus, G. i. tenuirostris, and G. coprotheres, and with the latter taxa forming a polytomy in the combined cytB and ND2 analyses without CR (Fig. 3A). In the multi-locus dataset including the CR (Fig. 3B), G. i. tenuirostris and G. coprotheres are posited as sisters with only weak statistical support.  
Long-billed Vulture morphological analyses  
Although the two taxa long classified as subspecies of "Long-billed" Vulture (G. i. indicus and G. i. tenuirostris) are similar in overall size, they differ markedly in proportions(Table 2). The rostrum of tenuirostris is much longer than that of indicus (as shown by culmen length and bill length from gape), while in indicus the rostrum is deeper and broader (as shown by bill width, bill depth, and maxilla depth). The longer skull and mandibular symphysis of tenuirostris is probably also a reflection of its relatively longer bill. The nostrils (nares length) of indicus are much longer than tenuirostris (reflecting the ovate shape of the nostril of indicus vs. the round nares of tenuirostris). In wing proportions, the "arm" (ulna length) and alula of tenuirostris are longer than for indicus while the "hand" (wing length) is longer in indicus. Lengths of individual primaries measured from the carpal joint did not differ significantly between the taxa and are not presented here. For the pes, most elements of tenuirostris are significantly longer than those of indicus, with the exception of the claws of digits 1 and 2, whereas pedal elements of indicus are proportionately more similar to those of tenuirostris in width and breadth measures.  
In a Principal Components Analysis (PCA), Factor 1 was a highly significant (P ≤ 0.001) shape axis distinguishing indicus and tenuirostris specimens (Table 3). Variables with high positive loadings on PCA Factor 1 were lengths of culmen, bill from gape, mandibular symphysis, alula, tarsus, tarsus proximal, tarsus distal, toes (pes digits), and depth of the claw of digit III. These variables contrasted with the strongly negatively loading nares length, and to a lesser extent with bill width, outer rectrix length, and width of the claw of digit III. Although the first six factors had eigenvalues above 1, component loadings of indicus and tenuirostris were significantly different only on Factor 1. Nevertheless, on this axis they were significantly different and readily distinguished (Fig. 4).  
Discussion  
Our objective in this study is to resolve phylogeny and taxonomic uncertainties for Gyps taxa, in order to inform current conservation efforts. By using museum specimens as DNA sources along with tissues obtained from the field, we sampled representatives of all generally recognized Gyps taxa with emphasis on those geographically distributed in south Asia; the primary area experiencing recent, drastic population declines. Our analyses support two changes to the traditional taxonomy for Gyps. First, two individuals identified as G. f. fulvescens were most closely related to G. himalayensis (Figs. 2, 3). Relatively high divergence estimates among all G. fulvus individuals (1.5- 2.5%, Table 1) and relatively low divergence estimates between G. f. fulvescens and G. himalayensis (0.0-0.6%) reflect this phylogenetic result. Additional sampling and The phylogenetic relationships found among Gyps vultures were largely the same for the different methods and mt datasets. Despite our finding of monophyly for the majority of Gyps species, relatively small sequence difference estimates (0.5-3.8%; Table 1) separating some named species made determination of sister relationships difficult, and multiple relationships were unresolved due to low nodal support. This suggests that the Gyps study taxa stem from relatively rapid and recent diversification events. If we use a generally supported avian mtDNA divergence rate for coding regions ranging from 1.6 to 5.0% change per million years (see [41]), our mt cytB and ND2 sequence divergence estimates (GTR+G; 0.8-3.4%), indicate that the radiation of Gyps vulture study species occurred 0.2 to 2.1 million years ago. These estimates must be considered with caution as they assume clock-like rates of sequence change, which is known to be violated in comparisons of some avian taxa and genes (e.g. [42 - 45]). However, we were not able to reject a hypothesis of clock-like behavior for our particular Gyps sequence dataset using a log likelihood ratio test (-ln Lclock = 3743.13, - ln Lnon-clock = 3731.94; 2Δln L = 22.38; d.f. = 18; P> 0.05).  
PatFenliongdutu i2rfro efsrotr4si cseoxrtesr noaf l Pmr ienncsipuarlalC cohmarpaoctne rnstsofA Gnyaplyss iinsd Ficaucstoarnsd 1G. Plot for scores of Principal Components Analysis Factors 1 and 2 for external mensural characters of Gyps indicus and G. tenuirostris. Gyps indicus and G. tenuirostris are significantly different (P ≤ 0.001) on Factor 1. Individuals with strongly positive scores on Factor 1 are tenuirostris, which have longer tarsi and toes, but narrower and longer rostra relative to indicus.  
Even if we assume that the above divergence rates are too high (see [45]), a lower rate (e.g., 0.6% per million years) still yields divergence times that are quite recent (<5.7 million years).  
These divergence estimates do not necessarily correspond with geographic proximity or the current distributions of species. For example, divergence estimates between G. indicus and both G. coprotheres and G. rueppellii are relatively low (0.9-1.3%; cytB & ND2 combined), yet the species compared occupy different continents. In contrast, divergence estimates between species with geographically proximate distributions, G. coprotheres and G. africanus in Africa and G. i. tenuirostris and G. himalayensis in South Asia (see Fig. 1) are relatively high (2.9-3.2% and 2.8- 3.1%, respectively).  
The historic radiation of this genus likely evolved in environmental conditions that no longer exist to the same extent throughout their current distributions. Gyps species are unique among Old World vultures in that they feed exclusively as scavengers, whereas other vultures are also known to kill their prey on occasion or, rarely, to feed on fruits (i.e., Gypohierax angolensis; [2,3,21]. This specialization in feeding behavior among Gyps vultures is thought to have evolved due to their close association with ungulate populations, particularly migratory populations in Africa and Asia. In fact, the observed temporal and geographic diversification of Gyps vultures coincides with the diversification of Old World ungulates, especially in the family Bovidae [46 - 50], and the expansion of grass-dominated ecosystems in Africa and Asia (see [51]). These close associations likely played a significant role in the adaptation and rapid diversification of Gyps vultures. Indeed, Houston [2] proposed that their large body size and ability to soar over large distances in search for food are related to the associated migrant distributions and seasonal fluctuations in mortality of ungulates, and that they have consequently become incapable of actually killing their own prey (see also [52]).  
Conclusion  
Both molecular and morphological data provide strong support for considering the "Long-billed" Vulture (G. indicus) to be comprised of two species, the Long-billed Vulture(G. indicus) and the Slender-billed Vulture (G. tenuirostris), with both considered critically endangered by the IUCN [1]. We found non-monophyly for our set of Eurasian Vultures, with both G. f. fulvescens individuals appearing more closely related to G. himalayensis than to G. f. fulvus, suggesting a topic for further analysis. Our phylogenetic analyses indicate the oldest divergence among Gyps species to be between G. bengalensis and the others, and conservative estimates suggest the diversification of Gyps taxa to be within the past 6 million years.  
The scavenging lifestyle of Gyps vultures and the decline of their historical food sources has likely contributed to their increased dependence on habitats heavily impacted by humans (see [3]). Many Gyps vulture populations have become increasingly dependent on domesticated animals, especially cattle, and this has contributed to their catastrophic decline in Pakistan and India, due to their secondary exposure to the veterinary pharmaceutical drug diclofenac (see [12,13,15,53]). Gyps bengalensis was fairly recently described as the most abundant large bird of prey in the world [4], yet, in as little as ten years, this species has become exceedingly difficult to find in the wild (see [54] for current trends).  
Determining genetic and evolutionary distinctiveness for Gyps lineages is increasingly important as a captive-breeding program is being established to prevent G. bengalensis extinction and other Gyps taxa are considered to be at risk or of uncertain status. Diclofenac susceptibility has been previously demonstrated for four Gyps species (G. indicus, G. fulvus, G. africanus, G. bengalensis [12 - 15]), and the relative recency of diversification and the phylogenetic position of these four known susceptible species each forming a sister relationship with at least one of the remaining taxa in this genus, support concern that the other Gyps taxa may be susceptible as well (see also [11,14]). The most obvious long-term solution to prevent their extinction is the immediate removal of diclofenac as a veterinary drug for domestic livestock. A recent study reported on findings suggesting that an alternative drug called meloxicam may serve as a surrogate to diclofenac without causing harm to Gyps vultures [11]. Fortunately, India has since banned the manufacture and use of diclofenac [55]; however, the drug is still available for veterinary use in Pakistan and vulture populations continue to decline.
description: Published as part of Jeff A Johnson, Heather RL Lerner, Pamela C Rasmussen & David P Mindell, 2006, Systematics within Gyps vultures: a clade at risk, pp. 1-12 in BMC Evolutionary Biology 6 on page 1