Evolutionary history of the Galápagos Rail revealed by ancient mitogenomes and modern samples

The biotas of the Galápagos Islands are probably one of the best studied island systems and have provided a broad model of insular species’ origins and evolution. Nevertheless, some Galápagos species remain poorly characterized, such as the Galápagos Rail Laterallus spilonota. This bird species is one of the less explored groups of endemic vertebrates on these islands, due to its elusive behavior, cryptic plumage and restricted distribution. To date there is no genetic assessment of its origins and sister relationships to other taxa, and importantly, there is no data on its current genetic diversity. This lack of information is critical given the adverse fate of island rail species around the world in the recent past. Here we examine the genetics of Galápagos Rails using a combination of mitogenome de novo assembly with multi-locus sequencing (mtDNA+nuDNA) from both modern and historical samples. We show that the Galápagos Rail is part of the ‘American black rail clade’, sister to Black Rail L. jamaicensis, with a colonization of Galápagos dated to 1.2 Mya. The separate analysis of cytb, ND2, and RAG-1 markers demonstrates shallow population structure across sampled islands, possibly due to elevated island connectivity. Additionally, birds sampled from Pinta possessed the lowest levels of genetic diversity, most likely reflecting the impact of past bottlenecks due to habitat loss caused by invasive goats grazing on sensitive habitat. The data presented here highlights the low genetic diversity in this endemic rail species and suggests the use of genetic data (both modern and historical) to guide conservation efforts.


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Studies of island biotas provide insights into the origin of species and the associated factors promoting it.

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The endemic Galápagos Rail Laterallus spilonota is an example of both this extreme adaptation to an 69 isolated system of oceanic islands and the fate of inconspicuousness, being the least studied land-bird species 70 on the Galápagos Islands. The historical distribution of Galápagos Rails has been documented by collectors 71 and naturalists since the early 1900s, allowing a reconstruction of the impact and decline of populations 72 compared to present data [22]. The introduction of rats and goats in the 18 th century, by mariners and early 73 colonists using these islands for water and food supply [23][24][25]

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Galápagos Rails are one of the least studied land bird species on the Galápagos: i) there is no genetic 85 assessment of its phylogenetic relationships to other rails, ii) there are no estimations of the time since its 86 colonization, iii) no inference of its phylogeographic patterns and inter-island genetic relationships, and 87 importantly iv) there is no assessment of its current genetic diversity. This last effort is critical if we 88 acknowledge the adverse fate of rails around the world in the recent past and thus, the uncertainty of its 89 evolutionary potential in the future.
Here, we focus on alleviating these aspects and bringing to light the evolutionary history of this 91 enigmatic endemic land bird of the Galápagos. We present data from a combination of fresh tissue samples 92 and century-old historic museum specimens collected by the California Academy of Sciences expedition to 93 the Galápagos in 1905-1906. The phylogenetic relationships for the Galápagos Rail proposed here for the 94 first time are based on high-throughput sequenced de novo assembly of its mitochondrial genome 95 (mitogenome), placing this species into a phylogenetic context. We also infer the timing of long-distance 96 colonization and focus on genetic diversity and relationships between islands. We focused the large-scale phylogenetic reconstruction on the generation of mitochondrial genetic data 100 from natural history collections. DNA extracted from toepad tissue allows mitochondrial genome assembly 101 at relatively low sequencing depth compared to DNA from nucleated avian blood cells, as the latter contain 102 significantly fewer mitochondria. While blood samples can be readily extracted from live birds, museum 103 specimens offered a better opportunity for destructive tissue sampling. Therefore, a series of Galápagos Rail 104 specimens deposited at the California Academy of Sciences (CAS) collected on Santa Cruz island in 1905-105 1906 were accessed and toe pads sections from ten individuals were loaned. Additionally, modern samples 106 (blood samples) were obtained from several islands to complement the genetic assessment of Galápagos

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Rails. The field work was carried out in four islands (May-July, 2017) and we concentrated our efforts to 108 the highlands (> 500 m). We sampled the islands of Santa Cruz, Santiago, Pinta, and Isabela. On each 109 locality we used playbacks to confirm the presence of rails as well as to define their territories. Birds were 110 captured using the V-netting with playback trapping method [38], which consists in the arrangement of 111 mist-nets forming a "V" and placed at ground level. Birds were lured inside the "V" using playback and led 112 into the mist-nets by two people that monitor and adjust dynamically to bird responses. From each 113 captured bird, we collected blood samples from the brachial vein in the field and blood was preserved on

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We mapped reads from the remaining samples to the circularized GR9 reference assembly in two steps 142 with the Geneious mapper. First we enriched for mitochondrial reads in a step using the setting 'low 143 sensitivity' with minimum mapping quality 15 and maximum mismatches per read 10% in two iterations.

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We then took the mapping reads and re-mapped them with the 'highest sensitivity' setting allowing up to  In addition to the high-throughput sequencing described above, we sequenced two mitochondrial and one 150 nuclear marker from a larger number of contemporary field samples. From each of the four visited islands 151 we sampled 60 individuals in total to provide more detailed population structure and intraspecific patterns 152 of diversification. Genomic DNA was extracted from blood cards using the Qiagen DNA Blood & Tissue

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Kit and following the manufacturer's protocol. We focused on the mitochondrial genes cytochrome b 154 (cytb) gene and nicotinamide dehydrogenase 2 (ND2), and the nuclear recombination-activating gene 1 155 (RAG-1). Amplification and sequencing of cytb was done using primers L14990-H16065 and primers 156 L5143-H6313 for ND2 following the protocols described in Bonaccorso et al. [43], and for RAG-1, we 157 used primers R52-R53 as described by Johansson et al. [44] (Table S1). Gel electrophoresis was used to   crown Gruiformes (i.e. the root of the tree) following an exponential distribution with rate parameter 8.5 and offset 52 million years, that was fitted from the oldest fossils records from different parts of the world.

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We used the fossil data compiled in Stervander (Table S2). The complete GR9 reference de novo assembly 219 produced by MitoZ was circularized and 17,045 bp long, with an average read depth of 61×. The GC 220 content was 42.4% and it contained 13 protein-coding genes, two rRNAs, 22 tRNAs, and a 1,526 bp long 221 control region ( Figure S1). For the four remaining samples, the average number of reads per sample that 222 mapped to the GR9 reference assembly was 81.9-345.7 (Table S2). Three samples (in addition to the complete mitochondrial assemblies whereas two samples (GR2 and GR7; ORN 259 and 268 CAS catalog 225 number repectively) contained ≤0.1% missing data.

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The five mitogenomes contained 27 variants, comprising 23 single nucleotide polymorphisms (SNPs) 227 and 4 insertion/deletion (indel) polymorphisms, distributed in protein-coding genes (15), control region 228 (11), and 16S rRNA (1; Table S3). Out of the 15 SNPs in protein-coding genes, 4 were in codon position 229 1 and 11 in codon position 3, with 12 being synonymous mutation and 3 non-synonymous (Table S3). All 230 variants were private to a single sample, with two exceptions: a synonymous SNP in COIII grouped GR7 231 and GR9 versus the three remaining samples, whereas an indel in the control region grouped GR9 and GR5 232 (reference haplotype), GR2 and GR8 (1 bp insertion), and GR7 (2 bp insertion; Table S3). Finally, there 233 was mononucleotide length variation in the beginning of 16S rRNA which was ambiguous and unresolved 234 due to low mapping success/coverage.  (Table 1). The MRCA of all four species was estimated 245 at a median age of 6.1 (stem) or 6.5 Mya (crown; for 95% HPDs, see Table 1), about a million years younger 246 than the ages estimated from the mtCDS dataset.    also ran alternative analyses in which the Rallidae calibration was applied to the crown node, with 262 yielding small differences of estimated node ages (see Table 2). Nodes that were time-calibrated are

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We report overall nucleotide diversity (p) of 0.0006 for cytb, 0.0007 for ND2 and 0.0001 for RAG-1.

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Values for each island for both Hb and p are reported in Table 2  Belgirallus fossil was considered a crown or stem rallid for the calibration of the dated tree (Table 1)

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Supplementary Information: The following are available as supplementary Information (pdf): Figure S1:

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Sequencing characteristics of mitochondrial genome,