Limited Introgression Supports Division of Giraffe into Four Species

Abstract

17All giraffe (Giraffa) were previously assigned to a single species (G. camelopardalis) and nine 18 subspecies. However, multi-locus analyses of all subspecies have shown that there are four 19 genetically distinct clades and suggest four giraffe species. This conclusion might not be fully 20 accepted due to limited data and lack of explicit gene flow analyses. Here we present an 21 extended study based on 21 independent nuclear loci from 137 individuals. Explicit gene flow 22 analyses identify less than one migrant per generation, including between the closely related 23 northern and reticulated giraffe. Thus, gene flow analyses and population genetics of the 24 extended dataset confirm four genetically distinct giraffe clades and support four 25 independent giraffe species. The new findings call for a revision of the IUCN classification of 26 giraffe taxonomy. Three of the four species are threatened with extinction, mostly occurring 27 in politically unstable regions, and as such, require the highest conservation support possible. 28 29 30 31 Keywords 32 Conservation, gene flow, hybridization, speciation 33 34 35 36 37 38 39 *

A conservative approach for species delimitation based on multi-locus DNA sequences: a case study of the genusGiraffa(Mammalia, Cetartiodactyla)

“…The program STRUCTURE was previously used to infer genetic structure in giraffe populations, using either genotypes from 14 microsatellite loci of 381 individuals [29] or PHASED alleles of seven introns for 105 giraffes [38] or rather the extended dataset of 21 introns for 137 individuals [12]. Brown et al [29] suggested the existence of at least six species, but the optimal K was not determined using either the method of Evanno et al [50] or that of Pritchard et al [44], and their results are not reproducible, because the microsatellite data were not made available.…”

“…Seven giraffe datasets were generated for our analyses using the sequences available in the NCBI nucleotide database: the mtDNA-G507 dataset, which contains a mitochondrial fragment covering the whole cytochrome b ( Cytb ) gene and the 5‘part of the control region (length = 1742 nucleotides [nt]) for 507 individuals (listed in Appendix B1.1), and its reduced version including only the 82 different mitochondrial haplotypes, named mtDNA-GH82; the mtDNA-GH82O3, in which the mtDNA-GH82 dataset was aligned to three outgroup species: Bos taurus (NCBI accession number KT184464); Ovis canadensis (NC_015889) and Okapia johnstoni (JN632674) (length = 1776 nt); a nuclear dataset, named nuDNA-G274, including 274 phased alleles of 21 introns (ACP5, C1orf74, CCT2, COL5A2, CTAGE5, CWF19L1; DDX1, DHX36, IGF2B1, MACF1, NOTCH2, NUP155, OTOF, PLCE1, RASSF4, RFRC5, SAP130, SOS1, UBN2, USP33, USP54) for 137 giraffes (accession numbers LT596685-LT598170, MG257969–MG262280); the nuDNA-G274O6, in which the nuDNA-G274 dataset was aligned to the alleles of three outgroup species: the okapi ( Okapia johnstoni , published sequences [12,38]), and two bovid species, (i) Bos taurus , for which the sequences were extracted by BLAST from the whole genome version UMD3.1.1 (http://bovinegenome.org/) or, in case of unavailability of certain genes, from the genome of Bos mutus available on NCBI (SAMN08580377); and (ii) Ovis canadensis , for which the sequences were extracted by BLAST from the genome available on NCBI (CP011888.1); the nuDNA-G137 dataset, comprising the alignments of original consensus sequences of 21 introns for the 137 giraffes (length = 16968 nt), which were recovered by detecting heterozygous sites in Geneious R10 (Biomatters, Auckland, New Zealand); the nuDNA-G137O3 dataset, in which the nuDNA-G137 dataset was aligned to the three outgroup species mentioned above (length = 17276 nt); the nuclear haplotype dataset, named nuDNA-GH274, which was inferred from the nuDNA-G274 dataset using the PHASE v2.1 algorithm implemented in the software DNASP v5.0 [39] (length = 1362 nt; it contains only the sites found to be variable between giraffe haplotypes). All alignments generated for this study were deposited in DRYAD (entry doi: XXXXXXX).…”

“…the nuDNA-G274O6, in which the nuDNA-G274 dataset was aligned to the alleles of three outgroup species: the okapi ( Okapia johnstoni , published sequences [12,38]), and two bovid species, (i) Bos taurus , for which the sequences were extracted by BLAST from the whole genome version UMD3.1.1 (http://bovinegenome.org/) or, in case of unavailability of certain genes, from the genome of Bos mutus available on NCBI (SAMN08580377); and (ii) Ovis canadensis , for which the sequences were extracted by BLAST from the genome available on NCBI (CP011888.1);…”

“…Indeed, the maternal inheritance of the mtDNA genome can be misleading for species delimitation because females and males have generally different dispersal behaviours (female philopatry versus male dispersal) [5,6], and because interspecific hybrid females are generally fertile, whereas hybrid males are often sterile (Haldane’s rule), facilitating mitochondrial introgression between closely related species [7–9]. To overcome these limitations, most recent taxonomic studies dealing with the delimitation between cryptic mammal species have focused on multi-locus datasets [10–12], as the use of multiple independent DNA markers has been shown to provide a strong and reliable signal for deciphering relationships among closely related taxa [13–14]. However, interpreting the results from multi-locus datasets can be difficult, especially when the DNA markers show low genetic variation or conflicting relationships between them.…”

“…However, the taxonomy of giraffes has been challenged by recent genetic studies: based on the analyses of mitochondrial sequences and 14 nuclear microsatellite loci, Brown et al [29] proposed a minimum of six species, corresponding to Giraffa angolensis , G. giraffa , G. peralta , G. reticulata , G. rothschildi , and G. tippelskirchi (N.B. the subspecies camelopardalis , antiquorum and thornicrofti were not included in their study); whereas Fennessy et al [38] and Winter et al [12] suggested a division into four species, i.e., G. camelopardalis , G. giraffa , G. reticulata and G. tippelskirchi , based on multi-locus analyses of 7 and 21 nuclear introns, respectively.…”